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

Input:
Title: THE BRAIN-DERIVED NEUROTROPHIC FACTOR VAL66MET POLYMORPHISM INCREASES SEGREGATION OF STRUCTURAL CORRELATION NETWORKS IN HEALTHY ADULT BRAINS
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The authors have satisfactorily responded to all my questions and made the necessary changes to the manuscript. I have no further comments.


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

Input:
Title: THE BRAIN-DERIVED NEUROTROPHIC FACTOR VAL66MET POLYMORPHISM INCREASES SEGREGATION OF STRUCTURAL CORRELATION NETWORKS IN HEALTHY ADULT BRAINS
Review round: 2
Reviewer: 2

Output:
Basic reporting: N/A
Experimental design: N/A
Validity of the findings: N/A
Additional comments: N/A


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: MEDIAL KNEE CARTILAGE IS UNLIKELY TO WITHSTAND A LIFETIME OF RUNNING WITHOUT POSITIVE ADAPTATION: A THEORETICAL BIOMECHANICAL MODEL OF FAILURE PHENOMENA
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The Authors present a study that aims to understand and predict cartilage failure as a function of time in a theoretical manner. They utilize state-of-the art modeling methods that combine motion (gait) analysis, musculoskeletal modeling of joint contact forces and discrete element modeling coupled with phenomenological modeling of material (here articular cartilage) failure. The reviewer commends authors on their approach which accounts for some limitations of previous approaches. However, the Reviewer does not fully agree with the conclusions that the authors present. Although, there are some issues that should be considered before this manuscript can be accepted for publication, the reviewer thinks that the results of this paper are very interesting.

Specific comments:

Introduction
Lines 64-67:  Why authors want only to focus on modeling tissue fatigue/failure? This is not necessarily the best approach as this neglects the fact that cartilage has at least different constituents that can fail (collagen or proteoglycan matrix). This is actually very relevant aspect as it is not currently known which component damages first (and/or the initiation can be OA phenotype dependent). The reviewer suggests that authors should also account for and present briefly some of the recent modeling approaches that actually aim at simulating and predicting the progression of OA. In these approaches, the compositional properties of specific tissue constituents (collagen fibrils, proteoglycan matrix) are assumed to degenerate. Some good examples are e.g.  Hosseini et al. 2014 (https://www.ncbi.nlm.nih.gov/pubmed/24185112), Mononen et al. 2018 (https://www.ncbi.nlm.nih.gov/pubmed/29150953) and Orozco et al. 2018 (https://www.ncbi.nlm.nih.gov/pubmed/30348953). It would also be beneficial to discuss the differences bwtween the approaches presented by the authors and these papers in the discussion section.

Methods:

This is a suggestion but the reviewer would prefer a general overview/methodological figure which illustrates the whole workflow and the geometries used. The reviewer finds it hard to think how the geometry of cartilage and meniscus looks in the model when seeing the figure 1.

Lines 130-136: Authors use non-linear elastic constitutive relationship (which is apparently a function of the logarithmic (or true) strain), however it seems that this relationship does not account for any rate-dependency (i.e. poro or viscoelastic behavior) so this reviewer thinks that saying this stress-strain relationship accounts for the rapid deformations is incorrect (although it is better suitable for large deformations as the authors mention).

What is composite modulus in your model? I think it is better to say dynamic/instantaneous/equilibrium modulus of cartilage/meniscus as this is what you are modeling and it avoids confusion.

Also, it is not clear why you have two indexes (i and j) for the number of elements. Further, I guess these indexes actually refer to the stress and strain matrix indexes, and this confuses the reviewer. I suggest revising the equations for clarity.

Lines 158-168: The authors fit Weibull cumulative probability function on data presented in Reimenschneider et al. 2019. As this fit is fundamental part of your paper, it would be good to mention R2-value and plot the resulting fit (with original data). I tried to plot the e.q. 4 and the result seemed odd, so this would be good to verification (it is also possible that the reviewer made a mistake). How unique was your fit (i.e. by varying initial values did you receive a similar fit and parameters) and what approach was used for fitting?

Also it is not clear for the reviewer if is there a "feedback" i.e. is modulus change if damage is occurring? If this is not present, it should be mentioned as a limitation as it would present a different course in the progression (most likely faster "complete" failure). And how was the adaptation actually incorporated, immediately in the initial material properties of the model or during the analysis?

Methods (Implementation and statistics): It is not clear for the reviewer how the motion was simulated as a function of time. Did you simulate one full gait cycle (or just stance phase) several times?

Discussion:
Lines 311-313: This sentence is difficult to grasp, could this be revised?

Lines 335-336: I guess it would be also good to mention that one (quite big) limitation is that authors neglect poro/viscoelastic properties of cartilage and meniscus that contribute to the rate dependency of the deformation.

Lines 352-363: The statements that the authors present is true only for the modeling approach that authors are using, and this should be clearly said.

Authors also mention that "running places high stresses on cartilage, and in a
358 healthy state these stresses trigger an adaptation response that extends the fatigue-life of cartilage". Although this might be correct, I am quite certain that this is not necessarily so simple as the fluid pressurization is neglected in the model. What were the estimated strains in cartilage (on average) during walking vs. running in these subjects, same or different? If fluid pressurization would be included I would assume that with otherwise similar material properties (lets assume, for simplicity, isotropic linear elastic) during running loading rate is faster and, thus, fluid pressurizes more (and fluid pressure inside tissue is greater) compared to walking, which could be harmful for cells (or even cause apoptosis) in the tissue or damage collagen network.

Further, I find it very challenging to accept that tibiofemoral radii would increase/decrease so substantially as the authors present (was this a change that was observed in running vs. walking people). Further, subchondral bone can remodel and adapt but I would assume that the bone mineral density (and thus modulus) is changing based on the over-/under loading of the cartilage and subchondral bone.


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: MEDIAL KNEE CARTILAGE IS UNLIKELY TO WITHSTAND A LIFETIME OF RUNNING WITHOUT POSITIVE ADAPTATION: A THEORETICAL BIOMECHANICAL MODEL OF FAILURE PHENOMENA
Review round: 1
Reviewer: 2

Output:
Basic reporting: The writing was clear and well structured. The figures were clear and easy to interpret.

The literature on the short-term training effects of running on cartilage was well summarized. However, no literature was mentioned that cross-sectionally compares runners to non-runners in terms of anatomy or bone densitiy which may give some understanding to the long-term adaptations that are relevant to OA.
Experimental design: Generally the study was well designed with clear hypotheses and subsequent evaluation.
Validity of the findings: The conclusions are adequately supported by the computational experiments performed in the study. The study obviously lacks experimental validation aside from a minimal comparison to epidemiological rates of OA, however this limitation is clearly stated in the discussion.
Additional comments: G1) The first hypothesis is based on contact stress (no difference between peak stresses in walking and running), whereas the second hypothesis is based on contact strain (no difference between damage in walking and running, where damage is a function of strain, Eq 4 & 5). The adaptations used to test the third hypothesis are evaluated based on damage (strain), but some would have opposite effects for stress (increasing elastic modulus decreases strain, but increases stress for a given input contact force). A better discussion on whether cartilage stress or strain is the important metric to OA development, and more explicit description that the damage model is based on strain could help clarify the reasoning behind your hypotheses.

G2) When comparing cartilage loading mechanics between walking and running, I would expect the viscoelastic properties of the tissue to be important due to the impact loading during running. The complex fluid-solid interactions that occur within the extracellular matrix ultimately determine the chondrocytes mechanical environment and biologic response [1]. Some justification should be provided that the joint contact model is of high enough fidelity to address the hypotheses being tested.
[1] Halloran, J. P., Sibole, S., Van Donkelaar, C. C., Van Turnhout, M. C., Oomens, C. W. J., Weiss, J. A., ... & Erdemir, A. (2012). Multiscale mechanics of articular cartilage: potentials and challenges of coupling musculoskeletal, joint, and microscale computational models. Annals of biomedical engineering, 40(11), 2456-2474.

G3) It appears that the geometry of the contact model does not scale with subject size, and that the absolute medial contact forces are applied to the joint contact model. It is generally assumed that joint contact forces should be compared across subjects by normalizing to body weight. In your method, if runner A weighs twice runner B, but they run with a similar peak knee contact force of 4x BW, you would apply double the force to the same geometry in the contact model, and thus expect double the stresses.


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: MEDIAL KNEE CARTILAGE IS UNLIKELY TO WITHSTAND A LIFETIME OF RUNNING WITHOUT POSITIVE ADAPTATION: A THEORETICAL BIOMECHANICAL MODEL OF FAILURE PHENOMENA
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript reads very well and structured very well. It meets PeerJ and any scientific journal standards.
Experimental design: 1)	Page 7 Lines 92-93: Maybe a little more information on the capture marker set, kinematics and inverse dynamics modelling is desirable.

2)	Methods: A data and modelling work flow chart might also be good.

3)	Page 7 Lines 113-116: Please provide a plot of the model estimated medial-lateral force with those from instrumented implants: I suggest doing this on your current figure 2. Please let the reader make a decision about the performance of the NMSK knee joint model.

4)	Page 8 Section 2.5.1: It would be nice to see how well the damage model fits Riemenschneider et al. (2019) data on a figure. Maybe this could be done in supplementary files. This would also give the reader a feel for the damage model being used.

