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: FAST AND AUTOMATIC ESTIMATION OF TRANSITION STATE STRUCTURES USING TIGHT BINDING QUANTUM CHEMICAL CALCULATIONS
Review round: 1
Reviewer: 1

Output:
Basic reporting: A) The article is well written, and mostly, citations to the directly related works are given. In the introduction, one should add a citation to H.B. Schlegel’s WIREs article (DOI: 10.1002/wcms.34), which gives a good overview over different TS optimization techniques. Furthermore, some of the TS optimization techniques mentioned therein might be worth citing as well – for sure the one that has been used in the DFT re-optimization using Gaussian should be cited here (not just the Gaussian suite itself).

B) The introduction itself could be longer in the sense that the importance for high throughput barrier calculations could be highlighted (kinetic model generation for example). Something like this is written at the bottom of p.5, (below “Comparison of xTB barrier estimates and DFT barriers”) and could be also given in the Introduction.

C) The color codings should be explained in the figure captions of figures 3 & 4.

Minor:
a) The abbreviation RMSD-PP is actually never introduced, i.e., that “PP” stands for push-pull. In the same context, I find “Grimme’s RMSD-PP semiempirical tight binding reaction path method” in the abstract too coarse, as there are two components here:
The path search mechanism based on an atomic RMSD-based bias potential and the underlying electronic structure method, which has been the semiempirical GFN2-xTB method in Grimme’s work as well as this one. It would not harm to be more explicit, like “RMSD-PP in combination with the semiempirical tight-binding method…”, since we could, in principle, also combine RMSD-PP with DFT.
b) It says “GFN-xTB […] models” in the summary, but the paper reports exclusively GFN2-xTB. Were other methods tested? If not, I would just use GFN2-xTB here as well.
c) The x-axes in Figure 6 and 7 should be more explicit (“DFT barrier” or so).
d)
p.1: There is a period after “Grimme” in the introduction that needs to be removed.
p.2: “haven” -> “having”
p.4: “TS structures proposed by Zimmerman was further analysed”, change “was” to “were”.
Experimental design: I consider this paper an important contribution to the computational chemistry community.
A) The “2-4 optimization steps without any bias” on p.1 need to be explained further. How many states are exactly taken or what is the criterion for 2 vs 4 steps. It could be emphasized that this is not a path optimization, but an unconstrained geometry optimization, is that right?

B) Is the interpolation (p.2) done in Cartesian coordinates? Depending on how far the next neighbor is, this could be a bad choice, maybe not for the systems in this benchmark here, but for rotating substituents (phenyl for example).

C) p.2: “the endpoints of the IRC are geometry optimized, and these structures checked by the same procedure”. This needs further clarification. Due to the computational cost, I am assuming that this means that the structures are all optimized at the GFN2-xTB level. If so: does one actually ever need the adjacency matrix scheme or couldn’t this reoptimization procedure replace that entirely?

D) p.4: the “random ‘initial distortion parameter’” needs further explanation. Why can this not be done more deterministic?

Optional:
E) Did the authors test their procedure in combination with GFN2-xTB and an implicit solvation model (like GBSA)? This may perform better for some of the charged species – this is just a suggestion.
Validity of the findings: The authors analyze their data quite well and give explanations for each reaction in the supporting information.

A) This is a point that needs further elaboration:
Figure 7 shows promising results (DFT singlepoints on the xTB geometries), but it is not discussed in the text at all. I don’t know why. Here it is also important to note what that means. Are xTB geometries used throughout or only for the TS estimate, it looks like the latter, since the deviations all indicate an overestimation of the barrier. Maybe using xTB geometries (for the minima) could help here. I think this deserves more attention as it might give access to fast screening (a DFT singlepoint is mostly bearable).

B) The discussion/methodology around Figure 8 could be elaborated a bit more. Particularly, how are “wrong TS” classified if they fulfill all the 3 criteria? For example, are they higher in energy than the ones presented by Zimmerman, or is there a possibility that they actually found a lower TS?

C) The systems are mostly too small for this to be relevant, but the authors should mention that there exists an difference in the included physics: GFN2-xTB contains a London dispersion correction, while the DFT level does not. I understand that for consistency with Zimmerman, a dispersion-devoid model was used. But the authors should clarify that this could expectedly(!) lead to differences between both levels of theory for larger systems.

D) Is the tool/workflow available to the community?

Optional:
E) For the TS geometries in Figure 5: Did the authors try to compute S^2 expectation values with UB3LYP on both geometries? It would be interesting to know if the DFT geometry is spin-contaminated or if the xTB geometry has related issues (GFN2-xTB always favors low-spin configurations). Maybe a simple S^2 expectation value on both geometries shows something here (maybe not).
Additional comments: The manuscript “Fast and automatic estimation of transition state structures using tight binding quantum chemical calculations (#50973)” by Rasmussen and Jensen elaborates on an earlier presented reaction path scheme and assesses its performance for transition state structure prediction using a previously presented benchmark set. The manuscript is well written. It needs, however, attention in a couple of aspects. I suggest publication after the addressing the above raised issues.


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: FAST AND AUTOMATIC ESTIMATION OF TRANSITION STATE STRUCTURES USING TIGHT BINDING QUANTUM CHEMICAL CALCULATIONS
Review round: 1
Reviewer: 2

Output:
Basic reporting: 2)	Thank you for providing a repository with data at https://sid.erda.dk/sharelink/EPvv68fOTp. The Supporting Information statement indicates that these are “data resulting from this study”. However, I note that:
a)	Only reactant and product structures are provided, whereas (approximate) transition state structures are the key data resulting from this study. Would it be possible to include those? Including both the TS geometries obtained with the method and those optimized with DFT would allow other researchers to properly evaluate the work and results.
b)	It is not described whether the provided structures are DFT geometries obtained from Zimmerman’s work, or geometries reoptimized with GFN2-xTB by the authors of this work. Please add this important meta-data.
c)	I note that only 77 reactant structures (*r.xyz) and 91 product structures (*p.xyz) are included in the reactant_product_structures.tar.gz archive. They include also the reactant and product structures for the 5 reactions from Zimmerman’s set that were not used in this study (reactions 94, 95 and 102-105). This means that many reactant and product structures are missing.

3)	The results for the barrier height prediction are not very clearly presented/quantified.
a)	The authors state that “the RMSD-PP barrier is a good approximation for the corresponding DFT barrier for reactions with DFT barrier heights up to about 30 kcal/mol”, but they provide little/no direct quantification of this. They do state “The correlation between xTB and DFT is considerably better for these reactions.”, so please include a measure for this correlation here.
b)	The presentation of Figures 6 and 7 is not optimal. I assumed that the six plots are for the data obtained from the 6 runs, with Runs 1-3 (obtained with parameter set 1) shown on one side, and Runs 4-6 (obtained with parameter set 2) on the other. This is probably the case (as indicated in the text), but the Figure legend itself states: “Figures (a), (d), and (e) correspond to runs 1, 2, and 3”, and it is not clarified what panels b,c,f show. This would be made much clearer by adding labels of “Run 1” etc. above the individual panels. Further, axis titles should be clarified, e.g. “Barrier xTB” --> “Barrier estimate from GFN2-xTB” and “Barrier” --> “DFT Barrier”. Further, the different ranges on the y-axis make visual comparison between runs difficult. Could the same range be employed throughout (e.g. 0-200), perhaps with outliers beyond 200 kcal/mole indicated using arrows?
c)	The authors could consider placing the full Figure 6 and 7 in the Supporting Information (in which case point b) above could be ignored), and replacing them with plots for 1 run only, where they can show two plots for each: the total set of reactions as well as the reactions with DFT barriers below 30 kcal/mole only. This may help to better deliver a main message of the paper (“the RMSD-PP barrier is a good approximation for the corresponding DFT barrier for reactions with DFT barrier heights up to about 30 kcal/mol”, but not/less so for those with higher barriers). This will have the additional benefit of making the labels better readable (as they are currently quite small).

