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 RELATIONSHIP BETWEEN (SUB)TROPICAL CLIMATES AND THE INCIDENCE OF COVID-19
Review round: 2
Reviewer: 3

Output:
Basic reporting: See attachment.
Experimental design: See attachment.
Validity of the findings: See attachment.
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: SERUM BDNF LEVELS AND THE ANTIDEPRESSANT EFFECTS OF ELECTROCONVULSIVE THERAPY WITH KETAMINE ANAESTHESIA: A PRELIMINARY STUDY
Review round: 1
Reviewer: 1

Output:
Basic reporting: None
Experimental design: None
Validity of the findings: None
Additional comments: This paper conducted a study that have investigated whether serum BDNF levels predict a rapid antidepressant response to ketamine used alone as an anesthesia during ECT in Chinese patients with TRD. There are several problems to be addressed:
(1) please notice the format of the abstract in the submit system
(2) In the result of “abstract”, please add the statistic values and P values.
(3) I checked the informed content and found that the protocol only associated with source of patients. Collecting the information from healthy volunteers also needs the informed content.
(4) A small sample size could lack enough statistic power to get the stable results. Authors should provide sample size estimation. I believe that authors can attain more healthy volunteers. The ratio (case: control=1:4) may have a higher statistic power.
(5) Line 115-117. Please provide the references to identify the feasibility of half-age method.
(6) Line 123. Please use “were” rather than “was”.
(7) Line 132. What is the meaning of “a single blood sample”?
(8) Line 157. Please use “baseline serum BDNF levels” rather than “serum BDNF levels”.
(9) Please specifically introduce the changes in HAMD-17 and BPRS scores following ECT treatment between remitters and non-remitters.
(10) I suggest that supplementary table S1 should be placed in the manuscript and Figure 1 should be placed in the supplementary materials.


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: SERUM BDNF LEVELS AND THE ANTIDEPRESSANT EFFECTS OF ELECTROCONVULSIVE THERAPY WITH KETAMINE ANAESTHESIA: A PRELIMINARY STUDY
Review round: 1
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: 1. The paper has some language issues. For example, line 6, a problematic sentence. Line 31, “major mental disorders” seems like a lay term in psychiatry, please make it clear.
2. The study has negative findings. One major limitation is the small sample size, 7 remitters were compared to 23 non-remitters and 30 TRD patients were compared to 30 healthy controls. Lack of statistical power should be a major reason for these negative findings. The conclusion seem also over-stated. Please consider to tone down it due to these limitations.
3. Line 58-79, the organization of the two paragraphs could be improved. The current review seems confused. I suggest the authors to review the biomarker roles of BDNF, either for antidepressant effect prediction or for disease severity, and review BDNF as a biomarker in ECT treatment, alone or combination with other anesthetics. For these reviews, it is important to emphasize more on positive findings on the biomarker roles of BDNF, because the authors hypothesized a positive role of BDNF in their studies. Inconsistent or negative findings on the biomarker roles could be moved to Discussion, because you have negative findings too!!
4. Line 98, there is no MDD or major depressive episode in ICD-10. Please check this.
5. Please clarify the clinical settings of patient recruitment, outpatient or inpatient?
6. Line 108, how patients and healthy controls were matched?
7. Line 113, in addition to the treatment sessions, please specify how long was the treatment delivered?
8. Line 121, clinical assessment. Safety outcomes are also important for clinical trials, but the authors only had efficacy outcomes.
9. In the supplementary study protocol, there were three treatment arms, but in this report, there was only one arm, ECT + Ketamine. Please specify why the two control groups were not included into the analysis.
10. Figure 2, please check the data of non-remitters. Scores of non-remitters and remitters are very similar to 6.
11. For outcome assessment, the authors defined treatment response, but no corresponding results reported.


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: SERUM BDNF LEVELS AND THE ANTIDEPRESSANT EFFECTS OF ELECTROCONVULSIVE THERAPY WITH KETAMINE ANAESTHESIA: A PRELIMINARY STUDY
Review round: 2
Reviewer: 1

Output:
Basic reporting: None
Experimental design: None
Validity of the findings: None
Additional comments: Thank you for your revision.  However, when I read your reply，I found that you told reviewers that patients and healthy controls were matched one by one. So Did you use individual matching？ If so，student’s t-test, chi-square test did not good choices. Pair t test and McNemar chi-square test are better ways to analyze the Paired-design 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: SERUM BDNF LEVELS AND THE ANTIDEPRESSANT EFFECTS OF ELECTROCONVULSIVE THERAPY WITH KETAMINE ANAESTHESIA: A PRELIMINARY STUDY
Review round: 3
Reviewer: 1

Output:
Basic reporting: None
Experimental design: None
Validity of the findings: None
Additional comments: None


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: QUANTIFYING UPPER LIMB MOTOR IMPAIRMENT IN PEOPLE WITH PARKINSON’S DISEASE: A PHYSIOLOGICAL PROFILING APPROACH
Review round: 1
Reviewer: 1

Output:
Basic reporting: Background literature should be increased concerning the measures of upper limb impairment in patients with PD. See, as an example, Proud et al., Arch Phys Med Rehabil, 2015.

Similarly, the obtained results should be compared to those already available in literature in the discussion section.
Experimental design: No comment
Validity of the findings: No comment
Additional comments: It is a well-conducted study on a crucial topic. The manuscript is clear and well written. I have just some comments that could improve the manuscript before publication.

1.	My main concern is about the “key tests for assessing upper limb function in people with PD”. Was the weighting system already proposed in the literature? In that case, a reference should be provided. Otherwise, it should be clearly stated that the system was specifically proposed for this study. Moreover, I consider the use of reliability, as measured in a cohort of healthy subjects, incorrect. Reliability should be measured in the investigated cohort since it can be deeply influenced by the specific characteristics of the investigated sample.

2.	The authors should describe in better detail why they decided to use two different samples as “control group”, i.e., the age- and gender-matched group, and the 50+ group, since it can be misleading for a reader.

3.	As reported, patients were performed all the tests with their dominant hand. What about the differences between the most and the less affected side?