5)	Section Damage and Repair models:

     a.	Rate of loading will affect the material properties since cartilage is poroviscoelastic structure. Running has a higher loading rate than walking, resulting in stiffer cartilage response, i.e. changing in the dynamic cartilage elastic modulus (see for example, Nia et al. J Biomech 48, 2015, 162–165). How will this affect Damage and Repair as modelled?

     b.	It would be good to see some figures showing how the Damage and Repair performs in isolation of the walking or running loading. Are there any experimental data that can be used for benchmarking?

6)	Page 10 Lines 224-225: Given that poroviscoelastic effects on cartilage dynamic elastic modulus and the effects on loss and increase in GAG’s, with the associated effects on cartilage elastic modulus, are not cartilage strains more important than stress and hence the third hypothesis is possibly different?
Validity of the findings: 1)	Page 11, line 258-259: I suggest change “Model validity was…” to “The validity of various sub-model components was…” Also see comments 4 and 5 in in Experimental design section: showing the performance of Damage and Repair models with literature would be beneficial.

2)	Page 11, Lines 268-269: How do you running loads compare to those reported in literature.

3)	Page 11, Lines 257-273: Peak knee loads from yours and other models may occur at different timings and knee flex-extension angles. Knee angles is important since this means a different region of the cartilage is loaded.

4)	Page 11, lines 274-275: Th first sentence of this paragraph is a Figure caption. I suggest rewrite to “The time to estimated medial tibiofemoral cartilage failure depended on how much walking and running was undertaken and the inclusion of repair (Fig. 4).” Or something similar.

5)	Page 11, lines 274-289: Rate of loading will affect the material properties since cartilage is poroviscoelastic structure. Running has a higher loading rate than walking, resulting in stiffer cartilage response. How will this affect the results given the relative mix of walking and running is examined, i.e. Fig. 4.?

6)	Page 12, Discussion, First Hypothesis: Focus on stress rather than strain – see comments 5 and 6 in Experimental design section.

7)	Page 12, Line 313: I think this should be Fig 5. not Fig 4.

8)	Page 13, Lines 364-387: The fact of dynamic elastic cartilage modulus, and its possible differences between walking and running could also account for reduced risk of cartilage damage in running. This fact may not require a repair model to be included – please comment.

9)	Fatigue model of damage: There are other proposed models of cartilage damage. For example, cartilage has hydrodynamic lubrication from fluid exudation which lowers friction and cartilage-cartilage contact preventing shearing off of cartilage surfaces.
Additional comments: Generally, this manuscript is where analyses should be taken to understand the development of knee OA, and it will add to the literature. I really enjoyed reading it. However, I encourage the authors to address the above 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: MEDIAL KNEE CARTILAGE IS UNLIKELY TO WITHSTAND A LIFETIME OF RUNNING WITHOUT POSITIVE ADAPTATION: A THEORETICAL BIOMECHANICAL MODEL OF FAILURE PHENOMENA
Review round: 2
Reviewer: 1

Output:
Basic reporting: -
Experimental design: -
Validity of the findings: -
Additional comments: The authors have done excellent job in their response and revision. I have no more comments. I am pleased to see this paper to be published in PeerJ.


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: EXPRESSION OF ASPERGILLUS NIGER GLUCOSE OXIDASE IN PICHIA PASTORIS AND ITS ANTIMICROBIAL ACTIVITY AGAINST AGROBACTERIUM AND ESCHERICHIA COLI
Review round: 1
Reviewer: 1

Output:
Basic reporting: This paper explains about cloning and expression of the enzyme glucose oxidase in the methylotrophic yeast Pichia pastoris and its applications. The experiments have been well framed and executed. Inhibition of the growth of Agrobacterium and Escherichia coli by varying the concentration of glucose oxidase and glucose has been well explained. The article however misses to explain key points relevant to the study. The materials and method section, results and discussion section needs to rewritten. The structure of the article conforms to the PeerJ standards. Nevertheless, the article needs to be further improved and lacks clarity. Figures and raw data has been provided. References should be checked once again as per PeerJ standard.
Experimental design: The objective of this article falls within the scope of the journal. The research questions have been well defined, however the presentation of results and methods needs to improved.
Questions to the author:
1.	The title may be modified as “Construction of Aspergillus niger glucose oxidase -transgenic Pichia pastoris and its antimicrobial properties”.
2.	What was the growth rate of GS115 and GS115-His-GOD? It is better to mention the growth rate in the abstract.
3.	The introduction part of the manuscript lacks earlier reports on GOD production in P. pastoris.
4.	Line 52-55 needs can be rephrased as “In this study, the gene coding for glucose oxidase from Aspergillus niger ZM-8 was expressed under the control of inducible alcohol oxidase 1 (AOX1) promoter in yeast Pichia pastoris. The antimicrobial property of the glucose oxidase enzyme was studied”.
5.	Line 57 – Title should be renamed as Strains and Plasmids
6.	Line 61 should start with “The linearized vector was inserted in frame ……… promoter in pPIC9k (invitrogen) that resulted in expression vector pPIC9k-His-GOD”.
7.	Line 66 – The electroporation was performed using a Gene pulser (Eppendorf) at 1.5 kV….”
8.	Line 75-81 should be rephrased as “One colony was picked among several high copy clones obtained from the plate containing P. pastoris GS115-pPIC9k-His-GOD. GS115-pPIC9k was used as a negative control for the experiment. Clones were inoculated in Buffered Glycerol-complex medium (BMGY) [1% (w/v) yeast extract, ………. , glycerol] and cultured at 30°C until an OD….”. The culture was then transferred to Buffered methanol-complex medium (BMMY) and cultured at 30°C. The absorbance of growing culture was measured every 3 hours”.
9.	Lines 83-89 should be rephrased as “The pichia strains were cultivated in BMGY medium at…. . Biomass was generated after an initial growth phase with glycerol as carbon source. Finally, to induce AOX 1 dependent protein expression, the methanol fed-batch phase was started with methanol ….. .  …………    The ultrasonication conditions used was ………..”.
10.	Lines 90 to 104 under analysis of glucose oxidase activity needs to completely rewritten.
11.	Lines 106-109 should be rephrased as “Protein concentrations of cell-free liquid, ………were determined by Brandford method. Absorbance was measured at 615 nm ….”.
12.	In Line 110 – Agrobacterium should be in italics
13.	Line 112 to be rephrased as “The concentration of glucose used were 1, 2.5 and 5 mg/ml respectively. Line 115 – …………pPIC9k as control. Concentration of GOD used was 1, 5 amd 10 U/mL.
14.	Maintain consistency for ml. It should be either ml or mL throughout the manuscript.
15.	Rephrase lines 120 -136
Validity of the findings: 1.	Line 140 - ……. described in materials and methods section to yield …….
2.	Line 143-144 – A positive transgenic clone, designated as P. pastoris GS115-His-GOD1 was grown on the YPDS plae with a high… and was considered for subsequent experiments.
3.	Line 146 should be rephrased as “Hydrogen peroxide, one of the products produced by GOD, injures living cells. In line with this statement, growth of the GOD-transgenic strain, P. pastoris GS115-His-GOD 01 was analyzed”.
4.	Line 147 should be rewritten as “Compared to P. pastoris GS115-pPIC9k ………. slightly decreased during 51st hour of incubation under GOD uninduced condition.
5.	Line 177 – 184 should be rephrased and written clearly. Fig 4 title and its explanation should be clear. What is A to F should be clearly mentioned .
6.	Did the author deduct the GOD activity of the control from the original value of the recombinant strain ?
7.	If glucose oxidase was produced by the GS115 host itself, what was the rationale behind choosing glucose oxidase from another organism ?
8.	Authors should try to explain in detail the novelty of the work in the discussion section. In what areas does this work standout from previous report on glucose oxidase and its antimicrobial properties? Compare with previous studies and discuss.
9.	Quote latest references. Some of the references are too old.
10.	Check references for PeerJ standards.
11.	In figure 1 remove 3h, 6h, 9h, 12h etc. from x-axis. Instead just mention 3,6,9,12….
12.	In figure 2, x-axis should be named as Cell-free supernatant, supernatant of cell lysate and precipitate of cell lysate. Activity of the supernatant of cell lysate is 18.2 U/ml but in fig. 2a it is around 16 U/ml only. Please check the bar graph. Error bars are missing in the bar graph.
13.	Error bars or Standard deviation is missing in figure 3.
Additional comments: It is a good work and the quality of the article can be improved with clarity in the explanation. The english language should be improved to ensure that an international audience can clearly understand your article. Therefore, I would suggest the author to submit the article for English language correction.