4)	The presentation of the reactions is not very clear / may contain mistakes.
a)	In the main text, Figures 3 and 4 would be much improved by including standard (electron pushing) arrows to describe the reaction. At least, the following should be added to the legend:
"Bonds broken in the reaction are indicated in red, bonds formed in blue".
b)	Table S2 (and deposited data file reactions.txt) contain several identical reactions, i.e. reactants and products are identical (e.g. 1 and 2; 4,5 and 6; 10, 11, 14). This needs to be corrected. Further, the minus signs indicating atoms with formal negative charge are often difficult to spot. Can they be enlarged, or (ideally) be placed in circles (as in Figure 3)?
Experimental design: 5)	Some further clarification/information on the methods could be added.
a)	I very much appreciate the short description of the RMSD-PP method, so that original paper(s) by Grimme describing the method do not have to be studied in detail. To clarify further how the method works, as relevant to the current manuscript, can you please describe: 1) How are points on the path defined (i.e. the 'distance' between them, and/or 2) how is it defined how many points there are on the full path?
b)	Could there be a short indication/description of how the k_pull and k_push and alpha values used in the parameter sets were arrived at? Trial-and-error? Based on previous work?
Validity of the findings: 1)	The authors correctly describe their method as ‘fast’ in the title, however, exactly what this means is not described or reported clearly.
a)	Table S5 indicates (for just two reactions) the wall-time required for TS estimation by their procedure, which is impressive. However, no reference is made to this table in the main text, nor is any approximate timing given for TS estimation. I suggest that the authors include somewhere (perhaps even in the abstract), that TS estimation can be obtained “within a few minutes on a single CPU”. Where such a statement is added to the text, Table S5 can be referred to. Further, it would be better if the CPU used for the Table S5 timings is described (e.g. speed in GHz, perhaps also type).
b)	In the abstract and summary, the authors only describe that “RMSD-PP barrier heights [..] can be estimated at the cost of a single energy minimisation”.  What this means is not very well defined, and is certainly hard to interpret by reading the abstract only. With RMSD-PP barrier height, to the authors mean barrier height estimation/calculation after TS estimation, or the whole procedure to obtain the estimate TS? With “single energy minimisation”, do the authors refer to a single energy calculation or a single structure optimisation, or the procedure of "start with the reactants, turn on push/pull potentials, minimize, and check, whether the system falls into products"? If energy calculation or optimisation, at what level of theory? This statement should be changed/clarified.
Additional comments: Rasmussen and Jenssen describe and evaluate a fast and automated procedure to determine transition state structures (TSs) for elementary reactions. The procedure is based on the RMSD-PP method from Grimme, together with his semiempirical tight-binding method GFN2-xTB. The work appears well-designed and conducted, and the methods and results are generally reported clearly and succinctly. I highly commend the authors for their efforts to produce a successful automated method for TS optimization that allows taking full advantage of the speed enhancements possible from Grimme’s methods, and making their code openly available and well-documented. There are, however, several things that should be clarified and some errors/omissions that should be corrected before the work can be accepted (see review sections).

The github repository is well structured and the code is extensively commented, which is highly appreciated. A suggestion: currently, the implementation can only be used with SLURM job scheduler. It would be nice to refactor the implementation to allow running the scripts with other job schedulers or at least directly on a local machine.

Finally, minor points regarding textual/language errors and small clarifications are annotated in the attached PDF. I would appreciate if the authors could address these too, to further improve the readability. In my view, it is not particularly useful for the community to make this annotated PDF available online; all main points raised are clearly described in the review text.

I hope this review will helpful to improve the presentation of your work.

Marc W. van der Kamp


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 SIR DYNAMIC MODEL OF INFECTIOUS DISEASE TRANSMISSION AND ITS ANALOGY WITH CHEMICAL KINETICS
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: The manuscript examines the analogy between differential equations and their solutions in chemical kinetics and epidemiological models. This goal is accomplished satisfactorily. The discussion could be further improved by giving engineering/chemical kinetics analogues to the extensions of the SIR model, if such analogues exist.
Perhaps quarantine in the SEQIJR model could be related to the presence of an absorber material in a reactor?
I am unsure how time-varying parameters and births, death, and loss of immunity could be related to the chemical kinetics/engineering, but perhaps the author has an idea.
Validity of the findings: no comment
Additional comments: I found the analogy between kinetic and epidemiological models very unexpected, but also very interesting. The manuscript presents this analogy in a very instructive way and I think it is a valuable read for anyone involved in physical chemistry in some way.


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 SIR DYNAMIC MODEL OF INFECTIOUS DISEASE TRANSMISSION AND ITS ANALOGY WITH CHEMICAL KINETICS
Review round: 1
Reviewer: 2

Output:
Basic reporting: This paper offers a pedagogical introduction to the Susceptible-Infectious-Recovered (SIR) epidemic model for readers familiar with chemical kinetic models.  By drawing analogies between chemical kinetic and compartmental epidemic models, the Author aims to foster the exchange of ideas between disparate research communities.

The paper is well-organized, clearly written, and technically accurate.  I would be happy to share a published version with my colleagues to use in lectures for senior undergraduate chemical engineering students. Moreover, the highlighted extensions of the simple SIR model may serve to stimulate research professionals in the area of chemical kinetics to seek opportunities to apply their expertise in “new” domains.
Experimental design: This paper is a review of prior work.
Validity of the findings: The presentation of the SIR model is clear and accurate.
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: AGGREGATION KINETICS OF BINARY SYSTEMS CONTAINING KAOLINITE AND PSEUDOMONAS PUTIDA INDUCED BY DIFFERENT 1:1 ELECTROLYTES: SPECIFIC ION EFFECTS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The paper appeared with interesting results and new messages are given. The paper is of a satisfactory level, quite clear and well organized.
Experimental design: All the methods used are well established and the discussion part is complete.
Validity of the findings: The contribution of this piece of research is valuable
Additional comments: The aim of this work was to study the aggregation of "mineral-bacteria" mixture colloids of “kaolinite-P. putida” induced by LiNO3, KNO3 and CsNO3. Dynamic light scattering studies on the aggregation kinetics of mixture colloids containing kaolinite and P. putida were conducted in this study. The experimental study is well organized, providing new insides in the field of colloidal aggregation behavior of binary systems.
Minor comments
1. The XDLVO theory should be used to explain the aggregation between kaolinite-kaolinite, kaolinite-P. putida, and P. putida-P. putida.

2. L123: The hydrodynamic diameter of P. putida was measured as 1600±100 nm. How did you measure the diameter?
3. L143: ….indicating the P. putida contents were 0%, 144 3.33%, 6.67%, 16.67%, and 33.33%. Based on weight? Please explain.

4. Eqs. 1 and 2: vT(f0) does not appear in the equation 1. Present clearly the vo/to(f0).

5. The procedure for the Kinetics of “kaolinite-P. putida” aggregation, should be described in the methods section.

6. Please correct the typo mistakes throughout the text. E.g. scattering, Oncsik, et al(Oncsik et al. 2015).

7. L261: We can speculate that there may …….. three different conditions since different electrostatic repulsion … kaolinite-kaolinite, kaolinite-P. putida, and P. putida-P. putida. Please use literature findings to support the statements appeared in the text.

8. L282: Based on this, we can deduce that the aggregation behaviour…….average effect produced by the various colloidal …….of separate colloidal particle. Please use literature findings to support the statements appeared in the text.

9. Please consider also the publications in this field: doi:10.1029/2010WR009560, doi:10.1016/j.colsurfb.2011.01.026


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: AGGREGATION KINETICS OF BINARY SYSTEMS CONTAINING KAOLINITE AND PSEUDOMONAS PUTIDA INDUCED BY DIFFERENT 1:1 ELECTROLYTES: SPECIFIC ION EFFECTS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Clear English;
Good literature;
Professional article structure, figures, tables. Raw data shared;
Experimental design: Well enxperimental design
Validity of the findings: This paper shows the novelty;
All underlying data are robust, statistically sound, & controlled;
Conclusions are well stated;
Additional comments: This study showed dynamic light scattering studies on the aggregation kinetics of mixture colloids containing kaolinite and Pseudomonas putida(P.putida) . This is a very good topic to study. The results showed that the aggregation behavior of mixture colloids or binary systems was determined by the average effects, rather than the specific component, of binary systems. The experiment design is good and English is good. I suggest that this paper can be accepted.


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: AGGREGATION KINETICS OF BINARY SYSTEMS CONTAINING KAOLINITE AND PSEUDOMONAS PUTIDA INDUCED BY DIFFERENT 1:1 ELECTROLYTES: SPECIFIC ION EFFECTS
Review round: 1
Reviewer: 3

Output:
Basic reporting: In this paper, the authors investigated the colloidal interactions between P. putida and kaolinite in the different mass ration by dynamic light scattering. The results showed that aggregation happened in kaolinite colloidal particles, and between kaolinite and bacterial cells, but not happened in bacterial cells. This study provides interesting data for understanding the aggregation phenomena in soil and sediment. However, several aspects should be improved before publication.
Experimental design: This paper is original research within the Aims and Scope of the journal.
Validity of the findings: Speculation is welcome, but should be identified as such.
Additional comments: Firstly, all the aggregation data were obtained by dynamic light scattering in 30 min. The authors don't tell us why 30 min, and is it enough for aggregation experiment? And what would happen if conducted a more prolonged test, for example, 60 min or 120 min?
Secondly,  P. putida is a gram-negative, rod-shaped bacteria, and with the polar flagellum.  Generally, bacteria prefer to survive in cell groups but not a single cell. Furthermore, plenty of positively and negatively charged surface sites existed on the cell surfaces. Therefore, it is hard to understand why the bacteria could not aggregate in the present study. The authors need to provide more explanation on this point.
Thirdly, the aggregation between mineral colloid and bacteria could be the first step in forming soil aggregates. It is essential but not enough to explain the total aggregation phenomena in soil. More aggregation work is needed to conduct, e.g. at different pHs, to explain the aggregation better.


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: AGGREGATION KINETICS OF BINARY SYSTEMS CONTAINING KAOLINITE AND PSEUDOMONAS PUTIDA INDUCED BY DIFFERENT 1:1 ELECTROLYTES: SPECIFIC ION EFFECTS
Review round: 1
Reviewer: 4

Output:
Basic reporting: no comment
Experimental design: The experimental design is thoroughly described
Validity of the findings: The conclusions are very well stated
Additional comments: peerj (#47222): Aggregation kinetics of binary systems containing kaolinite and Pseudomonas putida induced by 1:1electrolyte.