4.	In the statistical analysis, considering the number of testing, did you apply a correction method?

5.	It is unclear why the number of repetitions of each item was different. Muscle strength: the best of three trials in each test was used as the participant’s test score (line 90);  Unilateral movement and dexterity: the average of two trials, one in each direction, was used as the participant’s test score (line 102); Position sense: with the participant’s score being the average of five trials. Skin sensation: three trials were performed in each direction, with the average of the average second and third trials calculated as the test score (line 123). Bimanual coordination, Arm stability, and Functional performance: unspecified, I suppose just one repetition. Please justify these differences among test repetitions.

6.	Table: to improve clarity, I suggest merging Table 2 and Table 4.

7.	Figures: to improve clarity, I suggest merging Figure 1 and Figure 3, as well as Figure 2 and Figure 4, reporting in each graph the following categories: “Off state”, “On state”, “Control”, “Control minus Off state”, “Control minus On state”,

8.	Please amend the following line: lines 188 and 200 (Table 4.1 to  Table 1); line 193 (spell out 27); line 194 (time information); line 332 (remove the first “in”).


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: QUANTIFYING UPPER LIMB MOTOR IMPAIRMENT IN PEOPLE WITH PARKINSON’S DISEASE: A PHYSIOLOGICAL PROFILING APPROACH
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1. As this is a validity study, I suggest you refer to the COSMIN checklist for guidance on the requirements for high quality studies of measurement properties. (https://www.cosmin.nl/wp-content/uploads/COSMIN-study-designing-checklist_final.pdf#). You are missing some important details. See below:
Define the type(s) of validity you are testing throughout the manuscript.
Lines 36 -40. Clearly state your hypotheses. What were you expecting?
It is also important to make clear in the aims that you have two separate questions – a comparison with selected age and sex matched controls and comparison with normative values.
Line 62. Give details of whether the PPA has been validated in people with PD
2. Introduction: Although you describe the cardinal impairments in PD, you need to provide a rationale for why this broad impairment-based assessment tool may be appropriate for the assessment of people with PD.
3. The reporting of results is difficult to follow particularly the section at Lines 213 and 226. Perhaps you need another heading at Line 213 as this is a different comparison and different question.
4. Results. In validity testing it is usual to place a greater emphasis on the strength of the correlations and less emphasis on statistical significance. Define and discuss further.
5. Discussion. Include more discussion of the findings in relation to other studies, and the clinical implications of the results. Strengthen your reasons as to why the PPA tests would be a valuable addition to the currently available tests.
6. You have inconsistencies in language throughout the manuscript. Also be careful in your use of terms like ‘function’.
e.g You have described the DASH as a measure of upper limb function in Line 62 and upper limb impairment in Line 202. Maintain accuracy and consistency. (Refer to Drummond, A. S., Sampaio, R. F., Mancini, M. C., Kirkwood, R. N., & Stamm, T. A. (2007). Linking the disabilities of arm, shoulder, and hand to the International Classification of Functioning, Disability, and Health. Journal of Hand Therapy, 20(4), 336-344)
3. Minor points:
P33-35. See comments below about defining ‘on’ and ‘off’.
Line 75. Check your wording. Reliability relates to the repeatability of a test.
Line 88. seated, not sat
Line 97. Use academic language ‘sychronised with’, not ‘synced to’ (informal language)
Line 154. Have you previously defined this abbreviation (IQR)?
Line 193. It is a writing convention to spell out numbers at the start of a sentence.
Experimental design: Line 47-50. Were there exclusion criteria relating to other upper limb neurological or musculoskeletal disorders which could affect test performance? If not, include this in the limitations of the study. Also did you include a cognitive test?
Line 52: Explain why you chose to match with two controls and describe how closely the participants and controls were matched in years.
Line 161-2: You introduce a new question and sample here. Are you making a comparison with published normative values? It isn’t clearly mentioned as one of your aims. Explain why you chose scores for people aged 50+ when the mean age of your participants was 68 years. Performance on many of these tests is influenced by age as shown in reference 19.
Line 167: ‘participants were unable to provide a test score’ – do you mean they were unable to complete these tests? Do you have a supporting reference for why you chose this method to obtain a score.?
Line 175. Explain why you chose not to use available participant scores which were available from the testing.
Line 194. As you mention in the limitations you have two different samples at different points in their PD medication cycle which may perform differently. Please justify why they are not treated as separate groups.
Line 313. I would challenge the notion that the test battery is quick to .
Reference 19 Table 5 indicates that there are statistically significant differences for some PPA tests, with women mostly scoring better. The PD sample has relatively fewer women and this may influence the results. I would suggest separate analysis for men and women for this question.
Line 353.  impairments and functional limitations
Validity of the findings: Line 312. Define the criteria for acceptable validity. Are you sure you mean external validity here? Also Line 354 This is not external validity.
Line 221. Be careful about generalising your findings to the PD population. You can only know the results from this sample.
Line 288. The PPA tests do not document UL function, some tests are moderately associated with a questionnaire which assesses impairments, activity limitations and participation restrictions. Discuss the strength of these correlations. Are they similar to other studies? Why or why not?
P254 also Line 268. Ref 19 describes the reliability of the PPA in healthy adults. Reliability is specific to the individual population tested. Has reliability been evaluated in a PD sample? This is also relevant to Table 5
Line 278. Dyskinesia is also an important impairment. The figure shows that some participants had poorer performance in the on-phase arm stability test. Could this be associated with dyskinesia?
Line 282 Expand on this. Is there evidence from other studies that these deficits are present in people with PD?
Line 289. Discuss whether you would you expect this result given the basis of the HY.
Line 300 Need a bigger sample for this. Check studies of response to dexterity
Line 312. Acceptable external validity What do you mean here. Ext val refers to the
Table 4. While some of the score differences are statistically significant they are very small. Discuss in relation to measurement error.
Additional comments: Thank you for giving me the opportunity to review this manuscript. It is important to have valid measures for measuring the upper limbs in Parkinson’s disease.


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

Input:
Title: QUANTIFYING UPPER LIMB MOTOR IMPAIRMENT IN PEOPLE WITH PARKINSON’S DISEASE: A PHYSIOLOGICAL PROFILING APPROACH
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: I recommend this paper for publication.