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

Input:
Title: EXPRESSION OF ASPERGILLUS NIGER GLUCOSE OXIDASE IN PICHIA PASTORIS AND ITS ANTIMICROBIAL ACTIVITY AGAINST AGROBACTERIUM AND ESCHERICHIA COLI
Review round: 1
Reviewer: 2

Output:
Basic reporting: The authors reported “ Construction of Aspergillus niger glucose oxidase transgenic Pichia pastoris and antimicrobial activity”
I regret to inform you that the work described in this manuscript does not greatly differ in properties from other glucose oxidases, and therefore the results are not very novel, although solid. All the text should be rewritten in good English. In fact, MS grammars and syntax should be revised with exact English usage.
Experimental design: No comment
Validity of the findings: No comment
Additional comments: No comment


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

Input:
Title: EXPRESSION OF ASPERGILLUS NIGER GLUCOSE OXIDASE IN PICHIA PASTORIS AND ITS ANTIMICROBIAL ACTIVITY AGAINST AGROBACTERIUM AND ESCHERICHIA COLI
Review round: 1
Reviewer: 3

Output:
Basic reporting: See general comments for the author
Experimental design: See general comments for the author
Validity of the findings: See general comments for the author
Additional comments: The manuscript " Construction of Aspergillus niger glucose oxidase-transgenic Pichia pastoris and antimicrobial activity" submitted by Wang et al. to publication in PeerJ describes interesting data about the expression of Aspergillus niger glucose oxidase in Pichia pastoris and especially antimicrobial activity of recombinant enzyme against Agrobacterium and Escherichia. But the manuscript is extremely poorly written and needs to be largely reworked before considering for publication in PeerJ. I saw many defects in terms of scientific writing and English grammar. Some sentences were written confusing and difficult to understand. The manuscript is checked with the help of an English language expert or service after that it resubmits for review process.
The Introduction must contain informative data about current work and not general and well-document data. Authors have to review following references in Introduction section: Aspergillus niger glucose oxidase was expressed in yeasts Saccharomyces cerevisiae (Journal of Biological Chemistry, 1990; 265:3793–3802; Applied Microbiology and Biotechnology, 2003; 61:502–511), Pichia pastoris (Protein Expression and Purification, 2007; 55:273–278; Letters in Applied Microbiology, 2014; 58:393–400; Biotechnology and Biotechnological Equipment, 2016; 30:998–1005), Hansenula polymorpha (Yeast, 1993; 9:625–635), Kluyveromyces marxianus (Microbial Cell Factories, 2010; 9:4) and Yarrowia lipolytica (Molcular Biotechnoloy, 2017; 59:307–314; World Journal of Microbiology and Biotechnology, 2018; 34:128). Then they clearly mention their aims and novelty of current work at the end of the Introduction section and also in the Abstract.
I also recommend authors use the references for discuss their results in the Discussion section.


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: SURVIVAL, GROWTH AND CARBON CONTENT IN A FOREST PLANTATION ESTABLISHED AFTER A CLEAR-CUTTING IN DURANGO, MEXICO
Review round: 1
Reviewer: 1

Output:
Basic reporting: The English used presents some failures, but which are salvageable, when correcting the authors. Literature references are insufficient, especially in the discussion section, it also presents author's writing failures, years and format in general. If the authors pay special attention to this section, the manuscript can be improved. The article has professional structure and the results are correlated with the hypotheses.
Experimental design: The work is original and is necessary for many species in Mexico.The methodological part requires specifying some details of the experimental design and other characteristics indicated in the writing.
Validity of the findings: In addition to the comments made in the original text of the article, the potential for carbon sequestration of both species would be lacking. The authors can achieve this by incorporating the accumulation rates of biomass-C, based on the amplitude of the growth rings and providing these values with reference to the hectare. This so that part of the conclusion is not speculated and the payment of environmental services is magnified by this concept.
Additional comments: Authors should be careful in the journal format, as the manuscript must be substantially improved.


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

Input:
Title: SURVIVAL, GROWTH AND CARBON CONTENT IN A FOREST PLANTATION ESTABLISHED AFTER A CLEAR-CUTTING IN DURANGO, MEXICO
Review round: 1
Reviewer: 2

Output:
Basic reporting: The paper addresses the role of reforestation (forest plantation) in carbon sequestration or climate change mitigation, which is an important subject in forestry. However, the authors did not completely develop the research insights in the Introduction, rather they included not relevant information (see specific comments below). They should state clearly the research question, for example, are there differences in the contribution of individual species in carbon sequestration? Is it reasonable to expect any synergistic response of both species together in the reforestations? In this sense the Introduction section is not complete.
The hypothesis is rather general and it was not fully proved since their findings does not suggests that reforestation, by itself, are efficient way of mitigating climate change or increasing carbon sequestration, because species response varies with site conditions, seedling history, etc.
Experimental design: Not clearly stated in terms of trataments effect. Please provide research questions and an explanation on effects tested in ANOVA.

For data analysis (Anova) the authors should state clearly what treatments effects are testing for and why. For example, why comparisons among species was carried out for most variables but not for survival? The authors could improve the Discussion section considering that Pinus cooperi survival was clearly lower than P. durangensis, since this might have important implications for results in all other variables.
Validity of the findings: I suggest to restate the Conclussions since most statements are not based on the study results.  For example, total survival percentages does not show species performance.
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: IDENTIFYING AGRICULTURAL PESTICIDES THAT MAY POSE A RISK FOR BIRDS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Good reading and rational.
Experimental design: Descriptions are sound and good to follow.
Validity of the findings: The topic is appealing the authors present an interesting case. The general trends over such good length of time are invaluable. My main concern regards the generality of compounds used since pesticides are a rather diverse set of chemicals with different properties. However, the authors did track down the main compounds and their likely impact in the system considered recognizing the limitations of the study. My concern refers on the estimate of toxic load likely grossly over-estimating the studied effect. On the other hand the reliance on only LD50 and LD50 extrapolated from other species is also a key shortcoming of the method. Below I detail a bit more my concerns.
Additional comments: I liked the study and argument. The text is persuasive and engaging and the concept of “toxic load” explored is interesting. However, some of the values estimated sound strange and unrealistic – toxic load in the trillion range, but resulting in so few lethal responses (sublethal not even considered although they tend to be larger) is very hard to defend and even contextualize since is not particularly realistic. This is my key concern. The use of a general standard such a “toxic load” is useful and its relative determination helps, but the exaggeration distorts reality leading to bias. I would cautious against that. That said, the authors do make a clear point even at the Abstract, recognizing some important limitations of the approach, which is welcome.

Some minor points:

Lns 50-52: this is not a good reason to justify how rigorous is the EU legislation as virtually every country in the world requires regular re-assessments or reviews of registered chemicals for agriculture use. Thus, please revise providing a more compelling reason;

Lns 55-59: it is more than that, as even assessing impact lower amounts in complex system is also difficult. High or low are relative terms and depends on the context and ecosystem. Improvements are possible, perfection is not, but some longer ter-tests do exist and sublethal assessment and multiple-species assessments are also been considered for inclusion in protocols for registration;

Lns 59-62: The multiple chemical exposure is an excellent point!

Lns 75-77: The mode of action does not change with the species, but the secondary modes of action and their relevance may. Please consider reviewing for higher precision and clarify;

ln 84: prey consumption is not direct exposure;

ln 105-111: Although recognizing the relevance of the study, the sell of of a particular species as indicator or sentinel species based on alleged representativeness, I am skeptical on the subject and more in favor of broader surveys covering different species over time and recognized the most affected, if significant effect is detected in such assemblage-level studies.

Lns 115-116: the catch is really recognizing the rational behind the “potential killing” that serves as basis for the “toxic load” to prevent value overestimation;

Ln 129-130: LD50 is a sound toxicological endpoint, but lethality-based and not accounting for indirect effects nor sublethal effects, both of which are important and likely more important than lethality itself. This shortcoming need to be recognized upfront since it is a conceptual oversimplification;

Ln 141-150: methodological OK I guess, but subject to gross mis-estimations since the data is not really known of the species under investigation;

Ln 155-158: This is bound to drastically overestimate the effect pursued. This is so because a rather small percent of the applied compound will actually reach the target assessed up to the point of causing its death. The “potential” here is with great capital letters…I do like a standardize way to draw attention to the load applied and potential effect, but the distortion here bother me as a gross over-simplification piled over the the species extrapolation; I do appreciate that the intent is not estimate bird death, but still the grossly biased estimation is a concern since it is far from realistic and prone to generate the wrong kind of controversy.

Just an example of the shortcoming: if LD50 is considered, mortality estimates based on pesticide load applied should reflect the “kill” of 50% of the population, not 100%...And this is just one issue on such estimates;[of course it does not make much different when one talks about the trillion range…; still, trillion…]

The qualitative trends observed seem OK and intuitive matching my own experience. I also appreciate the fact that the authors were candid in recognizing some of the limitations of the crude analysis provided. However, I believe my concerns remains valid.


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: IDENTIFYING AGRICULTURAL PESTICIDES THAT MAY POSE A RISK FOR BIRDS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Generally well written article - a few small grammatical errors.

This reviewer would have liked perhaps more discussion around pendimethalin and possible adverse effects - while it might be limited, it would help to make the link between the results of the assessment of toxic load and real adverse effects.
Experimental design: This is an interesting methodology for assessing the impacts of pesticides on non-target species over time.  There are obvious limitations to making specific conclusions on acute toxicity to non-target species - but the authors are careful to note the limitations.  The methodology is solid given the limitations.  Although the methodology related to extrapolation of LD50 values from other bird species to the bunting is clear, it might have been helpful to have provided an example calculation, say for chlorpyrifos or pendimethalin.
Validity of the findings: Within the limitations of the study, the approach provides an interesting way to assess the historical impacts of pesticide use on non-target species.
Additional comments: In the same vein as earlier comment about relevance of toxic load vs. real effects, it would be useful perhaps to tie results to specific actions that could be take to help better define true risk - e.g., what specifically should be done to further evaluate the true risk of pendimethalin use on non-target species.