This study examines the interactions between kaolinite colloidal particles and P. putida. Certainly, the research topic is of great interest because the migration of a mixture of clay particles and bacteria in environmental systems is not fully understood. The manuscript is very well written and organized. The experimental procedures are thoroughly presented. The figures are clear. Consequently, this reviewer recommends publication of this manuscript in PeerJ after a relatively minor revision. The following is a short list of minor suggestions:

(a) The abstract should clearly list all the novel contributions of this work.

(b) The introduction section of the manuscript is very informative; however, the interaction of kaolinite colloidal particles with various biocolloids as well as numerous contaminants has been studied extensively in the literature. Therefore, the introduction should be expanded to mention previous studies such as the works by Chrysikopoulos and Syngouna (Colloids and Surfaces B: Biointerfaces, 92, 74–83, 2012), Bellou et al (Science of the Total Environment, 517, 86–95, 2015) and Fountouli et al (Environmental Earth Sciences, 78, 152, 2019), Fountouli et al (Environmental Earth Sciences, 78, 152, doi:10.1007/s12665-019-8147-x, 2019).

(c) Note that Vasiliadou et al (Colloids and Surfaces B: Biointerfaces, 84(2), 354–359, 2011) have studied the interactions between different structured kaolinite materials and P. putida. Also Vasiliadou and Chrysikopoulos (Water Resources Research, 47(2), W02543, doi:10.1029/2010WR009560, 2011) investigated the interactions between kaolinite and P. putida during transport in porous media.

(d) How do the measured zeta potentials compare with literature values?

(e) The captions could be more descriptive. The captions for Figures 1-3 need more attention.

(f) Can Figure 6 contain some quantitative information? Currently Figure 6 is not very informative.


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: AGGREGATION KINETICS OF BINARY SYSTEMS CONTAINING KAOLINITE AND PSEUDOMONAS PUTIDA INDUCED BY DIFFERENT 1:1 ELECTROLYTES: SPECIFIC ION EFFECTS
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: All of my concerns from my previous review have been addressed. I recommend an accept with some improvement of writing and language polishing.


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

Input:
Title: MOLECULAR DYNAMICS SIMULATIONS OF THE INTERACTIONS BETWEEN TRIOSE PHOSPHATE ISOMERASE AND SULFONAMIDES
Review round: 1
Reviewer: 1

Output:
Basic reporting: The hypothesis for the study is not clear based only on the introduction. The authors should revise the last paragraph of the into to include a clear hypothesis and/or a summary of main results/conclusions.
The English is unclear in certain places. I have tried to note most of them below.
1. Line 52. you should change curing to treating
2. Line 67. put parentheses around Figure 1B
3. Line 67. Delete the semicolon
4. Line 79. There is a typo or missing word or two in this sentence because it does not currently make sense as it is.
5. Line 64. Add the PDB codes after listing the structure of hTPI and pTPI.
6. Line 81. Delete ". This is" to combine the two sentences
7. Line 83. change it to it's
8. Line 113. Add 'the' after "The docking scores for "
9. Line 118. I believe B3LY0 should be B3LYP for the level of theory
10. Line 124. This sentence is missing a verb.
11. Line 126. remove the comma
12. Line 153. Delete the comma
13. Line 169-173. Equations 4 and 6 are never referenced in the text of the manuscript.
14. Line 171. You skipped Equation 5. There is an Eqn 4 and 6, but no Eqn 5.
15. Line 177. This sentence is incomplete. Or maybe the word 'that' needs to be deleted. It is unclear to the reader.
16. Line 182. Delete the word represents
17. Line 187. Delete the semicolon and change 'and' to 'an' following where that semicolon is currently.
18. Line 196. The I for ionic strength needs to be capitalized.
19. Line 197. It should be “water, respectively) and…”
20. Line 229. (Figure 3, right panel) should just be (Figure 3B)
21. Line 256. kJ/mol does not need to be italicized
22. Line 264. “more restrain” is not grammatically correct, even if I’m reading it the way the authors intended
23. Line 268. “thus repulsion the ligand sulfaE” – this doesn’t make sense and is likely a typo or incorrect form of the word repulsion
24. Line 323. Remove the ‘and’ after the parentheses
25. Lines 517-521. This reference is not correct. At the very least, no year is listed. The issue and volume may also be incorrect, but maybe it’s just a new reference that is online only currently?
26. Line 391. The use of 'buy' in this sentence is not correct.
27. Figures 1 and 2. The amino acids being explicitly displayed in Figures 1 and 2 should be labelled accordingly.
Experimental design: I have ordered the flawed sections of the experimental design below by priority (1 being highest priority).
1. The authors should run simulations of the apo (i.e. no ligand) pTPI and hTPI. Several comments in the text (see lines 225-227, 236-237, 245-247), make conclusions on what happens to the structure of TPI when the ligand binds. However, since the authors did not simulate the apo TPI structures, they do not have a set of simulations to compare with their ligand-bound simulations. Thus, the conclusions based on those findings cannot be validated.
2. Why do the authors only run 100 ns for each simulation? With the size of the systems, the authors could easily run each to at least 1000 ns. The RMSD plots (Figure 3) suggest that none of the simulations have reached a stable structural conformation with the bound ligand. Instead, the RMSD values continue to increase as the simulation progresses up until the end (100 ns). This suggests the protein-ligand complexes have not reached a stable structure (i.e. converged). Thus, the authors should extend their simulations well beyond 100 ns until the structures reach a stable conformation that the authors can trust and verify.
3. The authors should perform a hydrogen bonding analysis on their simulations. On multiple occasions (Lines 361 and 349) they mention the stabilizing interactions as polar and/or hydrogen bonds, but the authors do not support these statements with the corresponding analysis. The authors should report percent occupancies and lifetimes for any hydrogen bonds relevant to protein-ligand stability.
4. Why was sulfaE chosen as the representative ligand to test? The authors should make this clear. And if there is no obvious reason, the reviewers should run additional simulations to include the other two ligands mentioned from the previous work.
5. It is unclear how many frames/snapshots were used for MM-PBSA calculations for each simulation. Line 210 says that 20 snapshots were used. from each simulation. However, line 217 says that about 2000 conformations were used for MM-PBSA (is this per simulation or total across the four simulations?). If only 20 snapshots were used per simulation, that's only 80 snapshots. So where did the 2000 come from? In my experience, a minimum of 200 frames must be analyzed to get reasonable results from MM-PBSA.
6. At the very least, they authors should explain why they chose not to include entropic calculations in their methods. On lines 338-340, the authors mention the potential effect of entropy on ligand affinity. The authors should calculate the entropy of the systems using snapshots from their simulations to support their conclusion here.
7. Line 143 states that 6000 water molecules were added for each simulation. Was it precisely 6000, or is that an approximation? If it is not exact, the authors should clarify.
8. Line 192. The Poisson-Boltzmann equation. Did the authors use the linear or non-linear form of the PB equation? And why? The authors should justify the reasoning for their choice.
Validity of the findings: Most of the conclusions drawn from the current data meets the journals standards. However, there are certain areas where conclusions are drawn and/or statements are made that do not have data to back up the claims. These areas have all been highlighted in the comments on the Experimental Design, but I will restate them here for completeness.
1. I am skeptical of the validity of one of the simulations and/or the MM-PBSA results. Specifically, the simulation with sulfaE bound to the hTPI dimer interface. The MM-PBSA results state the average binding free energy is +35.86 kJ/mol. This value suggests the ligand and protein are incompatible and are energetically unfavorable for binding. If that is the case, it would be expected that the ligand should dissociate from the binding pocket of the dimer interface to mitigate this unfavorable bound state. However, the authors make no note or mention of the dynamics of this simulation, or the physical meaning of this energetic unfavorability. The polar solvation term is clearly the leading cause of this energetic instability. However, the authors do not explain why the polar solvation would tend to be so positive for these complexes, and especially so for the hTPI dimer interface simulation. Such a high positive binding free energy suggests 1) something is fundamentally wrong with the simulation, or 2) the MM-PBSA calculation did not use the proper variables to accurately represent the complex. Without seeing the simulations up close, it's difficult to suggest which of these may be the culprit of these poor results. Either way, I'm highly skeptical of these energies as they are being reported.
2. Several comments in the text (see lines 225-227, 236-237, 245-247), make conclusions on what happens to the structure of TPI when the ligand binds. However, since the authors did not simulate the apo TPI structures, they do not have a set of simulations to compare with their ligand-bound simulations. Thus, the conclusions based on those findings cannot be validated.
3. The RMSD plots (Figure 3) suggest that none of the simulations have reached a stable structural conformation with the bound ligand. Instead, the RMSD values continue to increase as the simulation progresses up until the end (100 ns). This suggests the protein-ligand complexes have not reached a stable structure (i.e. converged). Thus, the authors should extend their simulations well beyond 100 ns until the structures reach a stable conformation that the authors can trust and verify.
4. On multiple occasions (Lines 361 and 349) mention the stabilizing interactions as polar and/or hydrogen bonds, but the authors do not support these statements with the corresponding analysis. The authors should report percent occupancies and lifetimes for any hydrogen bonds relevant to protein-ligand stability.
5. On lines 338-340, the authors mention the potential effect of entropy on ligand affinity. The authors should calculate the entropy of the systems using snapshots from their simulations to support their conclusion here.
Additional comments: This study is interesting and has the potential to be intriguing to many people in the field. I believe the research has high impact capability. However, I believe the manuscript currently is not ready to be accepted because it has a number of flaws. The experimental design is lacking many important steps that would help make the project complete. Additionally, several of the conclusions are stated without proper evidence to support them. Hopefully, the suggestions I have given seem reasonable and you are able to perform the necessary revisions to complete the manuscript.