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

Input:
Title: WHAT ARE THE LIMITS ON WHALE EAR BONE SIZE? NON-ISOMETRIC SCALING OF THE CETACEAN BULLA
Review round: 1
Reviewer: 1

Output:
Basic reporting: See "comments" for full review.
Experimental design: See "comments" for full review.
Validity of the findings: See "comments" for full review.
Additional comments: This paper compares the relationship between tympanic bullae length and width measurements and bizygomatic width (a measure of skull size that correlates with body length in whales) across a large dataset of whales from “archaeocetes” to modern clades. The methodology seems sound, and the results are expected as per previous studies, but there are some issues with the paper discussed below that, if addressed, will make it acceptable for publication in PeerJ.

The three most important issues with the paper are: 1) the lack of contextualization of the paper (i.e., why was it done in the first place?), 2) lack of complete referencing; and, 3) lack of discussion of development as a possible constraint on bulla size in adulthood.

Expanding on the first point (lack of contextualization): It is never clearly stated in the introduction (nor circled back round to in the discussion) why the project was implemented, and what the implications of the results might be in the broader context of the widely and long-studied evolution of whale ears. A suggestion for improvement in this area might be to reframe the introduction. More clearly state that body size is probably the most important influence on the biology of animals, but almost nothing in organisms scales proportionally/isometrically. Given this, what sorts of results would you have expected to find? If the authors don’t expect body size to correlate with bulla size, why? What exactly is the role of the bulla, and what about whale bullae should be different or unusual compared with other parts of the skull? The size of the inner ear labyrinth (within the petrosal, not studied here) is strongly correlated with body mass (Ekdale 2015 J Anat, Racicot et al. 2016 BJLS), so why would one expect other parts of the ear not to be?

In terms of the second point (citations): The in-text citations are often irrelevant or likely incorrect – several, but not all, examples are listed below in the detailed line-by-line comments. Upon first read one gets the impression that the authors have not read any of the many papers on whale ear morphology that have been published over the years. When one reaches the bibliography, one finds that many of the important papers are listed, with quite a few unusual inclusions and omissions. For the omissions, a non-thorough list of alternative papers is included in the line-by-line comments. Given that I cannot thoroughly correct the paper for citation errors and exclusions, the authors should thoroughly review and correct citations in the paper.

Lastly, in terms of the third point (the lack of discussion surrounding development): The authors hint at the precocial nature of ear bone development in whales (actually this is true for all mammals) with one sentence in the introduction, but it is not discussed again. Based on the results of the paper, developmental constraints on ear bone size seem like a logical explanation for why the trend in baleen whales isn’t the same as the other groups. Similarly, precocial development and impacts of paedomorphism on the evolution of skulls may explain some of the trends seen in the groups of odontocetes that are known for possessing paedomorphic characteristics, e.g., see Galatius and Kinze 2003 Can J Zool, Galatius et al. 2006 Acta Zool, Galatius 2010 BJLS, Galatius et al. 2011 J Morph for discussions and quantitative analyses of phocoenids.

Below please find line-by-line suggestions and corrections:

Abstract:
The authors write “only about 70%” – 70% is a large proportion of the data that can be explained by the proxy for body size. Consider rephrasing.

The final sentence as written is a bit confusing. First, the results, rather than the dataset, would ‘suggest’ “that auditory functional capacity is dependent on congruent osteological morphology rather than allometric scaling”. Second, the quoted line is a bit confusing as worded. Please consider rewriting – I don’t have a suggestion, as I am not clear on what is meant.

Introduction:
It might be worth citing Ketten papers (most are in the bibliography) and Ekdale 2015 J Anat that include discussion of the correlation between ear size and body size.

Line 36: Citations listed here are not papers about hearing in whales.

Line 39: Several additional citations could be included here, but one of particular interest is: Lancaster et al. 2014. Mar Mam Sci

Paragraph starting with Line 43: Quite a few more citations on the evolution of whale hearing and anatomy are needed throughout this paragraph. Further, could it be explained somewhere how the tympanic bulla is specific to whales? It is homologous to the tympanic bulla in other mammals, so if there’s something the authors want to point out as special about it, it would be good to go into some specifics about how it functions compared to other mammals. It would also be interesting if the authors could explain why or how its size might affect auditory capacity.

Line 53: I am not sure if the Yamato & Pyenson paper discusses fossil-related markers of acoustic evolution, and the Geisler et al. 2014 paper does not discuss ear bones in the main text. It might be worth mentioning somewhere here that other researchers have been using the periotic/petrosal, which is a lot more robust than bullae and contains a great deal more anatomical information, including the internal anatomy, to understand the evolution of whale hearing (among other aspects of sensory evolution). A nonexhaustive list of additional papers that should be included here are: Ekdale & Racicot 2015 J Anat, Galatius et al. 2019 BJLS, Mourlam & Orliac 2017 Curr Biol, Park et al. 2016 Biol Lett, Park et al. 2017 Proc Roy Soc B, Park et al. 2019 BMC Evol Biol, Racicot et al. 2016 BJLS, Racicot et al. 2018 J Anat, Racicot et al. 2019 Biol Lett. Although the periotic may be more phylogenetically and anatomically informative regarding hearing, an advantage of the present paper is that the measurements used might be quick and easy to replicate, assuming you have complete skulls with completely preserved bullae.

Methods:
Did the authors need to do any correction for damage to the bullae (bullae are a bit more fragile than petrosals) or skulls, particularly in fossils? Or were all specimens used in completely perfect condition? Is it possible to decide that a fossil is undamaged? Were none of the fossils used distorted during the fossilization process?

How was bulla length and width measured? Were specific anatomical landmarks used? If so, please include this information. Figure 1 is not particularly informative regarding these questions.

Results:
Starting from Line 148, sentence beginning “These non-“: This part of the paragraph should go into the discussion. Log transformation is standard for these types of analyses. The line “increases in magnitude” is unclear in meaning.

Line 154, sentence beginning “However, as body size increases”: This belongs in the discussion.

Line 156 sentence beginning “We conducted…”: Suggest reword to “Our PIC analysis yielded no genus-level...”