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: IDENTIFYING AGRICULTURAL PESTICIDES THAT MAY POSE A RISK FOR BIRDS
Review round: 1
Reviewer: 3

Output:
Basic reporting: Several factual errors and serious gaps in the review of the seminal literature lead me to believe that the authors do not have a good grasp of the subject - pesticide effects on birds. I have included some of those details in 4. below.
Experimental design: Given the title, I was looking forward to reviewing this paper. Unfortunately, I have serious reservations. Beyond some possible errors in the choosing of toxicity values for the different pesticides (see detailed comments below), I have a more fundamental problem with the approach and believe the conclusions are simply wrong-headed and misleading. Fundamentally, I do not agree with the authors’ conclusion that: “… this is a useful approach to highlight pesticides that might be worth closer study with regard to possible impacts.” By impacts here, the authors mean lethal impacts on birds - as predicted by a lethal-toxicity-to-bird measure. Avian LD50 and amounts of pesticides used are the only two variables in play.

Indeed, this conclusion is already negated by this cautionary statement – with which I do agree: “We do not know the proportion of each compound that will be exposed to corn buntings or other non-target birds, therefore it is important to restate that this is not an attempt to estimate actual bird deaths. (At line 158)”

I should point out that, despite this caution, “potential corn buntings killed” is the tabulated measure and the paper purports to indicate which of the registered pesticides present the highest risk to birds on that basis. But this is more than semantics, and simply finding a different name for the calculated ‘toxic load’ would not satisfy me. The problem is more fundamental.

This analysis styles itself after similar ones on the toxic potential of pesticides to bees (Goulson et al. 2018; DiBartolomeis et al., 2019). In defense of the ‘toxic load’ approach in bees, it could be argued that, bees being insects, most (all?) insecticides applied in fields at levels necessary to kill insect pests are indeed likely to be causing some degree of toxicity and mortality in bees. Therefore, translating weights of chemicals applied into potential lethal doses is not quite as problematic – not ideal but tenable. The same cannot be said for the application of the concept in birds. By simply totaling up the number of LD50s, a massively used pesticide of low to middling toxicity could return a ‘risk value’ that is as high or higher than an acutely toxic pesticide used only in small amounts; e.g. carbofuran which probably was excluded from the analysis because only a small amount was used in the UK historically.  As used in this paper, the ‘toxic load’ approach is especially troublesome because all pesticides are included whether or not a toxic effect is even remotely possible. This includes herbicides (28.5% of the overall ‘toxic load’) and fungicides (15% of the toxic load). Some of these (e.g. pendimethalin – apparently the second-most dangerous pesticide to birds after chlorpyrifos), are used in large quantities but have never been shown to present any semblance of a direct risk to birds – even though lethal effects and debilitation are the only effects that can be predicted by an LD50 value.

I therefore do not agree that the method exemplified in this paper is a reasonable way to set priorities for looking at safety of pesticides to birds. Worse, the proposed method is likely to obscure which compounds do represent a real direct risk to birds – e.g some neonicotinoid seed treatments for which there already is field evidence.

I might be willing to believe the authors truly realise the limitations of their approach except that they do not seem to be aware of more serious attempts to quantify lethal bird effects as a function of pesticide toxicity. None of the key papers are cited. See for example my own contribution to the field:

“Mineau, P.  2002. Estimating the probability of bird mortality from pesticide sprays on the basis of the field study record.  Environmental Toxicology and Chemistry 24(7):1497-1506.”
“Mineau, P. and M. Whiteside. 2006. The lethal risk to birds from insecticide use in the U.S. – A spatial and temporal analysis. Environmental Toxicology and Chemistry 25(5):1214-1222.”

I should note that the approach documented above was used in the EU to ‘calibrate’ lethal risk assessment calculations based on LD50; viz.:“Anonymous. 2008. Scientific Opinion of the Panel on Plant protection products and their residues on a request from the EFSA PRAPeR Unit on risk assessment for birds and mammals. The EFSA Journal (2008) 734, 1-181.”

By reviewing the various toxic effects of chlorpyrifos, the authors also seem to be suggesting that their measured toxic load says something about sublethal toxic effects of pesticides. Unfortunately, there is no good evidence for this. Many of the more 'interesting' effects of pesticides on embryonic viability, for example, do not correlate with toxicity. There are too many numerous examples to cite - starting with DDT which has a very low acute lethal toxicity to  birds and other vertebrates!

The paper ends (Lines 254 onward) with a discussion of indirect effects of pesticides on birds – through insect removal. Although I do not disagree with the importance of insect removal for birds, this is – as stated by the authors – beyond the scope of this paper which looks at lethal toxicity to birds only as an endpoint. What I do object to is that the issue of indirect effects appears to be used here to justify the ‘toxic load approach’ espoused in the paper; e.g. why should pendimethalin have the second highest risk to birds after chlorpyrifos…. Viz: " ... studies on soil organisms found that pendimethalin reduced reproduction capability and number of offspring in springtails (Folsomia
250 candida), and reduced weight gain in earthworm (Eisenia fetida; Belden et al., 2005)" (At Line 248).

This is totally mixing apples and oranges and is nonsensical. The potential impact of pesticides to reduce the insect food supply of birds will look a lot more like the honeybee toxic load analysis – with a yet to be designed calculation of insect loss through vegetation control - a doubly-indirect effect. I am willing to bet that the results will look very different.

The issue of indirect effects is certainly no justification for the approach presented in this paper.
Validity of the findings: As outlined in 2. above, I do not believe that the findings have any validity and may seriously mislead and/or hamper the search for truly damaging pesticides; Viz: “Our analysis suggests that some groups of pesticides that generally receive little attention with regard to risks to birds should be given more scrutiny, including herbicides such as pendimethalin, fungicides such as mancozeb, and molluscicides such as metaldehyde." (At line 275)
Additional comments: Line 65. “(DDT) and its metabolite dichlorodiphenyldichloroethylene (DDE), a seed treatment commonly used between the 1940s and 1980s…”
I believe this is factually incorrect. DDT was not used for seed treatments – it certainly was not its main use. A number of cyclodiene organochlorine insecticides – e.g. Aldrin, dieldrin, chlordane – were used as seed treatment insecticides.
Lines 66-67.  “It was shown to cause eggshell thinning in raptorial birds (Rattner, 2009) and several marine species were also lethally affected (Pašková, Hilscherová, & Bláha, 2011).”
I am assuming you mean marine bird species here? Otherwise, it is off-topic. If so, the statement is also  largely incorrect. Eggshell thinning also extended to a number of marine species in order Pelicaniformes. Lethal effects with DDT were rare – only seen in cases of extreme starvation or in a few cases of very high application rates and earthworm contamination – e.g. American robins. Most of the lethal effects from organochlorines were from cyclodienes. It was the combined effect of DDE eggshell thinning and the acute toxicity of cyclodienes that proved catastrophic for a number of bird species.
By the way, I would argue that the effects of OPs on birds probably predated the DDE/eggshell story which took a while to unravel. Lethal effects on birds from OPs were documented very early indeed – see Silent Spring.
Line 74: ‘were’ banned
Lines 75-76. “While the mode of action of pesticides on target pest species is often well understood, little is known about their effects on other wildlife such as birds.”
Having published on the impacts of pesticides on birds for the last 40+ years, I would argue this is a ridiculous statement to be making as worded.
Lines 87-90. Not a proper sentence
Line 107. There is direct evidence in birds that size has an effect … but not how Peters envisioned it. Actually the opposite … so am not sure what part of Peters you are citing.  See: “Mineau, P., B.T. Collins and A. Baril. 1996. On the use of scaling factors to improve interspecies extrapolation of acute toxicity in birds. Regulatory Toxicology and Pharmacology 24:24-29.” This is before any consideration of the importance of body size on food intake.
Aaah. I see you do cite that paper later on. So you are aware that scaling does not follow the mammalian 2/3 or ¾ rule as envisioned by Peters and which forms the basis of most mammalian toxicology extrapolations.
Line 130. “Whenever available, we used 48 h oral acute toxicity LD50 values”
There is no such test for birds. 48 h is often used in the aquatic field to denote exposure time for measurement of LC50s.
Line 134. “For 54 pesticides, we used an average of LD50s from multiple unspecified bird species given in Mineau et al. (2001).”
Actually, I do not provide average values in that publication. Are you using the median or the HD5? Also, what are you using for the other 240 chemicals in the analysis? – presumably a single value given that you apply bodyweight scaling!? If you read the paper you are citing (Mineau 2001), you will see why it is difficult to compare toxicity among compounds with differing amounts of test data. As described, the methodology used is not at all clear.
Line 144: “In 1996, they calculated factors for 36 pesticides and found the average slope to be 0.5mg/kg.”
The 0.5 mg/kg value is not a slope term …not sure where it comes from. In fact, in our 1996 sample, we obtained an average slope of 0.15. In the larger 2001 sample, we got 0.239. … Viz: “For the 130 pesticides with n ≥ 4, the regressions were positive in 99 cases and the overall mean slope was 0.239; this was slightly more extreme than the value of 1.15 (corresponding to 0.15 when expressed on the basis of mg/kg) reported by Mineau et al. for a subset of 36 pesticides.(Mineau et al. 2001)”

So … the methods section leaves me wondering what procedure was used when several LD50 values were available. How could you do a body weight correction for the 54 pesticides for which a (median?) value was obtained from our 2001 paper? Is this comparable to the values you generated for the remaining 240 pesticides? How so?
Line 224. “However, we cannot be sure that applications to farmland in the UK are causing harm to birds.”
There are a number of field studies that do show impacts from chlorpyrifos when used as a spray. If memory serves, the supplementary material reviewed in the 2002 article cited above should have those references. Those studies were also reviewed by EFSA in 2008.