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

Input:
Title: MOLECULAR DYNAMICS SIMULATIONS OF THE INTERACTIONS BETWEEN TRIOSE PHOSPHATE ISOMERASE AND SULFONAMIDES
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1. Professional English is used throughout the manuscript. There are some grammatical and formatting errors in the manuscript.

2. The citations are sufficient and the information presented in the introduction is broad and somewhat vague.
Experimental design: 1. The research is within the aims and scope of the journal.

2. The research question is well-defined but it is not particularly meaningful, and it does not fill any knowledge gap as far as I know.

3. The methods are sufficiently described.
Validity of the findings: The focus of the work is to explore the interactions between pTPI/hTPI and sulfonamides using docking and MD simulations. There is no experimental evidence that 'sulfonamides' in general and in particular the 'antimalarial sulfonamides' used in the work can inhibit or bind to TPI. Without such premise, using modeling to explore interactions quite frankly does not contribute any new useful knowledge or hypothesis.

If the compounds were being consider as potential inhibitors of TPI, it would be better to use modeling and/or experimental tools to compare them with known inhibitors and non-inhibitors.
Additional comments: No additional comments


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

Input:
Title: MOLECULAR DYNAMICS SIMULATIONS OF THE INTERACTIONS BETWEEN TRIOSE PHOSPHATE ISOMERASE AND SULFONAMIDES
Review round: 2
Reviewer: 1

Output:
Basic reporting: The writing is much better in this revision, but there are still some grammar and/or typo issues. I have tried to note most of them below.
1.	Figure 1B, 1D, 2B, and 2D need explanations of the color scheme being used for the electrostatic potential surfaces.
2.	Some of the residue labels in Figure 2A and 2C are not easily readable, especially the D49.A because of the red text over red VDW atoms.
3.	Line 159-160 – Add the word ‘for’ between the words ‘enhances affinity’ and ‘sulfonamides’
4.	Line 270 – ‘that’ should be ‘than’
5.	Line 277 – delete the word it’s
6.	Line 293 – delete the word ‘for’
7.	Figure 4F appears to be missing some data. The graph does not fully extend all the way to 400 ns.
8.	Why are only 400 ns of the simulation data shown in Figure 4? Based on lines 292-293, I assume the first 100 ns of the simulation are not shown, I just do not understand the reasoning behind this omission.
9.	For consistency, all the graphs in Figure 4 should have the same range for the y-axis.
10.	The authors should be consistent with how they identify specific amino acids. For example, in lines 132, 135, 383, 444, 471, and several times in lines 409-415, the authors use one-letter codes and residue number (e.g. Y48). Contrarily, on lines 339-344, the authors use all-caps 3-letter codes with residue number (e.g. TYR48). This same inconsistency is found in the figures.
11.	Lines 349 – ‘and’ should be ‘are’
12.	Figure 9 is probably best represented as a table instead.
13.	The resolution of the graphs in Figures 10 and 11 is poor and should be improved prior to publication.
14.	Line 359 – The authors refer to their energy decomposition as ‘Pairwise’, which would suggest the energies correspond to the interaction energy between two different residues. However, the data appears to show a ‘per-residue’ decomposition since the energies are reported as the energy of a single residue, and not it paired with another residue. This should be clarified by the authors.
15.	Line 373 – enzyme should be enzymes
16.	Line 388 – IC50 needs to have the 50 appropriately subscripted
17.	Line 402 – “…formation 4 hydrogen…” should be “…formation of four hydrogen…”
18.	Lines 445 – remove ‘an’
19.	Lines 463 – Plasmodium should be italicized
Experimental design: Overall, the experimental design is very straightforward and reasonable. And the methods are explained well by the authors. I have only one main criticism of the experimental design, and that is concerning the details of the MM-PBSA calculation, as noted below.

1.	For MM-PBSA calculations, why were exactly 625 snapshots chosen? And why only 625 snapshots? And why randomly? And how were the frames “randomly” chosen? And why did these snapshots only come from 200-300 ns of the trajectory (as described on line 319).
Validity of the findings: Most of the conclusions drawn from the current data meets the journals standards. However, I found one area where conclusions are drawn that may be missing part of the explanation based on the data presented.

1.	Lines 331-332 – The authors mention the importance of the desolvation free energy term contributes to the observed enhanced binding for pTPI over hTPI, but the authors neglect to mention that the ΔEvdW term also contributes significantly (Table 2), and possibly more so than the desolvation free energy.
Additional comments: This study is interesting and has the potential to be intriguing to many people in the field. I believe the research has high impact capability. The authors have done a fantastic job of responding to my previous comments. I am very impressed with the amount of work the authors put into the revisions and the paper is significantly better than the previous version. I applaud their work, especially knowing it is being performed at a teaching undergraduate university (I also work at a PUI). I have only minimal comments/suggestions for the current version, and once those are addressed, I emphatically support the publication of this manuscript. Great work!


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

Input:
Title: CHEMICAL SPACE EXPLORATION: HOW GENETIC ALGORITHMS FIND THE NEEDLE IN THE HAYSTACK
Review round: 1
Reviewer: 1

Output:
Basic reporting: This paper cites only 18 references even though it addresses a rather 'classic' problem using rather 'classic' methods. 6 of the 18 references correspond to potential energy methods which are not the point of this study. Comparison to performance of other optimization runs published in design papers in the literature is lacking entirely. As such, I am skeptical about the scholarship level of this manuscript. For example, Geerlings and co-workers published some best first search molecular design results a couple of years ago where they also found surprisingly rapid convergence in chemical space. The authors offer little to no context regarding this and other related work in the field. All other boxes (English/Structure/self-contained) check.
Experimental design: I see two problems with this manuscript. First, the research question is, to the best of my understanding, not entirely well defined. Typically, when performing molecular design, some target property value should be given, and the optimisation algorithm has to search chemical space in order to minimise deviation from target property. This is what the authors do for the absorbance. It is less clear to the reader what is the actually target in their 'Graph-based approach' section. If it is just the Tanimoto similarity I do not really understand the point since the target is then known already and a simple greedy best first search algorithm should trivially lead to the result in (practically) no time. Or is this interesting because only a GA should be used? In any case, I do not think that this is sufficiently well explained.
Secondly, the example target values for the absorbance (200, 400, and 600 nm) seem ad hoc, and it is hard to decipher for the reader how 'hard' they really are. If the absorbance distribution of their molecular space had, for example, peaks at those three values, it should be relatively easy to rapidly find examples that get close. As such, it is not clear how such a hidden bias might have affected the results and the conclusions drawn. I think that a distribution plot of some representative sub-sample should be shown, and the authors should also include target values which lie outside of that distribution, in order to see if their conclusions also hold for the extremes.
All other boxes (primary research/rigorous investigation/methods) check.
Validity of the findings: The conclusions should be adapted in response to the changes requested before. All other boxes check.
Additional comments: This is an interesting study with interesting findings. There are some points which should be adressed, but I think that it can be easily done. It might also be worthwhile to include some speculations in how far the conclusions depend on the choice of GA as an optimization algorithm, and how they could change if other algorithms were used.


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: CHEMICAL SPACE EXPLORATION: HOW GENETIC ALGORITHMS FIND THE NEEDLE IN THE HAYSTACK
Review round: 1
Reviewer: 2

Output:
Basic reporting: The authors use precise English and the professional words. However, some mistakes still exist and the authors should proofread the paper carefully. The background may lack some proper description.
Experimental design: The problems the paper aims to tackle are clearly stated.
Validity of the findings: In this manuscript, the authors describe the genetic algorithms to find particular molecules in an enormous chemical space by considering only a tiny subset. The method has been proved exactly in authors’ previous work. Here, the rediscovery of specific molecules is discussed by strings-based and graph-based approach and the search for the target properties is also analyzed in detail. The explanation about particular molecules can be easier found by search algorithms.
Additional comments: (1)	The mutation rates used in string-based and graph-based genetic algorithms should be specified in manuscript.
(2)	It would be better to demonstrate that whether the search for molecules with target properties depends on the properties of initial population or not.
(3)	For each generation of different searches, the scores have different degrees of improvement. It would be better to show the processes of molecules changes.


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: TACKLING THE SARS-COV-2 MAIN PROTEASE USING HYBRID DERIVATIVES OF 1,5-DISUBSTITUTED TETRAZOLE-1,2,3-TRIAZOLES: AN IN SILICO ASSAY
Review round: 1
Reviewer: 1

Output:
Basic reporting: The article meets the standards of this journal.
Experimental design: The experimental design meets the standards of the journal.
Validity of the findings: Suggested improvements are given in the "general comments" section.
Additional comments: In this submission to this PeerJ journal, the authors present a series of calculations on ligand-target interactions using the Molegro MVD package. The authors also investigate in silico molecular couplings, so-called molecular docking using the MolDock score function from Thomsen and co-workers.