Discussion:
This section will need some restructuring to better explain and reframe the results as suggested regarding the introduction. Circle back to the main points of why the project was done, what the main findings were, what do they mean in the broader context of what we already know and what we can use them for moving forward.

Line 169: These citations should be modified. Perhaps the authors need only cite the Society for Marine Mammalogy Committee on Taxonomy list.

Line 170, sentence beginning with “Whale ear bones…”: The paper cited here (Slater et al. 2017) does not mention the words “ear bones” nor any related words, nor does it discuss or address ear or hearing evolution in whales. If the authors mean, “Whale body size”, then rephrase.

Line 175: This first sentence should actually be somewhere in the introduction. It can be restated in the discussion but there is no mention of function in the introduction where we really need an understanding of what the authors are investigating and why.

Line 175, whole paragraph: Here is where the authors should also mention developmental constraints. The three possible factors are all related.

References:
The reference list is quite impressive, although many important papers are not cited in the main text. Also, many can likely be excluded from the list. For example, Racicot & Berta 2013 – while pterygoid sinuses are related to sound reception, I would suggest citing the papers listed above that directly investigate the inner ears of different cetacean groups.

Figures:

Figure 1. The bulla measurements should be shown in the same way as the BZW measurements, i.e., with ticks at the ends of the lines rather than arrows. Otherwise, it is unclear where precisely the measurements were taken.

Figures 2–3. Both figures should include the r-squared and p-values for each regression line somewhere in the blank space of the plots.


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: WHAT ARE THE LIMITS ON WHALE EAR BONE SIZE? NON-ISOMETRIC SCALING OF THE CETACEAN BULLA
Review round: 1
Reviewer: 2

Output:
Basic reporting: What are the limits on whale ear bone size? Non-isometric scaling of the cetacean bulla, by Groves et al.

1. This ms studies the morphometrics of the cetacean tympanic bullae in a large set of different cetacean skulls, both extinct and extant. The tympanic bulla forms part of the cetacean peripheral hearing organ, and its size is of relevance for the auditory characteristics of an animal. This also seems to be the purpose of the authors, to provide data that would increase our understanding of the evolution of cetacean hearing better. The authors mention various issues that might lie behind the scaling results found for the tympanic bulla.

2. The cetacean tympanic bulla is found to be "non-isometric" when related to body size. I wonder why it was expected in the first place that the bulla would be isometric; many structures in different mammals have been found to be allometric, and positively allometric, like also in this ms, for example the middle ear structures that lie within the bulla, i.e., the middle ear ossicles and their parts (see e.g., Nummela et al. 1999, Scaling of the cetacean middle ear. Hear. Res. 133: 71-81)

3. Resolving the dilemma between phylogenetic vs. functional vs. ecological causes/constraints is not an easy task, and that was evidently not the task here either, however, the authors keep on talking about the different constraints without ever really taking the discussion any further.

4. Were you only concerned about the upper size limit for the cetacean bulla, or also about the lower size limit of the bulla? It might be fruitful to ask, how small a cetacean bulla can be, in order to still house the structures needed for its auditory task. In any case, I was expecting some more discussion on the functional aspects of size, at least in odontocetes.

5. Lines 164-165: "Our results demonstrate that all cetacean ear dimensions increase in a positively allometric pattern irrespective of taxonomic identity or phylogenetic history."  Did you measure "all ear dimensions"? I think this statement is somewhat an exaggeration.

6. Lines 172-174: "Such departures from linearity suggest that functional auditory capacity is not based on proportional congruences, but may instead be constrained by functional or biological auditory limits."  So, what you say here is that functional auditory capacity may be constrained by functional auditory limits. Could you rephrase the sentence so it would become more clear what you actually mean with this.

7. Additionally, related to this discussion when including mysticetes, it might be good to point out that the hearing mechanism of mysticetes has not yet been quite resolved, at least one can say that it is not the mechanism which odontocetes use, so drawing direct conclusions from the bulla size to its function is a bit uncautious.

8. Lines 178-180: "The pachyosteosclerotic bulla enhances the reception of sound underwater, and therefore may be bound within a functional size range."  Could you explain what you mean with this?

9. References
You list about 130 references for the ms, of which I could find only 21 among the text. This is certainly unintentional. For the Supplemental references, I didn't start checking. Please check these through and make the corrections needed in both reference lists.

10. Figures
All the figures are clear and illustrative, they show well how the measurements were taken, and what the results are.
Experimental design: Statistics
For each individual, did you use both bullae separately, or did you calculate the mean value for the left and right bulla? Aren't the left and right bulla size dependent on each other, as they are products of one and same individual?
Validity of the findings: This is an important work in that it adds to our knowledge on the morphometrics of the cetacean auditory structures, and given that cetaceans now after their evolution of over 50 million years probably have reached their maximun body size, it is fascinating to ask the question what constrains the size of their hearing organ, and of course their whole body size. The results of this ms are certainly useful for future pondering.
Additional comments: See Basic reporting.


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: WHAT ARE THE LIMITS ON WHALE EAR BONE SIZE? NON-ISOMETRIC SCALING OF THE CETACEAN BULLA
Review round: 2
Reviewer: 1

Output:
Basic reporting: The reporting is improved, a few issues with the citations that should be looked over. There are still some other issues.

1) The abstract still ends with an unclearly worded sentence.

2) Line 48, sentence starting with "The tympanic bulla's...": Technically the bony labyrinth is a space that contains soft tissue, not a bony element – consider rewording.

3) While the intro has more information about the bulla now, and I agree that not every paper "needs to be a magnum opus", it would still be nice to know what the questions of the study are and why the study was embarked upon. There should still be more specific information about the bulla, why it was chosen for the study, and how it helps to answer these questions. Was it simply a matter of, as I suggested before and the authors include in the methods, an easy thing to measure that maybe relates to hearing? If that is the case then maybe move that information to the introduction. Without this fundamental justification, the reader is not going be given much reason to carry on.

4) line 61, citations, see also Gearty et al. 2018 PNAS
Experimental design: Some improvement here, but as noted above the specific questions of the study are not outlined and it is not clear how the research fills an identified knowledge gap. There's perhaps some info in the discussion on this, so maybe some repetition from that area should be included in the introduction so readers know why the study was undertaken and how it can be useful.