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

Input:
Title: IDENTIFYING AGRICULTURAL PESTICIDES THAT MAY POSE A RISK FOR BIRDS
Review round: 2
Reviewer: 1

Output:
Basic reporting: .
Experimental design: .
Validity of the findings: .
Additional comments: My fundamental issues with the methodology persist. I tried to explain my thinking re. the difference between a 'toxic load' approach for insects exposed to insecticides only (our PLOS One paper) vs. birds being exposed to all sorts of products ... some of low toxicity but very high volume/popularity.

Unfortunately, this led to the following comment in your rebuttal statement: "We are somewhat puzzled by the reviewer’s negative response to this approach, since it is almost identical in approach to a paper Prof Mineau published in PlosONE last year" .... a comment I found a little disingenuous as I think a clear reading of my comments should have removed any puzzlement.

But I see I will not succeed in convincing you. I do believe you need to clarify your methodology however. As I pointed out, the largest degree of uncertainty comes from the wide variation in toxicity between species. The body weight scaling issue is a minor one in comparison. You deal with the latter - which is good - but it still is not clear what departure point you are using to set a toxicity value. In your rebuttal comments, you mention taking the bobwhite as the chosen species. This is not what the text says. In any case, using a single species approach brings much more uncertainty to the calculations. That was the reason behind our compendium and we provide the factors necessary for a (quasi) distributional approach where too few species have been tested... but it doesn't look like I will convince you of that either.

In any case, I would ask that you please remove the mention that I was a reviewer. This does give the impression of an endorsement.


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: REGIONAL DISTRIBUTION OF CHRISTENSENELLACEAE AND ITS ASSOCIATIONS WITH METABOLIC SYNDROME BASED ON A POPULATION-LEVEL ANALYSIS
Review round: 2
Reviewer: 1

Output:
Basic reporting: Comments:
Please review to improve clarity as noted below and see suggested figure modifications.
Line 17: change to ‘gut microbiota’
Line 23: “…analyzed by various bioinformatic methods” is ambiguous. Please restate or remove.
Line 25: “…a greater complexity of the co-occurrence network” is unclear. Please restate your meaning. More taxa co-occurred with Christensen	ellaceae? Greater than what?
Line 32-33:  “…which provides a good explanation for the negative relationship between body weight and Christensenellaceae.” Overstatement of correlative results; please reword to “which may explain the negative relationship.”
Line 37: “…application of Christensenellaceae as a probiotic.” Christensenellaceae is a broad and diverse family of bacteria, whereas probiotics are delivered as strains. Please restate as “investigation of strains within Christensenellaceae as potential probiotics” or similar.
Line 53: “Specific microbial contributions…” Microbial metabolites?
Line 60-61: where the type strain Christensenella minuta was first isolated from…
Line 80: The term sOTU commonly refers to ‘sub-OTU.’ Please provide a citation to the definition of ‘sequential’ OTU if that is your meaning, or modify to ‘sub-OTU’ throughout
Line 196: Please rephrase – suggests Christensenellaceae was a treatment

Figure 3B: Needs additional description to what this represents. Please indicate if only significant correlations are shown and what p-value. What do the numbers and colors on the circle mean?
Figure 4B: Would benefit from a legend that indicates the range of values the dots represent
Figure S1 E: Needs units – distance or % similarity? Why do four of the sOTUs appear to be 100% identical?
Experimental design: Comments:
Line 89: Clarify the criteria process for removal/inclusion. 6896 people were included (some removed for missing metadata?), then in Line 101 you state 6879 (additional missing metadata?)
Line 93: ‘diabetes and early symptoms’ (pre-diabetic?)
Line 112: Explain what is the ‘external standard’
Line 124: ‘quality scores were then discarded’ is unclear. If you mean filtered by the Phred quality score, then please state the threshold (e.g. q20).
Validity of the findings: Comments:
Line 152: Please check “17083 quality-filtered sequences were obtained.” Does that refer to the average sequences per individual? Or number of Christensenellaceae sequences? Could you add the average Christensenellaceae abundance of the Q4 group?
Line 163: Please state statistical test or R package in methods that generated the R2 and p-value (e.g. PERMANOVA)
Line 172: units = sOTUs? Please keep consistent throughout.
Line 174: Please describe the software or tool used to construct the evolutionary tree in the Methods section.
Line 211-212: Overstatement of correlative results. Please rephrase.
Line 216-218: More appropriately moved to the Discussion
Line 221: Add reference
Line 227: Make clear that these are the most abundant sOTUs, with reads greater than 1000
Line 293: Please reword. The methods applied are unlikely to resolve strains.
Line 305: The mean age between the groups was 2 years difference, and overall not an ageing or elderly population. This conclusion would be better supported if age were used in the correlation analyses (Fig 3A).
-Please check for inappropriate usage of term ‘probiotic.’ See Hill, C., Guarner, F., Reid, G. et al. The International Scientific Association for Probiotics and Prebiotics consensus statement on the scope and appropriate use of the term probiotic. Nat Rev Gastroenterol Hepatol 11, 506–514 (2014) for criteria.
-The lower abundance of Christensenellaceae found in urban populations is interesting, and not well-addressed in the discussion. Do the authors find any literature to support this finding? Are there obvious lifestyle or dietary factors that may help to explain?
Additional comments: This manuscript represents a sub-analysis of a previously published dataset from the GGMP study. The strength of this study is the very large subject number and sampling that occurred from different geographic regions in China. The analyses completed are straight-forward and highlight interesting correlations between the abundance of Christensenellaceae and MetS. A limitation of the study is that it is observational, and the associations to metabolic health are correlative. The authors have identified some potential targets that may warrant further testing in efficacy-type animal models.


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: REGIONAL DISTRIBUTION OF CHRISTENSENELLACEAE AND ITS ASSOCIATIONS WITH METABOLIC SYNDROME BASED ON A POPULATION-LEVEL ANALYSIS
Review round: 2
Reviewer: 2

Output:
Basic reporting: This manuscript is clearly written although there are some spelling and grammar mistakes to correct. The literature referenced needs to be completed as listed below.

The raw data are not presently listed and should be added prior publication.

INTRODUCTION:

The introduction misses to reference a number of important publications.
Line 50: the reference 2019 is missing
Line 53: on the topic of microbiota, diet and gene interactions, the excellent review by Zmora et al. published in 2019 in Nat Rev Gastro Hepatol could be added along Ussar et al 2015
Line 62: please replace the reference to the review (Waters and Ley) by the original work by Morotomi and co-authors (2012) who named the new Christensenellaceae family after Prof Henrik Christensen and correct spelling (not Henkry)

Please replace the word “race” by “ethnicity” lines 70 and 71. The link with ethnicity is actually very well illustrated by the study by Brooks et al 2018. This reference should also be mentioned along Deschasaux et al.

Line 73: relationships with BMI have been reported by many other studies. Add at least a few such as Peters et al. 2018, Fu et al. 2015 Circulation Research, Oki et al. 2016 BMC Microbiology

Line 74: evidence of associations between Christensenellaceae and IBD have also been reported more recently by Braun et al. 2019 in the American Journal of Gastroenterology

FIGURE 1D: Cladogram is too small to be read; increase size or remove

TABLE 1: Units are missing

SUPPLEMENTARY FILES:

A list of concomitant medication in the metadata table provided would be very useful
Experimental design: Major comments:

1- To perform a comparative study of 2 groups, the authors chose to divide the cohort between a group where no Christensenellaceae were detectable, named Q1, and another extreme of the population distribution, named Q4. The authors describe these 2 groups as 2 quartiles (lower and upper quartiles of the distribution), although these actually do not correspond to quartiles. The population stratification must therefore be clarified. In addition, a better description of the Christensenellaceae distribution should be added, including overall median value, standard deviation, maximum value…

2- The authors identified several OTUs within the Christensenellaceae family that they used to make an evolutionary tree. Yet, the evolutionary tree does not include any reference V4 16S of the known Christensenellaceae members. In particular, the tree should include the reference genome of C. minuta strain DSM22607, which is the original type species strain of reference. Other species to include are C. timonensis, C. massiliensis and C. hongkongensis.

The Christensenellaceae OTUs sequences must be published along the paper since they form a major part of the present manuscript.

3- The authors report interesting variations between various geographical locations within Southern China that they relate to variations in “economically thriving centres”. A map identifying each region showing such variations in Christensenellaceae along a marker of economical health, such as the degree of urbanisation, would be very useful to appreciate this result.