I consider this manuscript to be of interest to readers of this PeerJ journal, and I am supportive of publication with a minor note. There has actually be prior work using advanced methods (such as DFTB) for understanding ligand-protein interactions, which should also be mentioned:

J. Comput. Chem. 38, 1987–1990 (2017)
J. Chem. Theory Comput. 15, 2807-2815 (2019)

In particular, these prior works have shown that DFTB is more accurate than MD calculations for ligand-protein interactions, and is faster than DFT for large systems, which should be mentioned as previous studies relevant to this field. With this minor revision, I would be receptive towards 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: TACKLING THE SARS-COV-2 MAIN PROTEASE USING HYBRID DERIVATIVES OF 1,5-DISUBSTITUTED TETRAZOLE-1,2,3-TRIAZOLES: AN IN SILICO ASSAY
Review round: 1
Reviewer: 2

Output:
Basic reporting: This is highly requested that authors should increase the understandability of the sentences all over the manuscript. In the introduction section please mention the specific benefit for the substitution conducted in this work. In 64 – 67 please mention the abbreviation and please provide information about what kind of biological theoretical understanding is necessary and why it is important and improve the understanding from the previously published works. In the last paragraph of the introduction please arrange relevant information about your study and mention the methodology used in this work. Some of the general issues have to be corrected
Line 44- please update the number according to the present situation.
Line 114 – insert (-) sign
Line 139 – why SER144 is capital?
In table 1 – Please insert the unit accordingly in the main text.
This type of irrelevancy should be carefully removed.
Experimental design: I hope the authors don’t mind including more detailed information about the simulation techniques and the initial setup of the simulation. Further, in line 88 – 89, optimization was conducted at the UFF level, what is the reason for choosing UFF, authors should define the reason and explain the validity of the choosing such methods.
Validity of the findings: Electrostatic charge distribution on the surface of the 1,5 – disubstituted tetrazole – 1,2,3-triazoles is important for this study and read will like to see the distribution of the change which could provide easy understandings, authors can provide such information by making a surface grid figure at least for the co-crystallized, 1e, and P8 compounds.

A clear representation of co-crystalized ligand and the 1e ligand is necessary for easy understandings.

In figure 4 the reason for the repulsive interactions has to be explained, further, it is worthy to provide an explanation about the difference of the interaction energies for different target sites.
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: TACKLING THE SARS-COV-2 MAIN PROTEASE USING HYBRID DERIVATIVES OF 1,5-DISUBSTITUTED TETRAZOLE-1,2,3-TRIAZOLES: AN IN SILICO ASSAY
Review round: 1
Reviewer: 3

Output:
Basic reporting: This article was well written in english and was clear with out ambiguity . Technicalli it is looking sound and professional.
Experimental design: Methods were described sufficiently and followed simple straight proved and widely accepted protocols which makes consisitent
Validity of the findings: Results section authors have given the name recombinant strain
COV-2MA
Additional comments: Authors required to include the following references at appropriate places in the manuscript

https://www.sciencedirect.com/science/article/pii/S2352914820301805

https://www.tandfonline.com/doi/full/10.1080/07391102.2020.1770127


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: UV SYNCHROTRON RADIATION LINEAR DICHROISM SPECTROSCOPY OF THE ANTI-PSORIATIC DRUG ANTHRALIN
Review round: 1
Reviewer: 1

Output:
Basic reporting: Line58. "previously investigated"
Report references.
Experimental design: Lines 95-99.
The authors should explain why they use two different TD-DFT techniques:
1. TD-BRLYP/6-31+g(d,p), in Fig. 5 and Data_S2;
2. TD-CAM-BRLYP/aug-cc-pVTZ, in Fig. 3, Fig. 4, Tab. 1, and Data_S1.
Line 105. "Gaussian convolutions"
Please, add some details.
Validity of the findings: Line 71."Fig. S1"
Did the authors take into account the heptane solvent effect, e.g. via the PCM method?
Line 84. "58000-31300 cm-1"
But the paper reports observed data  down to 25000 cm-1.
Line 143. "Fig. 3"
Correct the caption as pVTZ.
Better oscillator strength instead of "f".
Line 148. "Table 1"
Footnote a: Data S3 are missing.
Footnote g: correct as Fig. 4.
Line 193. "Fig. 4"
Report the color meaning.
Line 198. "Geometry optimization"
Did the authors take into account the n-hexane solvent effect, e.g. via the PCM method?
Line 200. "Fig. 5"
Anthralin left results are different from those in Tab. 1, Data_S1, and Fig. S4. That's probably due to the different TD-DFT methods employed, and the authors must explain and comment on that.
Line 205. "Fig. S4"
Anthralin energies here are different from those in Fig. 5. Why?
Additional comments: This nice study reports anthralin absorption and fluorescence spectra, obtained and analyzed at the level of the present state of the art. I especially appreciated the assignment of the SRLD observed bands via TF-DFT calculations. The article should be accepted if the authors make some minor revisions, as commented above.


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: UV SYNCHROTRON RADIATION LINEAR DICHROISM SPECTROSCOPY OF THE ANTI-PSORIATIC DRUG ANTHRALIN
Review round: 1
Reviewer: 2

Output:
Basic reporting: This is a well written, carefully performed piece of work, that should be accepted for publication.

I would be much happier if the units used for energy (cm-1, nm and eV) were converted to the numbers usually used by spectroscopists in the relevant regions (i.e. nm for the electronic spectroscopy and cm-1 for the vibrational region) as I found it hard to compare everything and to compare with my own experience.

Figure legends should go below figures.
Experimental design: Well designed experiment to understand the nature of the spectrosocpy of the molecule.

Nothing is achieved in terms of mode of action - which is the motivation for the paper. However, the data will be useful.
Validity of the findings: Satisfactory
Additional comments: See above.


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: UV SYNCHROTRON RADIATION LINEAR DICHROISM SPECTROSCOPY OF THE ANTI-PSORIATIC DRUG ANTHRALIN
Review round: 1
Reviewer: 3

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: In this paper, the authors characterized by synchrotron radiation linear
dichroism (SRLD) spectroscopy the molecule Anthralin using samples in which the anthralin molecule was partially aligned in stretched Polyethylene. The obtained results were also compared by quantum chemical calculations using time-dependent density functional theory (TD–DFT).  This study allowed to assign the obtained spectral features.
The manuscript is clear and well structured, the introduction show the context is well referenced measurements, figures, calculations and results are well explained. Raw data is supplied.
The original primary research is in the scope of the journal, the research question is well defined, relevant and meaningful. The work is rigorous and was performed with high technical and high ethical standard. All data have been provided and it is robust. Conclusions are well stated and completely supported by data and calculations. The paper can be recommended for publication in the present form.


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: EFFECTS OF NUMBER OF PARALLEL RUNS AND FREQUENCY OF BIAS-STRENGTH REPLACEMENT IN GENERALIZED ENSEMBLE MOLECULAR DYNAMICS SIMULATIONS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The authors have written clear and unambiguous sentences, which fully meet the criteria of this paper. However, three minor fixes are required as follows:

1. The context on lines 74-76 "It is reported that ....." doesn't have the reference. By clarifying who did the study, you can avoid the confusion of readers.

2. "0.002 ps ns," on line 205 is a spelling mistake. I think this is "0.002 ps,".

3. The simulation condition t_vst of the data shown in figure 6 is different between line 389 of the text and the caption. I guess that the text is correct.
Experimental design: no comment.
Validity of the findings: Research questions well defined and their conclusions are clearly described. I'm convinced that this paper provides useful information to researchers who are using simulation methods similar to this study.
Additional comments: I require two corrections to improve the reader's readability as follows:

1. The types of lines in the line graph as shown in figure S6 should be reconsidered. It is difficult for readers to distinguish between the two dotted lines in the caption.

2. There are too many digits in the numerical data shown in the text such as PCC_PMF and P_native. Especially, there is no point in displaying the value of standard deviation with many digits.


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: EFFECTS OF NUMBER OF PARALLEL RUNS AND FREQUENCY OF BIAS-STRENGTH REPLACEMENT IN GENERALIZED ENSEMBLE MOLECULAR DYNAMICS SIMULATIONS
Review round: 1
Reviewer: 2

Output:
Basic reporting: I do not see any raw data in the SI. As much raw data as is practical should be provided.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The work appears to be carefully done, and the authors make clear recommendations based on an analysis of the data


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: EFFECTS OF NUMBER OF PARALLEL RUNS AND FREQUENCY OF BIAS-STRENGTH REPLACEMENT IN GENERALIZED ENSEMBLE MOLECULAR DYNAMICS SIMULATIONS
Review round: 1
Reviewer: 3

Output:
Basic reporting: The quality of writing is excellent through. The literature references are appropriate and sufficient. The figures look good and the paper is complete.
Experimental design: The authors report appropriate analysis of the efficiency of generalized ensemble simulations for protein folding. It answers some fundamental questions about the efficiency of these methods and the optimal simulation options for these models. The quality of the models is the norm for the field.  The calculations are done rigorously and the methods are well-described.
Validity of the findings: The findings are valid and are consistent with their reported data. The results are presented honestly and unambiguously.
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: STRUCTURE AND DIFFUSION OF MOLTEN ALKALI CARBONATE SALTS AT THE LIQUID-VACUUM INTERFACE
Review round: 1
Reviewer: 1

Output:
Basic reporting: 'no comment'
Experimental design: 'no comment'
Validity of the findings: 'no comment'
Additional comments: 1.The methods of the manuscript needs more detail. I suggest that you improve the description at lines 114- 120 to provide more justification for your study (specifically, you should give the
specific expression of potential function and detailed numerical value of the interaction parameters ).
2.In the manuscript, the simulation results lack a detailed calculation process. It is only based on similar methods in the literature, but the specific calculation method is not given. This makes it difficult for future readers to understand.
3.The description at lines110-112"Initial configurations were prepared by placing 1000 cations (according to the fractions in Table 1) and 500 carbonate anions randomly on a grid in a simulation cell with dimensions of 40 Å*40 Å*100 Å." Please give the answers to the following questions in the appropriate place in the manuscript.
a. What kind of random algorithm is used for random placement?
b. Are there multiple simulations and statistical analysis of the simulation results?
c. Are the results of multiple simulations consistent?
d. Whether the micro-simulation results can be extended to the macro-system?