It would be good to include in the methods information on whether the measurements were taken on physical specimens - it is unclearly worded currently, but perhaps implied. The methods figure provided may imply to readers that 3D surface models were used for measurements. If it was a combination of both, then this information should also be included somewhere.

I noticed that some of the measurements taken from the literature are from papers that were published before Tsai & Fordyce 2015 (the cited bulla measurement method). How are the authors sure that these earlier measurements were taken in the same way as their measurements? A sentence on justification would be good here.

The authors note in the methods why they elected to use the bulla. Some of this information would be better in the introduction (see above). There is also an implication that CT studies can't be replicated, which is incorrect. Again, if the reason for looking at the bulla is purely because it's easy to measure then include that info in the introduction.

Small note: Wording of first sentence of results needs adjustment.
Validity of the findings: Although the original research question(s) is still not clear, the discussion is still relatively informative and interesting. There are some citation errors, e.g., Galatius et al. 2019 is a paper that looks at the evolution of specialized hearing in whales, it does not talk about the paedomorphic cetacean ear. There are several papers that I suggested previously that do talk about how the organ of hearing in whales develops precocially, as with all mammals. It would be better to include those in this area, and perhaps the Galatius one in the part where other studies on hearing are discussed.
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: WHAT ARE THE LIMITS ON WHALE EAR BONE SIZE? NON-ISOMETRIC SCALING OF THE CETACEAN BULLA
Review round: 2
Reviewer: 2

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: The authors have addressed my previous comments very well, and I have no further comments at this stage. It might be good to check through the references once more, e.g. in the Intro, line 6, you refer to Nummela et al. 2017; do you perhaps mean 2007 instead of 2017?
I trust this work will find its place in the future, perhaps together with some further studies, contributing to our understanding of scaling between ear structures and animal body size, and their constrains.


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: VITAMIN C SUPPLEMENTATION REDUCES EXPRESSION OF CIRCULATING MIR-451A IN SUBJECTS WITH POORLY CONTROLLED TYPE 2 DIABETES MELLITUS AND HIGH OXIDATIVE STRESS
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: - Your introduction needs a reference to the end of the sentence on line 54/55;
- In line 88 of the introduction write what is the acronym PNM;
- Put at the bottom of table 1 what are the acronyms mentioned in the table.


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: VITAMIN C SUPPLEMENTATION REDUCES EXPRESSION OF CIRCULATING MIR-451A IN SUBJECTS WITH POORLY CONTROLLED TYPE 2 DIABETES MELLITUS AND HIGH OXIDATIVE STRESS
Review round: 2
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: SAMPLE SIZE: The research has a very small sample size. What was the reason why the proponents didn’t consider a larger sample size to make generalizations more reliable? The sample size was based on what computation?
SAMPLE SELECTION: The inclusion criteria were mentioned, but what about any exclusion criteria that were considered? What possible biases were committed in the selection of samples? How did the authors address such possible biases? Do the authors see the small sample size as a factor that may have prevented the determination of inter- and intra-individual variability.
LACK OF CONTROL: What research design was employed in the study? Observational, cohort study design, or experimental study design? If the former was used, there shouldn’t be an intervention, while if it was latter, a control would have been necessary.It will be interesting to note how the supplementation would have influenced the miRNAs in a non-diabetic.
METHODOLOGY: There was no discussion about how the following parameters were determined: phagocytosis/ PMN function and oxidative burst, and reduced oxidative stress. Were the parameters measured only during pre- and post- intervention (supplementation with Vitamin C)?
Validity of the findings: The authors were able to establish the novelty of this paper in terms of investigating the influence of Vitamin C supplementation to the expression of miRNAs in T2DM.
However, the authors have conflicting statements in the following lines.
In the lines 291-293, it was mentioned that there's a need to confirm and validate using a control group, however, this is in contrast to the conclusion which said that miR-451a might be used as a biomarker. With a small sample size and lack of control, such conclusion may be inconclusive.
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: VITAMIN C SUPPLEMENTATION REDUCES EXPRESSION OF CIRCULATING MIR-451A IN SUBJECTS WITH POORLY CONTROLLED TYPE 2 DIABETES MELLITUS AND HIGH OXIDATIVE STRESS
Review round: 2
Reviewer: 3

Output:
Basic reporting: Dear authors,
the manuscript is unintelligible in some parts of the text, in particular when a tentative explanation of the experimental design is presented. Besides these aspects, the general organization and structure appear well done.
The background is poor in the context of diabetes and their pathogenesis and complications, especially in the explanation of the relationship between hyperglycemia and  oxidative stress/antioxidant response, given the Vit-C a powerful antioxidant. So, I suggested in the file uploaded to introduce some references peculiar for framing miRs and their roles in metabolic derangement.
Regarding table and figures, they are kind, but lack of a clear explanation in discussion section.
Another important aspect is the validation of the miR-target gene. The authors should perform a luciferase assay in order to demonstrate that a specific 3'UTR of mRNA target gene binds with miR.
Experimental design: The experimental design would be ameliorated in the form of writing.
The diagnosis of diabetes should be described in the text, as well as the drug administered and the duration of the disease.
Validity of the findings: This argument is a vest but with a note of freshly due to miRNA approach. The introduction in this work of a time-point is very interesting, and add much information about the controlling mechanism of Vit-C on mirna expression in diabetics.
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: VITAMIN C SUPPLEMENTATION REDUCES EXPRESSION OF CIRCULATING MIR-451A IN SUBJECTS WITH POORLY CONTROLLED TYPE 2 DIABETES MELLITUS AND HIGH OXIDATIVE STRESS
Review round: 3
Reviewer: 1

Output:
Basic reporting: Please, check English by native speaker
Experimental design: Experimental design should be improved
Validity of the findings: without a validation of a target for mir-451a the manuscript appears only descriptive
Additional comments: Apart from the consideration that the authors did not consider my comment about the need to perform Luciferase assay, some parts of the experimental design are incomplete. For example, statistics to assess the effective correlation between glycemic parameters and mir-451a did not appear in the manuscript. So that, this miRNA not influence glycemic status, therefore diabetes status. However, also a statistical model more structured should ameliorate the analysis to find more robust data to present.
The levels of miR-451a were reduced in Vit-c supplementation, but the correlation shown in table 1 appeared borderline.
I recommend to seriously consider to identify a target to focalize the entire paper.