Minor comments in MATERIAL AND METHODS:

Line 92: clarify what “metabolic index” means; should this be replaced by “metabolism”?
Line 93: diabetes is cited twice
Validity of the findings: A few sentences in the results and discussion are overstated or in contradiction with some results as listed below:

RESULTS:

Line 196: change title to avoid overstatement. Correlation is not causality.

Line 211: this is also overstated, especially since there is no indication of an impact on LDL or on HDL/LDL ratio.

Line 281: “methane metabolism”; this is in contradiction with fig 1 that shows methanogens to be increased in Q4. Can the authors comment on this in their discussion?

Line 283: “low risk of diabetes mellitus”; this is contradicted by figure 3B showing a positive correlation between some OTUs of Christensenellaceae and T2D

DISCUSSION:

Line 293: “strains” and “species”; no strains nor species were studied in this work; replace “strains” by “OTUs classified within the Christensenellaceae family”; “key species” could be replaced by “keystone taxon”

Line 295: “metatranscriptomic and metabolomic analyses…” ; Neither metatranscriptomic, nor metabolomic analyses were performed in this work, so I suppose the authors are making some suggestions for future work here. Therefore this statement should be moved at the very end of the manuscript as a suggestion for future work.

Line 311: “contributes”; no causation relationship was evidenced in this study; this should be replaced by something along the line of “was significantly associated with…”

Line 315: Reference to Fu et al. 2015 Circulation Research could be added here along Peters and Waters

Line 325: Oscillospira and Ruminococcaceae have been regularly reported as positively correlated with Christensenellaceae in other studies such as Goodrich et al 2014

Line 338-340: This is an overstatement; the PICRUST analysis only predicts biochemical pathways potentially modulated in the microbiota population, not in the host. Therefore this statement is a shortcut between microbial metabolism and potential host targeted effects. mTOR stands for “mammalian Target Of Rapamycin”… This is a perfect illustration of an abusive use of the KEGG database by the Picrust software. The authors must adjust their analysis, either to remove pathways that are not found in microbes, or by adding a statement in the discussion to explain this odd association and remove reference to endogenous metabolic regulation.

CONCLUSION:

Line 353: “probiotics” is not fully appropriate here since many of the bacteria it refers to have not been developed as food. Suggested change is to replace with “beneficial bacteria”
Additional comments: Minor comments:

Line 152: “17083 quality-filtered sequences”: is this per individual?
Line 167: replace “genera” by taxa
Line 254: what is a “normal” relationship? Replace normal by “expected”
Line 270: “The first principal component…” This statement is incorrect. This only means that 71.20% of the variation in the dataset is captured by PC1. “Between” can be replaced by “within” to make this correct.
Line 275: suggestion to replace “altered” by “different”
Line 308: what is “bioavailability” referring to? This should be clarified
Line 323: suggested change: “microbiota was” by “bacteria were”
Line 327: this sentence is unclear; suggested change: replace “for host fat consumption” with “to limit high fat-induced body weight gain”
Line 330: “block” could be replaced with “alter”
Line 356: suggestion: replace “indicators” by “alterations in the gut microbiota community”


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: REGIONAL DISTRIBUTION OF CHRISTENSENELLACEAE AND ITS ASSOCIATIONS WITH METABOLIC SYNDROME BASED ON A POPULATION-LEVEL ANALYSIS
Review round: 3
Reviewer: 1

Output:
Basic reporting: This is a re-review. I will only comment on specific points already raised in review 1.

All points raised here in review 1 have been addressed.
Experimental design: All points raised here in review 1 have been addressed.
Validity of the findings: Line 211: this is also overstated, especially since there is no indication of an impact on LDL or on HDL/LDL ratio.
Response: We have restated the result to “The level of HDL was significantly higher in people with Christensenellaceae, indicating that the bacteria was negatively correlated with blood lipid level.”

This response is not satisfactory since an association with increased HDL is not an indication of a negative correlation with circulating lipid levels. I suggest to rather focus on the fact that low HDL levels are one of the criteria of metabolic syndrome and therefore, a positive correlation with HDL is indicative of a healthier metabolism.
Additional comments: Thank you for your consideration of my comments and for adjusting the manuscript accordingly.


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: TAXONOMIC AFFILIATION INFLUENCES THE SELECTION OF MEDICINAL PLANTS AMONG PEOPLE FROM SEMI-ARID AND HUMID REGIONS—A PROPOSITION FOR THE EVALUATION OF UTILITARIAN EQUIVALENCE IN NORTHEAST BRAZIL
Review round: 1
Reviewer: 1

Output:
Basic reporting: The basic structure of the ms is just fine.
Experimental design: The design of the study is interesting, but does suffer from some potential shortfalls:

Given that the authors only included "knowledgeable" participants, the exclusion of plants and applications that are only named by one participants considerably reduces the sample. IN case of lay participants this would be ok.In case of "expert informants" this is problematic.

The overall sample of the study is relatively small, as it has shown that in ethnobotanical free-listing a much higher participant number is needed to attain a complete knowledge overview.

The use of plant families is problematic, because family taxonomy has changed considerably over the last decades (see APG 2 vs APG 3 vs APG 4), i.e. the affiliation of a large variety of genera to respective families has been changed. Thus, the same data might give other results if a different taxonomic concept is used. This should be at least discussed.

In case of genera, the concept is ok. However, the authors should either exclude introduced species (as knowledge about them would have been introduced together with the species in many cases), or it should be at least discussed.
Validity of the findings: See the above discussed problematic of not including individual mentions, and the problematic of taxonomy and species mintroductions.
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: TAXONOMIC AFFILIATION INFLUENCES THE SELECTION OF MEDICINAL PLANTS AMONG PEOPLE FROM SEMI-ARID AND HUMID REGIONS—A PROPOSITION FOR THE EVALUATION OF UTILITARIAN EQUIVALENCE IN NORTHEAST BRAZIL
Review round: 1
Reviewer: 2

Output:
Basic reporting: While I support the publication of this work, there are some aspects needed to be addressed. Particularly, about the biocultural significance of the objectives of the study.

Introduction
Lines 46-56. In my opinion it is necessary a more extensive presentation on the subject that puts better focus on the problem of study. For example, a definition of local medical systems and their the biocultural significance are needed.
Experimental design: In my opinion, there are some aspects needed to be addressed.
For example:
Study area
Line 186 and line 481.  The four communities appear to be affected by various socio-cultural factors that make them very different from each other. The fact that they are rural, more than 10 km from the city and close to a natural area are not, in my opinion, sufficient reasons to make them comparable in the terms proposed in the article. Please justify better how these situations are really comparable.
Data analysis
The fact that only 64 species were left for statistical analysis does not seem to me to be representative of the medical systems of each place, so what is really being analyzed? Please justify the logic behind the quantitative analysis. The medical systems aren't being dissected much?
I think it would be interesting to test how the pairs vary by comparing the similarity data between the 4 communities. Perhaps the number of plants in common is lower but could add some light to the ideas being discussed.
Line 266. Please, include the definition of ethnospecies.
Validity of the findings: In my opinion, there are some aspects needed to be addressed.
Legend of Tables, replace sex by gender.
Line 403. The statement "revealing that the selection of medicinal plants does not occur in a random way" is not very obvious considering the existing literature about this topic? Please, re edit and explain the biocultural significance of this results.
Line 419. Why the authors think that this is impossible? Mass media, exchange by virtual communications are an important force of exchange of plant information worldwide that cross countries and borders.
Additional comments: This paper is creative and imaginative. It focuses a new light on some elements of the field, and will lead to new avenues of investigation in some areas of ethnobotany. The manuscript is a very interesting text that follows the line of work of the research group, very relevant because it allows the analysis of new theoretical frameworks for the ethnobotanical discipline considering ecological inputs as structural frameworks.


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: TAXONOMIC AFFILIATION INFLUENCES THE SELECTION OF MEDICINAL PLANTS AMONG PEOPLE FROM SEMI-ARID AND HUMID REGIONS—A PROPOSITION FOR THE EVALUATION OF UTILITARIAN EQUIVALENCE IN NORTHEAST BRAZIL
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have addressed all comments of this reviewer sufficiently and the ms is now acceptable for publication.
Experimental design: The authors have addressed all comments of this reviewer sufficiently and the ms is now acceptable for publication.
Validity of the findings: The authors have addressed all comments of this reviewer sufficiently and the ms is now acceptable for publication.
Additional comments: The authors have addressed all comments of this reviewer sufficiently and the ms is now acceptable 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: CONVOLUTIONAL NEURAL NETWORKS TO AUTOMATE THE SCREENING OF MALARIA IN LOW-RESOURCE COUNTRIES
Review round: 1
Reviewer: 1

Output:
Basic reporting: The authors presented the background very clearly, with up to date statistics. They reviewed widely and provided good summary of previous works on CNNs for malaria detection.

Figure 4 presents too much information, most of which not referenced in the main text. These figures may be presented as supplementary instead.
Experimental design: While the research area is not new, the authors defined a relevant gap and presented a sound machine learning pipeline.

The experiments done were very rigorous and commendable.

While the topic and application of resolution upscaling is interesting and good results were demonstrated, the motivation behind it is not clear in the text. The flow of the narrative would greatly improve if reasons are explicitly stated. Otherwise, the section seems a bit separated from the rest.