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

Input:
Title: STRUCTURE AND DIFFUSION OF MOLTEN ALKALI CARBONATE SALTS AT THE LIQUID-VACUUM INTERFACE
Review round: 1
Reviewer: 2

Output:
Basic reporting: The English require substantial improvements.  For example, line 36: are specifically; Line 81: they have identified; line 105: The considered three systems; line 112: were; line 113:was ; line 129: Visual Molecular Dynamics (VMD) ……
 In the introduction, paragraph 3 required references to validate the statement.

Line 229 to 245:  description of identifying the instantaneous interface should go to method part.
Experimental design: The purpose of this work is not very clear. Why are authors focused on liquid-vacuum interface not other interface?  The potential application of the findings should be described more specifically.

How the total number of ions in the systems are determined? What is the initial liquid density?

The description of the system set up is not very clear.  It is better to have a representative figure to show the system set up.
Validity of the findings: The simulation time is very short, and the equilibration of systems should be validated to ensure the sufficient sampling such as analysis of the density change with time

The observed peaks and drops are very small, and the fluctuation could be dramatic along the simulation.  The density profile evolved with time should be analysis and error bar should be added to time-averaged density profile.

It is not clear how the results are analyzed. Is it analyzed using the last frame in the simulation or averaged over all the frames?
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: STRUCTURE AND DIFFUSION OF MOLTEN ALKALI CARBONATE SALTS AT THE LIQUID-VACUUM INTERFACE
Review round: 2
Reviewer: 1

Output:
Basic reporting: 'no comment'
Experimental design: 'no comment'
Validity of the findings: 'no comment'
Additional comments: The author has revised the manuscript as requested by the reviewer.The reviewer suggested accepting the manuscript.


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

Input:
Title: STRUCTURE AND DIFFUSION OF MOLTEN ALKALI CARBONATE SALTS AT THE LIQUID-VACUUM INTERFACE
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: My comments have been addressed and I recommend 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: OUTLINE OF AN EXPERIMENTAL DESIGN AIMED TO DETECT PROTEIN A MIRROR IMAGE IN SOLUTION
Review round: 1
Reviewer: 1

Output:
Basic reporting: There are several places where a space is needed before a reference.

"MD simulationVorobjev et al."
"biologySchnell"
"task.Machuqueiro"
are three examples I found, but suggest a general check throughout.
Experimental design: According to the methods section, "As mentioned in the main text, all acid 110 (Asp, Glu) and base (Lys, Arg) residues were kept in the ionized state because their respective pKo’s 111 (3.5, 4.0, and 10.5, 12.5, respectively) are shifted by more than 2.5 pK units from the pH (7.0) at which 112 the calculations were carried out."

but pKa's can shift depending on the protein environment. Why was no check done to make sure these residues have pKas safely far away from 7? Seems like that should be straightforward given your other calculations.

Precisely how the MD simulations were set-up and processed is not detailed. Details are necessary for replication.
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: OUTLINE OF AN EXPERIMENTAL DESIGN AIMED TO DETECT PROTEIN A MIRROR IMAGE IN SOLUTION
Review round: 1
Reviewer: 2

Output:
Basic reporting: This paper uses computational predictions of a the structures of a histidine-containing mutant of the B-domain of staphylococcal protein A to propose an experimental test of as to whether this protein holds the generally accepted conformation or its "mirror image."

The literature review seems sufficient. I think the writing was clear, but the formatting should be improved, particularly because PeerJ uses parenthetical citations. There are several non-standard writing choices, like beginning a paragraph with "At this point is worth noting the following."
Experimental design: I believe the experimental design is too flawed for the results to be conclusive.

Also, the proposed experiment would distinguish between the conformations of a histidine-containing mutant of the protein as a function of pH. Protonation states can have a large effect on the conformation of a protein, so replacement of a glutamine residue with a histidine residue and then titrating the protonation state of the histidine.

Much of the generation of the protein conformations are done "by hand" and there is an acknowledged subjective element. e.g., "For the native conformation we used 1BDD. Gouda et al. (1992) For the mirror image we started from 2 points: a mirror-image conformation previously obtained by Vila et al, Vila et al. (2003) an a mirror image folded by hand. We discarded all the conformations obtained from this last starting point as their energies were much more higher than for the other two starting points.

The technical details of their molecular dynamics simulations are incomplete, but it seems clear that the simulations were only performed for 10 ns. Much longer simulations are necessary to calculate rigorous pKa differences. I suspect the observed differences might disappear if the simulations were performed longer. Also, pKa prediction methods of all types rarely more accurate than plus or minus one unit relative to the experimental values, so there difference of 6.1 vs 7.3 is within the margin of error of the calculations. The authors report a statistical error ~0.2-0.3 pK units, but this is likely a major underestimate due to limited sampling. At minimum, this result would have to be reproduced by other constant pH methods, such as the Brooks, Roitberg, Shen, or Roux methods. Simulation in an explicit solvent and demonstrating convergence are essential for this. Also, long-timescale simulations would be needed to show that the histidine mutant as the same conformation as the wild-type in both the A and mirror conformations as well as their relative Gibbs energies. It would also need to be shown that titration of the histidine and its fractional population in a protonated state does not induce a conformational change (which is unlikely to be true).
Validity of the findings: For the results I describe above, I do not believe it's possible to make any rigorous conclusions based on the modelling performed here.
Additional comments: My feeling is that there is not to much to be gained by further theoretical studies of this problem. Without experimental validation, it's difficult to be conclusive. The essential design of the proposed experiment introduces an amino acid that will undergo a change in charge as part of the experiment and is also fractionally populated. This could totally change the conformation of a small protein, so I don't believe the problem is well-defined. The computational pKa prediction methods would also need to be shown to be able to predict experimental values with a higher accuracy of any previous method. If this method can do that, there are many more exciting problems to study with that tool.


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: EFFECT OF FEMTOSECOND LASER PULSE REPETITION RATE ON NONLINEAR OPTICAL PROPERTIES OF ORGANIC LIQUIDS
Review round: 1
Reviewer: 1

Output:
Basic reporting: All comments are listed  in the Referee report (attached)
Experimental design: All comments are listed  in the Referee report (attached)
Validity of the findings: All comments are listed  in the Referee report (attached)
Additional comments: Overall the manuscript appears to be clearly and carefully written. The results presented are interesting for the field of pump probe technique. I think that the manuscript might deserve publication in Peer J Physical Chemistry after some points are dealt with and some missing details are added prior to 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: EFFECT OF FEMTOSECOND LASER PULSE REPETITION RATE ON NONLINEAR OPTICAL PROPERTIES OF ORGANIC LIQUIDS
Review round: 1
Reviewer: 2

Output:
Basic reporting: There are some typo that need to be corrected. For example:
In line 57: "nonlinear optical " should be abbreviated as "NLO";
in line  191, "The change in *2 was observed" should be "The change in n2 was observed"
Experimental design: no comment.
Validity of the findings: 1. The authors claim that the negative nonlinear refraction index (Fig. 3) is caused by the thermal lens effect induced by the highly repeated laser pulses. The authors should explain why the nonlinear refractive index is negative?

2. Also in Fig. 3, reversal of sign in the nonlinear index of refraction is observed in CH3OH when excited by high repetition rate laser. What's the situation with the material of CS2? More experimental results are needed.

3. As shown in Fig. 6 and Fig. 8, the authors have demonstrated the influence of the pulse-to-pulse separation on the nonlinear index of refraction. It’s reasonable that thermal effect plays an important role in the measurements. However, the repetition rate could also influence the pulse width and the averaged intensity of the pulse (as shown in Fig. 7). This influence should be considered in the measurement of the nonlinear optical response.
Additional comments: Generally, the findings in the manuscript is interesting and article is well organized. As mentioned above, some more experimental results are needed to support the author's point of view, and deeper discussion is needed to make the manuscript more readable. The reviewer suggests that the manuscript should be major revised before 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: EFFECT OF FEMTOSECOND LASER PULSE REPETITION RATE ON NONLINEAR OPTICAL PROPERTIES OF ORGANIC LIQUIDS
Review round: 2
Reviewer: 1

Output:
Basic reporting: Dear Editor,

Thank you very much for the opportunity to review the revised article entitled Effect of femtosecond laser pulse repetition rate on nonlinear optical properties of organic liquids" by Sandeep K Maurya, Dheerendra Yadav, Debabrata Goswami.

The authors have made satisfactory amendments to the manuscript in response to my previous comments and concerns. Overall the manuscript reads well and has clarified the work of the authors. In my opinion the manuscript contains now all information and is suitable for publication in journal PeerJ Physical Chemistry as a regular article.