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

Input:
Title: DNA METHYLOME SIGNATURES AS EPIGENETIC BIOMARKERS OF HEXANAL ASSOCIATED WITH LUNG TOXICITY
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Exposure to hexanal is an interesting issue and the use of epigenetics as a potential marker is intriguing even if the paper is only observational and limited to an in silico analysis of pathways and targets epigenetically altered.
Some minor revisions:
The authors should better explain the reasons for the three dosages: the definition of low, medium, high or even the dosage itself is not clear if not contextualized, if not related to dosages which are representative for humans, for instance. In this light, the authors should explain how, or if, their model fits into potential human exposure.
The groups consist of a few elements. Is the low number of elements counterbalanced by a low variability within every group?
At line 292 the meaning of the sentence is too generic: not always the presence of methylation is strictly related to low expression: the inverse correlation between methylation and expression is not always a rule but a possibility which depends also on the methylation density within the promoter gene.
There is no information about the possibility to restore the physiological epigenetic landscape after suspension of the exposition.
A translational purpose for this kind of study would be very interesting. The epigenome of rats is not the same as humans. The authors could compare, for instance, the presence of CpG islands in the genes which are epigenetically modified in rats by hexanal respect to the same genes and/or pathways in humans.


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: DNA METHYLOME SIGNATURES AS EPIGENETIC BIOMARKERS OF HEXANAL ASSOCIATED WITH LUNG TOXICITY
Review round: 1
Reviewer: 2

Output:
Basic reporting: In the current study, Cho et al has carried out methylome sequencing of hexanal treated rat lungs and made some relevant observations. The authors have demonstrated experience in these models and the studies follow similar pattern from their previous studies where they analyze mRNA levels. Overall, this study is of interest to the readers who study lung toxicity by volatile irritants.

The introduction Line no 40-58 are non-relevant and should be removed.
Grammatical errors and sentences without much information are found; Line 84-87.
Experimental design: The experimental set up is well thought out and the technologies used are recent and gives meaningful data.
Specific comments

In methods, mention reference/s for the protocol for methylated DNA extraction by fragmentation. Gel images for size determination should be shown as supplementary fig.
Line 207; add reference correctly.
Serum biochemical analysis data/ toxicological data is missing from the manuscript. Also, data not shown is not acceptable (lines 239,242). Add these data to supplementary info.
Validity of the findings: Fig 1 and Fig 2B are trivial. The readers would not gain much info from these figs.
There is no validation of the medip seq by bisulfite sequencing or methylation-specific PCR.
There is no validation of the genes correlated in medip vs mRNA array. qPCR should be carried out for at least some  top hit genes.
Discussion Lines 317 to 336 is rather vague and do not convey anything and can be removed. There is no discussion about individual genes in the discussion.
 Is there changes in corresponding levels of proteins after hexanal treatment? Discuss other studies.
Additional comments: The authors could rewrite the results and discussion to make the findings more clear to the readers.  An  illustration  about what  happens after hexanal exposure  at gene level could be depicted based on the GO and other comparative analyses.


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

Input:
Title: DNA METHYLOME SIGNATURES AS EPIGENETIC BIOMARKERS OF HEXANAL ASSOCIATED WITH LUNG TOXICITY
Review round: 1
Reviewer: 3

Output:
Basic reporting: Basic reporting
This paper is well-organized with no significant grammatical issues found. Sufficient background material is provided.

a) Minor Grammar Issues:
1) Line 123 should be changed to "in accordance with relevant guidelines"
2) Spelling error "diffierentiation" on line 309
3) Missing articles and Incorrect article usage throughout the paper. I suggest the authors re-run a basic grammar check.
4) line 373-4 "Using the DAVID functional annotation bioinformatics tool, we performed GO analysis was also  performed"
5) Did the authors mean Benjamini-Hochberg on line 221?

b) Background context

1) A good background material is provided throughout out the introduction and discussion.
2). The authors have done a good job of organizing the paper professionally. I suggest the authors provide Tables in CSV or excel formats in the supplementary material.
3) Is it possible to label some/all of 73 genes in figure-2?
Experimental design: The article Fits well within the Aims and Scope of the journal.
The following improvements should be made to the manuscript :

1) Authors should clarify for which values of r and p, they call something as an anti-correlated gene.

2) On line 352-3 "we identified the differentially methylated genes of hexanal exposure showing a 3.0-fold-change (p < 0.05). "
Did the authors perform multiple test correction? I could find the relevant text of selection criteria in methods
Validity of the findings: The authors have done a good job of providing all underlying data needed to replicate the study. The tables and figures are sufficiently clear to understand the results
Additional comments: I think the authors have done a good job of presenting data and results for their claims. While I appreciate the overall effort, authors should following clarifications to the paper:
1) Criteria for an anti-correlated gene
2) Whether FDR adjustment was done in differentially methylated genes analysis.
I also suggest the following minor edits:
1) Fixing grammatical issues
2) Adding labels to figure-2
3) Clarity on criteria in the methods section


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

Input:
Title: DNA METHYLOME SIGNATURES AS EPIGENETIC BIOMARKERS OF HEXANAL ASSOCIATED WITH LUNG TOXICITY
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have  significantly improved the manuscript.  No further comments.
Experimental design: No comment
Validity of the findings: No comment
Additional comments: No additional comments


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

Input:
Title: HEALTHCARECOVID: A NATIONAL CROSS-SECTIONAL OBSERVATIONAL STUDY IDENTIFYING RISK FACTORS FOR DEVELOPING SUSPECTED OR CONFIRMED COVID-19 IN UK HEALTHCARE WORKERS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Excellent
Experimental design: See below.
Validity of the findings: I think, overall, this is a well conducted study that provides potentially useful observations regarding health care worker risk.