The section on the integration to the mobile platform seems insufficient. One of the main ‘selling point’ is to be able to run this system in low-resource countries but very little weight was given to this topic. Perhaps additional implementation details and a review on other CV apps on Android may help strengthen the section.
Validity of the findings: Performance metrics were defined well and although previous works performed better, the authors provided sound reasoning as to why it was the case and why their model is still worth looking into.
Limitations were also stated.

The conclusion involves code modularity but this is not well-argued in the paper. While there are obvious components in the proposed platform, the replaceability of each is not well-established.
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: CONVOLUTIONAL NEURAL NETWORKS TO AUTOMATE THE SCREENING OF MALARIA IN LOW-RESOURCE COUNTRIES
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1.1	Professional standards of clarity, non-ambiguity, technically correct English

The introduction is very well-crafted English. However, in the methods, results, and discussion sections, there are occasional words missing. In addition, in these sections, there are several instances of subject-verb number disagreement (annotated in PDF).

1.2	Literature references including sufficient field background and context

The methods section is severely under-referenced and does not indicate the sources, either academic or commercial, of most of the algorithms and platforms used. Computing terminology is not consistently explained at a level appropriate for a medical/biological journal.

1.3	Professional article structure, figures, tables

Text is in the correct article structure. Figures and tables are interpolated in the PDF provided to reviewers. Correct procedure is to submit figures and tables as separate files; however, the manuscript PDF provided may have been integrated by the PeerJ content management system. No other issues.

1.4	Self-contained with relevant results to hypotheses.

It should be made clearer in the abstract and introduction that the primary goal is proof-of-concept of a smartphone-executed detection algorithm without need for high-speed internet connection(s). Otherwise no issues.
Experimental design: 2.1	Scope
The research is relevant to medical practice and is within scope.

2.2	Research question well defined, relevant and meaningful
The research goal is well defined and shown to be relevant and meaningful. A sharper emphasis on the exact knowledge gap (a smartphone-only based app suitable for field use in Africa) and best applications of the knowledge obtained would be helpful (see comments in PDF).

2.3	Rigorous investigation, high technical and ethical standards
No issues.

2.4	Methods described with sufficient detail & information to replicate
Some ambiguity in which algorithms, and which versions of which algorithms, were used. Insufficient references given in Methods section to fully source all algorithms. In a few cases, ambiguity existed on which dataset was used in which step.
Validity of the findings: 3.1	Novelty and replication
Novelty is assessed but only in terms of the goal parameters (development of smartphone-only algorithm). Meaningful replication is encouraged for further development of robustness.

3.2	Data integrity
No issues.

3.3	Sound conclusions
Conclusions do not extend beyond the goal of proof-of-concept, appropriate for the work performed.

3.4	Speculation identified
No issues.
Additional comments: This is a work advancing the field of automated malarial smear reading with the notable step of eliminating the need for cloud-based processing. The authors show acceptable performance within the limits of their training sets; however, they acknowledge that better datasets with wider variety and less prone to overtraining are needed for production of a usable tool in field medical work. The manuscript as currently published has nonstandard reference formatting and insufficient references, particularly in the Methods section. It also has insufficient explanation of computational technical terms to fit the journal’s intended scope. However, the methods and results are solid overall and the topic is appropriate and sufficiently interesting to merit 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: WHITE-HANDED GIBBONS DISCRIMINATE CONTEXT-SPECIFIC SONG COMPOSITIONS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The ms is concise and well written, with good coverage of the literature, and is structured appropriately. Part of the rational for the study, used in interpretation, involves two mss "in press", so are not available. In that sense, the ms is not fully self-contained.

A few minor comments on writing:

Line 57. Explain what these studies show.
63. "In linguistics"... OK, but what do your mean by "pragmatics" here.
160. what is the measure of variation?
202. Duration of notes, or duration of a series of notes?
258-262. Cut; this just repeats the Introduction.
268. "waoo" does not appear in the main text (or S4), as I recall, until this point. Yes, it is in the Supplement, but you need also to include it in the main text.
272. Join this "paragraph" to the previous one, which has a topic sentence also including this material. Possible trim the resulting longer para.

I'd really like to be able to hear some recording of the songs (they are a highlight of SE Asian forests), and strongly recommend a couple of examples of duet and predators songs in the Supplement.
Experimental design: The design is generally clear and appropriate, with good equipment and rigorous definitions of responses. The sample size is small, which limits some conclusions, but this is not unusual in studies of primates. Furthermore, despite the small sample, there are clear differences in the response to playback treatments.

I noticed three things missing in the methods: (1) There is no reporting of playback amplitude. The text 156-157 states that the playbacks were broadcast such that the "subjects always heard the recordings", and "with comparable natural audibility (tested at each playback with a decibel meter)." Without presenting data on the actual playback amplitudes, amplitude of real calls, or information on gibbon hearing/responses, none of these claims is currently supported. Please give appropriate data, or explain why it is not given and why you think it is OK not to give the data. (2) Responses to playback were videoed, which often gives the opportunity for blind scoring, but it is not explicitly stated that the videos were not scored blind (which I assume was the case). Please say clearly if scoring was blind or not, and if not explain why is was not done blind. (3) I assume that focal animals were sometimes out of sight during the videos. What did you do in that situation?
Validity of the findings: The rationale for the study is clear. The general finding, that there were behavioural differences in response to the two treatments appears sound (although I would like to know the answer to both methodological omissions above). The data are provided. The conclusions are stated and for the most part seem robust.

The supplement Table S4, and associated statistical analyses and conclusions are not fully supported. Failing to reject a null hypothesis of no difference, especially with small sample sizes, does not show that the playback duet responses and spontaneous duets are the same (and indeed one difference was found). Please modify the claims and conclusions about these analyses. S4 & analyses would be much more compelling if it also included quantitative data on predator songs, and so revel that responses to playback more closely resemble natural duets and than predator songs. I imagine that there would be some overlap with the paper "in press", but I also expect that the overlap would be minimal and so may not compromise those papers. I think Tables S4 would be better in the main results.

283- on. Gibbons looked down after playback of the "alarm" duets given to leopard prompt. This seems to imply that they have different alarms to terrestrial and aerial predators. Is this the case? And if they always look down, as if expecting a terrestrial predator, why bother being cryptic? A leopard couldn't chase a gibbon in the trees. Are gibbons also hunted by people? That might explain crypsis, much like in the Diana monkey findings.

305-307. Yes, but the lack of evidence might be because people have not looked at acoustic structure of notes. Without an analysis of the acoustic structure of notes, &/or appropriate experiments, you'll surely never be able to be sure that "syntax" is responsible for communicating meaning. That point could perhaps could be addressed more directly.

313-314. The claim the duet and predator songs differ is NOT shown in Table S3. See point about adding quantitative data on predator songs to Table S3.

319-325. It might be difficult to compare notes, but it seems worth trying, for reasons already mentioned.

331. This ms does not show the "complex coding abilities". I guess that will be in the "in press" articles?
Additional comments: I enjoyed reading the paper; it's nicely written and interesting.


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: WHITE-HANDED GIBBONS DISCRIMINATE CONTEXT-SPECIFIC SONG COMPOSITIONS
Review round: 1
Reviewer: 2

Output:
Basic reporting: all fine, although some suggestions for additional background information: please see 'General comments' below
Experimental design: please see 'General comments' below
Validity of the findings: please see 'General comments' below
Additional comments: The manuscript entitled: ‘White-handed gibbons discriminate context-specific songs composition‘ by Andrieu et al., tests the hypothesis that gibbons respond differently to the playback of duet songs versus predator songs, suggesting that gibbons perceive a difference in these song types. The authors suggest that this could be a result of the difference in note prevalence or timing of the songs, given that the two songs share syllable types. Playbacks in the field are difficult but particularly relevant to natural behavior. The authors further took care to match playbacks to the natural sound level and also the general location of natural calls in the canopy. They measured a range of vocal and nonvocal responses to playback, creating a richer profile of call-responsive behavior. The results are consistent with white-handed gibbons distinguishing different types of songs from neighbouring groups. The manuscript could be made more accessible to non-specialist reviewers by fleshing out the information included in the manuscript.

Comments:

Although presenting playbacks in the field is a challenging undertaking, why did the authors choose to present playbacks to 6 rather than all 8 of the groups they work with? Please describe this in the Methods.

Some of the information in the early supplemental figures (esp 1 and 2) would be useful in providing context in the body of the manuscript- can these be moved? Also, it would be helpful if supplemental Fig 1 included labels of all of the types of notes (the ‘waoo’ would be especially interesting).

In fig 1, it would be helpful to provide some kind of indication of time- do the songs take tens of seconds? Minutes? Additionally, it is difficult to find any information about the variability in the song sequences used for playback. There is some information in supplemental table 4 on the duet call, but I did not see information of the variability among predatory song playbacks. This information would be helpful in understanding the range of parameters that gibbons heard across playbacks.

Some of the information in the supplemental text would be interesting as part of the main manuscript, especially what is known about the function of duets from past work. Further, are there any functional studies of predator calls? Are the predator songs directed towards predators (as the wording here implies), for distraction, warning, what exactly? Are they a sort of alert to conspecifics for predator presence?