Best regards

Tarek Mohamed
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: BUILDING A SYNTHESIS OF ECONOMIC COSTS OF BIOLOGICAL INVASIONS IN NEW ZEALAND
Review round: 1
Reviewer: 1

Output:
Basic reporting: My comments are in given in detail in the below section on "additional comments".
Experimental design: My comments are in given in detail in the below section on "additional comments".
Validity of the findings: My comments are in given in detail in the below section on "additional comments".
Additional comments: This manuscript represents an economic assessment of invasive species for New Zealand over the period of 1968- 2020. Economic assessments are notoriously difficult to achieve, as they are full of unreported costs and assumptions. But the authors do a good job using a newly created database and acknowledge limitations in the analysis. The manuscript needs a careful and thorough review and tidy-up for the grammar and presentation in main manuscript, the figures, and the references. I also have some points for the authors to consider below. After revision I think the paper should be acceptable and would make a useful addition to the literature that would be well cited. The points to consider are:

Abstract. The abstract is fine, although doesn’t mention the multi-country analysis that you show on figure 4.

Introduction. The introduction is long and extensive, covering much of the relevant literature. You might want a sentence or two introducing NZ to readers that don’t know much about NZ: where it is, the size, and perhaps the fact that we have suffered extensively from biological invasions including a lot of extinctions. On line 38, I don’t think you need “(but see Crystal-Ornelas and Lockwood, 2020)”. Just say “however, despite GENERALLY widely known…”.

Methods. I thought the methods were presented in sufficient detail to enable the reader to understand what is happening. My only suggestion would be to expand  on what the log-log-linear analysis is testing and how to interpret that analysis. As I note in the section below on the references, these need updating. For example in the methods you state tha Leroy et al. (2020) just on bioRxiv. Has it not moved on from bioRxiv yet?

Results. I liked the results and the focus on the “highly reliable costs”. On lines 234- 235 you state that there has been a very limited total cost reported from health, public and social welfare sectors. It would be useful to follow that up in the discussion or additional analysis. My suspicion is that the invasive species we have in NZ haven’t had a major health or social welfare impact here. Note on lines 258 that NZ has “successfully” eradicated many of the invasive species you mention.

Discussion. The discussion is very long. As it stands I think it’s length dilutes your primary messages. Perhaps paragraphs such as lines 390- 404 could be removed. Despite and in contradiction of my comment on the length of the discussion, there are a couple small additions that you could consider. I don’t think your definition of taonga is quite there, as it is often considered ‘treasure’. In your discussion on management costs, you could talk about species such as rabbits, given they are an increasing cost because the government has put less money into managing them (no more rabbit boards, etc). The discussion on SARS-CoV-2 is interesting as it could well be considered an “invasive species”. How many billions has that cost us? The discussion is certainly an area that the punctuation and grammar needs work. It also has some VERY long paragraphs.

References. The references need careful inspection and revision.The first two references (Ahmed et al. 2021 and Allen et al. 2006), for example, are both incomplete. Please include page numbers issues, titles, etc.

Figure 1 is a nice idea and start, but needs more work. What are the units on the y-axis? Billions of NZ or US dollars? The units aren’t stated in the caption or on the graph. Please ensure the axis labels are in the right place.

Figure 2. Does this graph show the costs are leveling-off over time, or is that a function of the logarithmic scale? The horizontal error bars are weird. They are suggested to represent the variation around the decadal averages of economic costs, but the horizontal or x-axis is years. I think this graph presentation needs further thought.

Figure 3. The authors need to make it clear in the caption what the white sections of the plots are, which I assume to represent a lack of information.

Figure 4. This figure and analysis is not mentioned in the abstract. More detail is needed for the axis units. The areas of the graph highlighted in blue are the countries suggested to have “lower expenditures than expected by GDP”. How did the authors derived these expectations? Such a derivation is not mentioned in the methods and is highly subjective. I suggest removing it.

I hope these comments are useful for improving the manuscript.


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

Input:
Title: BUILDING A SYNTHESIS OF ECONOMIC COSTS OF BIOLOGICAL INVASIONS IN NEW ZEALAND
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript by Bodey et al. reviews the costs of biological invasions in New Zealand. This study is part of a larger global initiative, INVACOST, that is providing assessments across nations, taxonomic groups and socio-economic sectors. This is important to note, because the value of the study must be considered in this context: results are not only interesting by themselves, but could be compared with the rest of assessments published or in the process of being published.

The language, structure and figures of the paper are clear and the manuscript is easy to follow. The sources of data are indicated and readers are encouraged to even provide new evidence to this growing database.
Experimental design: The research question is well defined and meaningful. Indeed, New Zealand is a global reference for good practices in invasive species research and management. It is therefore important to quantify, and compare with other countries, the costs and benefits associated to management. Methods are well explained and replicates the statistical workflow of other Invacost papers, which should facilitate comparison.
Validity of the findings: The costs of biological invasions in New Zealand are estimated in N>$97 billion over the last 50 years. More than 80% correspond to damage costs. Yet, in comparison with other countries, New Zealand is investing considerably more on management.

I only have one criticism. The manuscript misses an important opportunity to quantify the economic benefits (or avoided damage) associated to the strong regulation of IAS in New Zealand, which is currently stated but not fully quantified. First, it is not clear when did regulations start. Even considering delays in the implementation of measures, it’d be very important to identify in temporal trends (e.g. Fig. 2) such important moments. Second, other Invacost papers have used methods to estimate the “avoided cost” of managing invasions (see Ahmed et al. 2021 https://orcid.org/0000-0002-8402-5111; Cuthbert et al. in press in STOTEN https://doi.org/10.1016/j.scitotenv.2022.153404). In spite of the limitations that the method may have, this seems more interesting than the simple comparison with other countries provided here, which is similarly subject to many limitations in data availability. Third, while I understand the use of this decadal time analysis (the one in Fig. 2), I wonder if other statistical approaches wouldn’t be more appropriate. For instance, piece wise regression analysis to identify potential inflexion points.
Additional comments: Page 8, line 71. “relatively early”. Can you be more specific?
Page 9, lines 73-75. “New Zealand has strict IAS screening protocols based on a rigorous evidence-based assessment of the risks associated with a given overseas port or on particular goods”. Since when exactly? Can you be more specific?
Page 9, line 75. How expensive? Can you be more specific?
Page 129, line 129. Can you explain the relevance of the number of records? Why would this information be as important as the total amount invested?
Fig. 1 could be nice as graphical abstract, but not very informative.
Fig. 2 could indicate the start of major IAS regulations.
Fig. 3 is confusing and little informative. Can you think of a different way to represent this information?


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: BUILDING A SYNTHESIS OF ECONOMIC COSTS OF BIOLOGICAL INVASIONS IN NEW ZEALAND
Review round: 2
Reviewer: 1

Output:
Basic reporting: Nothing more to add from the first review.
Experimental design: Nothing more to add from the first review.
Validity of the findings: Nothing more to add from the first review.
Additional comments: I’ve now read the revised manuscript and the author’s response to the referee’s comments. I’m now happy to recommend that the paper be accepted into the journal and have no additional comments. The authors have suggested the inclusion of an abstract in Te Reo or the Māori language, which I’d encourage the journal to allow. My only other suggestion would be to ensure the journal issue and page numbers are added at the proofing stage for the manuscript—there are several that need these if they are fully published in time.


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: NEIGHBORHOOD-BASED INFERENCE AND RESTRICTED BOLTZMANN MACHINE FOR MICROBE AND DRUG ASSOCIATIONS PREDICTION
Review round: 1
Reviewer: 1

Output:
Basic reporting: Studies on the response of pathogens to antibiotic treatment are limited by the time and resources necessary to attend each clinical case individually. A promising strategy is the computerized use of biological information available in databases and the development of bioinformatic tools, which is one of the main objectives of this study.

However, this type of machine learning has already been used for the diagnosis and treatment of diseases such as cancer, the background should include this type of information that allows the reader to have confidence and context in the use of computational approaches to the clinic.

On the other hand, the quality of the figures should be improved, in particular, figure 1 and 2.
The objective of this article is clear and relevant, the strategy used to test the system and validate it in a bibliographical way and with experimental data is acceptable. The results presented are consistent and in accordance with the objective of the investigation.
Experimental design: The topic of this article is clear, well defined and relevant. The application of this research can be significant and enriching in the study of antibiotic resistance and treatment of various pathogens. The methodology used fully meets the requirements for the computational study of interactions and the methodology used and developed is described in sufficient detail to be replicated and used by other researchers. Validation of the proposed strategy with other pathologies generated by non-emerging microorganisms would be useful.
Validity of the findings: The study is of benefit to the area of knowledge, the objectives and conclusions are well expressed. The data used are robust, however they can be enriched and validated with more experimental information.
Additional comments: I believe that this article can be accepted after improving the bibliographic information that supports the importance of predictive studies that use machine learning in the medical and microbiological area. In addition to improving the quality and resolution of the figures and including in the conclusions that the proposed proposal works efficiently in the cases described in the article but that it may depend on the databases or microorganisms analyzed.