 There are considerable challenges in conducting epidemiological research of this nature when the clinical performance of different PCR and serological diagnostics remain unclear  and their availability limited.

However, the use of the composite outcome concerns me considerably (see below). Until a  sample of individuals who have definitively suffered SARS-CoV-2 infection (PCR/serology) has been captured and the uncertainty within those diagnostic tools known, understanding the risk factors for COVID-19 is challenging.

I think these limitations need further acknowledgement, even if they can't be addressed.
Additional comments: The authors have undertaken a large survey based study of UK healthcare professionals to identify risk factors for potential of confirmed COVID-19 and provide helpful descriptive data from multiple healthcare domains including the number of days isolated, PPE training, provision and use.

The data is well presented and the methodology clear.

My only significant criticism of the study design is on the use of the composite outcome (see below).

Major points

With respect to the authors’ primary outcome, I believe that there are significant limitations with the inclusion of “suspected” COVID-19 cases in the analysis presented.

Using reportedly sensitive serological assays, the seroprevalence in symptomatic UK health care workers is around 30%. (Shields et al - Thorax doi: 10.1136/thoraxjnl-2020-215414), or using a composite PCR + equally sensitive flow cytometric-based serological assay, around 50% (Houlihan et al - Lancet doi.org/10.1016/S0140-6736(20)31484-7).

This suggests that many (perhaps the majority) of individuals did not isolate COVID-19. It is therefore likely that this study has enrolled considerable numbers (maybe more than 50%) of HCW who did not have COVID-19. Equally, the authors cite studies estimating up to 10% of individuals asymptomatically seroconvert - estimates from other studies are greater than this (e.g. Hains et al JAMA. 2020 Jun 16; 323(23): 2424–2425, Shields et al - Thorax doi: 10.1136/thoraxjnl-2020-215414, Eyre et al - eLife 2020;9:e60675)

It is of great importance that the occupational risk factors for COVID-19 in health care workers are better understood so countermeasures can be employed to reduce that risk. Although the authors acknowledge this issue (line 345), there is no consideration or quantification of how much of an impact this has on their analysis; it is likely to be considerable if 50% didn’t have disease in question.

The justification of the approach by comparison to other studies (line 348) is not particularly constructive.

Perhaps a more helpful piece of analysis is to only consider the PCR proven or antibody proven disease (n=459) within a logistic regression models.

2. There are a couple of interesting risk factors that merit further discussion - firstly - the authors show that there are non-hospital risk factors for their composite outcome (e.g. travel to work by public transport). This strongly suggests that not all COVID-19 in HCW is health care related, which is worth discussing further.

3. Related to above, the other significant limitation is that the survey distributed did not document when people had COVID-19. National data suggests that there is a temporal delay in the peak of community COVID and hospital admissions. The survey has collected data on exposure to infectious patients, but this may have occurred after their COVID-19 illness.

4. Current smoking emerges as a protective factor in multiple models - I have not seen this observation documented before and perhaps it could be discussed further.

Minor points

Line 66 - the authors may wish to consider the work by Shah et al - BMJ 2020; 371 in relation to health care worker risk. In my opinion this provides more robust evidence of the burden of COVID-19 in HCW than the Heneghan piece currently cited.

Line 69 - the statement that health care workers are exposed to “higher viral loads” is conjecture. Indeed, Ra et al, Thorax 2020 http://dx.doi.org/10.1136/thoraxjnl-2020-215042 provides evidence of equivalent viral load - determined by PCR CT - in asymptomatic and symptomatic individuals.

Line 84 - the authors should clarify specifically what they mean by “the nature of SARS-CoV-2 transmission”; aerosolised, droplet transmission

 Line 127 - Survey administration: I think the organisations that distributed the survey on behalf of the authors should be directly included in the methods sections, rather than in the appendix. It is also not clear what the reach of the survey was - I am unable to find how many people this survey was directly distributed to (e.g. via mailing lists). If the survey was publicly available, how did the authors confirm the respondents were health care professionals?

Line 317 - “Thus, despite similar susceptibilities to the disease” - not clear what you mean here. Are you proposing similar exposure to the virus, similar chances of being infected by the virus (which is not proven) or similar susceptibilities of infection becoming symptomatic illness?

eTable 9 - sufficient training in PPE has an Asterix but isn’t statistically significant. There are similar occurrences in other tables, which should be reviewed by the authors.


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

Input:
Title: HEALTHCARECOVID: A NATIONAL CROSS-SECTIONAL OBSERVATIONAL STUDY IDENTIFYING RISK FACTORS FOR DEVELOPING SUSPECTED OR CONFIRMED COVID-19 IN UK HEALTHCARE WORKERS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The paper is very clearly written, well motivated, and well executed. The raw data has been made available. Some very minor comments:

1. Do you mean 'another ethnicity' rather than 'another ethnic' on line 137?
2. Lines 151-155 - I think you should reiterate here that there was a free-text gender identity option.
Experimental design: 1. Your finding that smoking reduces the risk of the composite endpoint is suprising, and not in line with other findings e.g:

The effect of smoking on COVID‐19 severity: A systematic review and meta‐analysis,  Reddy et al (2020)

Is there a smoker’s paradox in COVID-19? Shariq Usman et al (2020)

I appreciate the efforts to understand if this is induced by collider bias, but think you could go further. Can you see if the association holds in hospitalised patients? If you still find this result then it warrants more coverage in your discussion, including putting the finding into context with the rest of the literature.

2. I can't find mention of what is adjusted for in the multivariable models, please clarify.

3. There is mention of model validation using the AIC and AUROC, but these are not presented in the results. Either these results should be included, or this claim removed (lines 183-185),
Validity of the findings: 1. It would be good to comment on how the respondent population compares to the UK population of healthcare workers, in order to understand how generalisable your findings may be. This also touches on point 2 of Experimental Design - without knowing what you control for in your multivariable models, it is difficult to assess whether your findings could be skewed by over/under representation of certain groups.