Why were only male nonvocal responses to playback measured, and not those of females? How was the primary male identified? Please state in the Methods

Methods: How much time passed between duet versus predatory playbacks for a given group? Were they on the same day? Separated in time?

What time of day they did playbacks occur? The introduction (line 82) states that “…mated pairs produce morning duets…” Is time of day significant for this specific vocal production?

Regarding the Bonferroni corrections, how many repeated comparisons were made and corrected for?

Given that the responses to duet playback occur before the duet songs are over, the authors make the point that neighbours must be responding to early parts of the duet song, potentially lending more weight to the difference in ‘ooaa’ syllables in allowing groups to distinguish duet from predatory songs. This is a very interesting point, and highly relevant to the manuscript’s premise that white-handed gibbons can distinguish among the two song types. Isn’t this information worth including in the main part of the manuscript?

Supplementary text, lines 47-50: ‘However, songs in response to duet playbacks contained more ‘waoo’ notes than spontaneous duets. Despite the early ‘waoo’ note composition difference, elicited duets in response to playback treatments could not be distinguished from natural duet songs.’ These two statements seem contradictory to each other- what am I missing?

Minor comments:

lines 104-105: the predictions at the end of the introduction are rather vague and could likely be made more specific (for example, what types of behavioural responses would be ‘adequate to the situation’ for predator or duet playbacks?).

line 138: what criteria were used to determine that a specific song was suitable for playback?

lines 222-223: This sentence is a little confusing: ‘In addition, eight non-neighbouring groups also produced duet songs in 3 of 6 trials, while none of them produced a predator song’ Does it mean that, of the eight groups, all eight produced duets in each of the 3 trials? Across these three trials? Other?

If non-neighboring groups responded to playbacks, were there any discernible differences in response based on proximity vs distance? For example, were closer groups more likely to respond?


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: WHITE-HANDED GIBBONS DISCRIMINATE CONTEXT-SPECIFIC SONG COMPOSITIONS
Review round: 1
Reviewer: 3

Output:
Basic reporting: The basic reporting for this article is of very high quality. Overall, this is was an interesting paper that contributes new insights into the communication system of white-handed gibbons. The authors rightly point out that gibbons are a bit of a blank spot in our knowledge of Hominoidae. I appreciate that this paper looks at responses to the songs and the contexts in which they were produced – and it seems to tie in with another study that includes predator presentations.

My main suggestions are to include more details in the methods, and to elaborate on some of the theoretical points in the introduction and discussion. The paper is very concise, but at some points that conciseness means that the reader doesn’t get all of the required information. I've made specific comments to the author in areas that I think require more detail.
Experimental design: The experimental design is based on well-established methods within this field, and was appropriate for the question being asked. The sample size was quite snall, but that is to be expected in playback studies. There were a couple of points that needed more detail in order for others to replicate this study, which I've included in specific comments to the author.
Validity of the findings: The authors provide all of the data required for their analyses, and their results are clearly stated. There was some phrasing in the discussion that I felt would have required a different analysis (i.e. acoustic comparison of call elements for each song type) in order to substantiate those claims, and have made alternative suggestions in the comments to authors. The authors later acknowledged the current methodological limitations of gibbon song analysis, and so it would be good to tie those two points together.
Additional comments: Introduction
General - Do you have any idea about the rough frequency with which gibbons hear the songs of their neighbours? It’s not crucial to this study, but would be cool if there’s a reference so that we can understand the ecological implications/potential adaptive value of overhearing different song types.
Line 53: What does it mean that the variations in calls are “meaningful to recipients”? It would be useful to the reader if you could explain and give a specific example (in addition to the species list that you already have).
Line 64: It is my understanding that the Gricean approach to meaning is not that “the signaller’s intention affects signal meaning”, but rather that a signal has to be intentional in order to have meaning in the first place. I think the authors are trying to say that we need to consider the context in which calls occur, and are perhaps arguing for a more Carnapian approach? Plonking Grice at the end is just a bit confusing for the reader, unless you could add a sentence to wrap up the paragraph saying which approach you favour.
Line 68: This paragraph is great and a good model for the types of examples that would be helpful to the reader in the previous two paragraphs. Clear writing and interesting content!!
Line 85: “its production is subject to social influences”, such as … - please elaborate on which social factors impact song production in which ways. This information will be useful for interpreting your results later.
Line 90: I didn’t realise that gibbons have predator songs. That’s really cool!
Line 101: To talk about meaning in the Gricean sense, you need to establish that the songs are intentional. That’s why I wonder whether you should say you’re taking a Carnapian approach to pragmatics, then you can talk about meaning without hashing out the Gricean argument.

Methods
Line 111: Are gibbons in groups of 1 adult male, 1 adult female and their offspring? A brief description of typical group structure in the intro would give the reader some context. It could also tie into an explanation later (line 166) of why you chose to target the primary male for video recording. I see that you’ve given the specific group details in a supplementary table, which is also helpful!
Line 134: Figure 1 is a very helpful visualisation of the two song types.
Line 141: This is a solid set of criteria! It must have been challenging to collect these stimuli. Well done!
Line 155: Were you able to ensure that the individuals from which you took the song recordings were not able to overhear the recordings of their own voices? i.e. was someone aware of the location of the neighbouring group while you were conducting the playback with the target group? This has ethical implications, but also affects whether the target group may have been aware of the neighbours in a different direction from the one you presented the playback. If you were unable to know their location, it is important to state that here.
Line 166: Why did you target the primary male for video recording?
Line 166: Which video recorder did you use?
Line 167: Which program did you use to code the videos? What variables did you code? It’s too vague for someone else to replicate.
Line 169: For a moment, I thought that you were extracting this information from the videos. For clarity, could you start the sentence with “We also conducted a focal scan of the primary male during one hour following the playback (5-min scan sampling, i.e. 13 scans per experiment; Table 1),…”
Line 176: Is this in the video coding scheme, or an audio coding? It’s a bit unclear where this data is coming from.

Results:
Overall the results are presented in a very clear way that is easy for the reader to follow.
Line 243: Please include the direction of the difference in text, so that readers don’t need to interpret the table right away.

Discussion:
General – at some point in the discussion, there should be a note saying that all of these responses are from the primary males, and another study would be necessary to see whether female gibbons also respond appropriately to the different duets (you could also hypothesise that they will or will not, based on preliminary observations).
Line 272: The study demonstrates that gibbons respond differently to each type of duet, but it doesn’t provide direct evidence that the structure of the duets leads to these different responses. The authors describe that the calls are the same but in different lengths and different orders, but there is no direct comparison of the call types within the sequences that were used as stimuli. (I apologize if this was included in a reference to another study earlier and I’ve missed it – if so, could bring it up again here?)
Line 279: “discriminate and respond appropriately to the context in which another group’s song was produced” – I’m not sure the wording properly expresses what’s happening, it sounds like they are directly responding to the context. Perhaps something like “Our data show that gibbons are able to use another group’s song to determine the context in which that song occurred, as demonstrated by the listeners responding appropriately to the context of the callers. They discriminate the context of the call without having witnessed the context themselves.”
Line 284: “once in visual contact” with a predator?
Line 291: I think “sometimes” would be more appropriate than “temporarily”
Line 323: Oh, this seems to tie back to my previous point about direct comparison of elements within each duet type. Could you elaborate in a sentence after this one about why it will be important to use these analyses?


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: ANTI-COVID-19 MULTI-EPITOPE VACCINE DESIGNS EMPLOYING GLOBAL VIRAL GENOME SEQUENCES
Review round: 2
Reviewer: 1

Output:
Basic reporting: This is a very comprehensive bioinformatic analysis of SarsCov2. the authors included many different immunobioinformatic tools to analyze the virus genome and were able to propose immunogens to be used for the development of a vaccine. The text is clear, professionally written, with few phrases to be rebuilt. The structure is good, well designed, and all data are shown by the authors. Some Figures, as shown in the pdf attached to this review, should have their definition improved. Tables are ok. The results of the bioinformatic analysis are sufficient to justify the author´s hypotheses. The discussion, however, is insufficient in some points and should be improved, mainly in some of the immunological aspects of the use of the new immunogen (please see the attached pdf file).
Experimental design: This work is original, interesting and is included in the journal´s scope.
The research question is well defined. All the aspects included in the study are pertinent.
The results are well presented. The bioinformatic tools used are valid and the association between them is remarkably interesting.
Methodology is sufficient.
Validity of the findings: The findings are interesting, originals. The authors present their findings in a very well structured way.
All data were supplied. The results of the bioinformatic analysis are well presented.
Discussion is not sufficient in several points (please see the attached pdf file).
Conclusions are well stated. Speculations are identified, and are not redundant or even excessive.
Additional comments: This is a very comprehensive and inclusive study. Several bioinformatic analysis were made, and they are well structured and presented.
In some points it would be better not to mention the Pakistani situation so much. Avoiding such a thing would give to the work a more global aspect.
The authors should be more specific when describing the B cell epitope analysis results and discussion. They even state the importance of humoral response, but they do not present these results or even discuss them sufficiently.
The discussion on TLR8 should also be improved.
The definition of some figures should be improved as well.
Please refer to the attached pdf file for more information.