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

Input:
Title: NEIGHBORHOOD-BASED INFERENCE AND RESTRICTED BOLTZMANN MACHINE FOR MICROBE AND DRUG ASSOCIATIONS PREDICTION
Review round: 1
Reviewer: 2

Output:
Basic reporting: It is an interesting manuscript in general well written and understandable, references are adequate.
However, bacterial names are not in italics and sometimes the first letter of the gender was not I capital letter, e.g. pseudomonas should be Pseudomonas.
Experimental design: No experiments were done, it is only a theoretical work, validated with existing information from the literature, it is well structured, the research question is well defined and enough details of the methods are provided.
Validity of the findings: The predictions of the developed model were validated using the literature, looking like a robust approximation.

Interestingly some information obtained and sowed in tables 5 and 6 is not validated by any study, like  the possible effect of ciprofloxacin over Streptococcus pneumoniae  serotype 4, Actinomyces oris and HIV virus, also the predicted antibacterial effects of minocycline over Salmonella enterica and other 3 bacterial species, it would be ideal that at least some of those non validated effects are tested to further validate the strength of the model predictions.
Additional comments: L 533 “Therefore, minocycline has been used for treating many diseases such as acne, neurodegenerative conditions and dermatitis (Nagarakanti & Bishburg 2016).

Please explain why if minocycline is an antibiotic it is used for neurodegenerative conditions and dermatitis

L 540 “the pseudomonas aeruginosa was” remove “the”, capitalize “P”

Also put scientific names in the reference in italics.


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: NEIGHBORHOOD-BASED INFERENCE AND RESTRICTED BOLTZMANN MACHINE FOR MICROBE AND DRUG ASSOCIATIONS PREDICTION
Review round: 2
Reviewer: 1

Output:
Basic reporting: The comments have been addressed according to the possibilities of the researchers based on the available literature.
Experimental design: The validation of the computer results with available literature that expresses experimental evidence was attended. Table 6 increased by 2.
Validity of the findings: The proposal presented here is relevant in the medical and informatics areas.
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: NEIGHBORHOOD-BASED INFERENCE AND RESTRICTED BOLTZMANN MACHINE FOR MICROBE AND DRUG ASSOCIATIONS PREDICTION
Review round: 2
Reviewer: 2

Output:
Basic reporting: it is now OK
Experimental design: it is now OK
Validity of the findings: it is now OK
Additional comments: I thank the authors for addressing my comments and for the modifications made to their manuscript.


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

Input:
Title: THE ROLE OF AUTOPHAGY IN HIGH-FAT DIET-INDUCED INSULIN RESISTANCE OF ADIPOSE TISSUES IN MICE
Review round: 1
Reviewer: 1

Output:
Basic reporting: In this study, the author demonstrated that high-fat diet treatment caused the suppression of late-stage autophagy, endoplasmic reticulum (ER) stress, and apoptosis in the adipose tissue of mice. The author also indicated that autophagy inhibition didn’t change the insulin signaling transduction but caused insulin resistance indirectly through ER stress or apoptosis.
This studying is meaningful. The manuscript is well written in English. The structure of the manuscript conforms to PeerJ standards. Raw data and supporting information were supplied.
However, PeerJ uses the 'Name. Year' style with an alphabetized reference list. Please format the in-text citations and reference section following the PeerJ guidance.
Experimental design: The experimental design is clear. The method is writing in detail. Here are two minor points that may need to be addressed.
1. Line 25: What is the Bafilomycin A1 working concentration?
2. Line 220-222: How to treat the cells with FFAs is suggested to be included in the Materials & Methods section.
Validity of the findings: The result section is not very clear. Some results are not matched with the figures. Some figure legends are not consistent with the pictures. That caused big confusion. Please rearrange these figures. I have some comments as follows:
1. In Figure 1F, Figure 5 E, Figure 6A, and Figure 6C, the general housekeeping reference protein should be included.
2. In Figure 7, there is no A or B labeled.
3. Figure 3 legend is not matched with Figure 3.
4. Line 230-231: “After treatment with CQ, we observed increased LC3II and SQSTM1/p62 protein levels in 3T3-L1 cells (Figures 3B, 3C). ” But Figure 3B showed LC3II and SQSTM1/p62 protein levels were no differences between the control group and CQ treatment group. In Figure 3C, the LC3B mRNA level was decreased in the CQ treatment group. The result is not clear here.
5. Line 232-234: lack of Figure 3D and Figure 3F.
6. Figure 4 legend is not matched with Figure 4.
7. Figure 5 legend is not matched with Figure 5.
8. Figure 6 legend is not matched with Figure 6.
Additional comments: NO 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 ROLE OF AUTOPHAGY IN HIGH-FAT DIET-INDUCED INSULIN RESISTANCE OF ADIPOSE TISSUES IN MICE
Review round: 1
Reviewer: 2

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: In the present study, Yovita Permata Budi and colleagues evaluated changes in autophagy and insulin signaling transduction in adipose tissue from C57BL/6 mice fed a high-fat diet (HFD) in order to elucidate potential mechanisms in autophagy-regulated adipose insulin sensitivity. The authors found that HFD induced late-stage autophagy inhibition, endoplasmic reticulum (ER) stress and apoptosis in the adipose tissue. Autophagy suppression did not influence insulin signalling via phosphatase expression, but indirectly caused insulin resistance through ER stress or apoptosis. This is an interesting study shedding light into the communication of autophagy and insulin resistance in the context of obesity. Nonetheless, some specific points require to be amended.

Specific comments

1.	Title: please change “diet induced” with “diet-induced”.
2.	Abstract: please include age and sex of experimental animals as well as the dose and time for chloroquine administration.
3.	Introduction, lines 62-63: this sentence is confusing since ER stress has been proposed as a central feature of peripheral insulin resistance and type 2 diabetes due to its ability to supress insulin receptor signalling (Ozcan U et al. Science 2004, PMID: 15486293)
4.	Introduction, line 77-78: In the study of Sossi et al. the authors state that adipocyte autophagy is attenuated in human obesity. However, it is important to explain that this condition conversely progresses to increased expression of adipocyte autophagy flux in insulin-resistant states, with patients with type 2 diabetes showing increased expression of autophagy-related proteins (ATG5, ATG7, Beclin-1 and LC3C) and autophagosome accumulation than induviduals with normal weight or with obesity and normoglycemia (Rodríguez A et al. DIabetologia 2012, PMID: 22869322; Kosacka J et al. Mol Cel Endocrinol 2015,  PMID: 25818883).
5.	Methods, line 154: please include the reference to Table 1 after “all target protein-specific primary antibodies”,
6.	Murine genes should be written according to the approved nomenclature. Murine gene symbols should be written in italics, with the first letter capitalised, without hyphens and greek letters (e.g. Actb for the gene encoding -actin).
7.	Please avoid informal abbreviations, such as doesn’t, didn’t, haven’t, throughout the manuscript.


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

Input:
Title: THE ROLE OF AUTOPHAGY IN HIGH-FAT DIET-INDUCED INSULIN RESISTANCE OF ADIPOSE TISSUES IN MICE
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript accomplishes all the essential criteria, meeting all the standards.
1.-Structure, figures and tables meet the criteria and show the raw data.
2.- They include the custom checks.
3.- All the uncropped westerns are also included.
4.- English seem to be well used (I am not an English native person).
5.- Literature references are OK.
Experimental design: They showed a correct experimental design.
Validity of the findings: They show robust results but I will tone down a bit the discussion and conclusions.
Additional comments: Dear authors,

Your manuscript is very well performed, it is thorough research about the HFD effect on Type II diabetes. However, a few details may be improved:
1.- In the in vivo assays, it would be interesting to know the inflammation after the surgery, which can be affected by the procedure and affects the autophagy results.
2.- Figures need to be checked as the letters of the legends don't correspond to the figures explained.
3.- Altered autophagy is shown already at 8 months, reanalyse the flux results.
4.- I would recommend testing the toxicity of the doses of the fatty acid, which seems a bit high.
5.- State the reason for using chloroquine instead of leupeptin or another lysosomal inhibitor in vivo.
6.- Tone down the discussion and the conclusions, they are a bit overreaching.


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

Input:
Title: THE ROLE OF AUTOPHAGY IN HIGH-FAT DIET-INDUCED INSULIN RESISTANCE OF ADIPOSE TISSUES IN MICE
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comments
Experimental design: No comments
Validity of the findings: No comments
Additional comments: The majority of the reviewer’s questions have been addressed. However, there are some minor points that should be addressed.
1. Line 225: The result should start from Figure 1A. All the Figures (A, B, C...) should be included in the context. Please check all the result section.
2. Figure 7 legend: “Western blotting (A) and quantitative data (B) of PTEN were presented” However, quantitative data of PHLPP1 and PTEN is A, and WB is B in this Figure. In Figure 7A, the author used the “control and CQ”, but in Figure 7B, the WB was labeled by PBS and CQ. Please correct it and make the labeling consistent.


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

Input:
Title: THE ROLE OF AUTOPHAGY IN HIGH-FAT DIET-INDUCED INSULIN RESISTANCE OF ADIPOSE TISSUES IN MICE
Review round: 2
Reviewer: 2

Output:
Basic reporting: I thank the authors for addressing all my comments/suggestions. The manuscript has been substantially improved, and it is my belief that it is now ready for publication.
Experimental design: The experimental design is well-defined and relevant, and the methodology is clearly explaines in the revised manuscript.
Validity of the findings: The results are clearly stated and robust, and the conclusion is linked to the original research question.
Additional comments: No additional comments