2. Lines 310 - is the difference in BAME hospitalised workers statistically significant?
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: HEALTHCARECOVID: A NATIONAL CROSS-SECTIONAL OBSERVATIONAL STUDY IDENTIFYING RISK FACTORS FOR DEVELOPING SUSPECTED OR CONFIRMED COVID-19 IN UK HEALTHCARE WORKERS
Review round: 2
Reviewer: 1

Output:
Basic reporting: Excellent
Experimental design: Previous concerns addressed
Validity of the findings: Well described, limitations acknowledged and an important contribution.
Additional comments: Thank you for the opportunity to review the submission following revisions.

My concerns have been addressed, in particular with respect to the additional analysis performed on the laboratory confirmed COVID-19 cases. The study is well conducted and the strengths and limitations acknowledged. I congratulate the authors on their tremendous efforts in producing this work which meaningfully contributes to the understanding of healthcare associated risk during a very challenging time for the NHS.


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: HEALTHCARECOVID: A NATIONAL CROSS-SECTIONAL OBSERVATIONAL STUDY IDENTIFYING RISK FACTORS FOR DEVELOPING SUSPECTED OR CONFIRMED COVID-19 IN UK HEALTHCARE WORKERS
Review round: 2
Reviewer: 2

Output:
Basic reporting: I'm satisfied with the authors' response to my comments.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: No additional comments


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

Input:
Title: GINSENOSIDE RB1, SALVIANOLIC ACID B AND THEIR COMBINATION MODULATE GUT MICROBIOTA AND IMPROVE GLUCOLIPID METABOLISM IN HIGH-FAT DIET INDUCED OBESE MICE
Review round: 1
Reviewer: 1

Output:
Basic reporting: L82: actually there are studies on the effects of SalB on the gut microbiome: Wu et al. (2018, Journal of Functional Food) and Li et al. (2020, Food & Function).

L164: with multi-panel figures, it is better to refer to specific panels instead of the whole figure in general following relevant descriptions in the main text.

The first part of the discussion (L234-259) contains largely background information unrelated to findings of the current work and should be removed or simplified and merged with the background section.

It would help if there was a figure showing the whole experimental design (L110).

The existing figures should also be improved as follows: in general, labels of figure panels should be placed on top of each diagram instead of under them; y-axis labels are missing in panels D and F of Fig. 2, keys in these panels are also redundant and should be removed; the five panels in Fig. 3 should be annotated as A-E as in other figures; the text in Fig. 4 and 5 is too small to read, especially that in panel C and E of Fig. 4; the cladograms in Fig. 6 are of poor quality with unreadable text (Consider removing them as the same information is already shown on the bar plots to the left); Fig. S1 is an important figure and should be made a main figure (L216).

The English language of the manuscript needs to be improved. In particular, the usage of “respectively” is incorrect throughout the manuscript (L30, Fig. 2 legend L5, Fig 4 legend L5 and L8).

Numerous scientific terms were not used accurately or precisely throughout the manuscript.
For instance, it should be “taxa” instead of “microbiome” (L273) or “phylotypes” (L287, L324) or “species” (L145 Fig. 6 legend L4), “OTUs” instead of “microbiome” (L203) or “species” (Fig. 4 legend L3-4), ‘indicator taxa’ instead of ‘phenotypes’ (L41) or “phylotypes” (L219) or “marked species” (L220), “distance matrices” instead of “measurement indicators” (L153), “diversity and overall structure, respectively” instead of “richness” (L212), “taxa at different taxonomic levels” instead of “species or microbial communities” (L221), etc. Also, scientific names and genus names should be in italics throughout the manuscript (e.g. L74-75, L224). The revised manuscript should be checked by a microbiologist or scientist with relevant background.

All abbreviations should be defined at their first use in the main text (E.g. L125). Please carefully check for the rest throughout the manuscript.

References are missing for some softwares/methods used, e.g. QIIME (L149), UniFrac (L152) and LEfSe (L156).

Add a sentence somewhere in the manuscript stating that the raw sequence data is available in NCBI Sequence Read Archive (SRA) under the accession PRJNA610166.
Experimental design: Methods of the molecular analysis (L131) are not described in sufficient detail. For instance, which kit was used in DNA extraction (L135)? Which region of the 16S rRNA gene was amplified (L135)? With which primers and what recipes and conditions? How were the PCR products purified (L136)? How was the library quality controlled (L136) and checked (L137)? Was the paired-end sequencing a 2 x 300 bp one (L137)? How were the paired-end reads assembled/merged and quality filtered (L138)? How were the chimeric sequences checked and removed (L139)? Which reference database was used in taxonomic identification (L142)? Which version of QIIME was used (L149)? Was the NMDS analysis conducted using QIIME as well (L155)?

Other method-related queries include: How many mice were there per cage (L91)? Were both tap water and food sterilised (L92)? With standard chow (L112)? Why these concentrations (L117-118)? Information of the kit used for HbA1c detection is lacking in the Reagents and equipment section (L129).

L25: change the section heading to ‘Background’ and add the incentive of the study or research gap
Validity of the findings: L37: according to Fig. 1A, SalB did not decrease the body weight significantly.

L44-45: this conclusion is not supported by the data (Fig. 1A and 2A). Also on L261-262.

L45: tone down the claim, e.g. by using “may be” instead of “is”. Also on L333-334.

Without inclusion of a naive control group for comparison, it is invalid to claim that the model group has “impaired” glucose tolerance (L179-180) or “dyslipidemia” (L188-189), or that a certain treatment could “reverse” an alternation of the microbiome (L285-286).

L182-183: however, there were significant differences in the baseline values between some treatment groups and the control group (Fig. 2E).

L198-201: all sequence datasets need to be rarefied to the same (smallest) number of reads before meaningful comparisons can be made. Otherwise, it is just normal for the Rb1SalB group which has the lowest no. of reads to have the lowest no. of OTUs!

L216: did you test this statistically with e.g. PERMANOVA? It seems that the 2nd panel has good potential to show significant groupings after the test.
Additional comments: L27: mention OGTT as well

L34: provide full names of the abbreviations here

L50: the ’2’ at the end of the sentence should be in superscript

L105: incomplete sentence

L131: “16S rDNA”

L132: ‘administration of drugs’?

L145: ‘bar plots’ instead of ‘accumulative histograms’

L148: “Alpha diversity analysis”

L149: “rarefaction curves” is the preferred term

L160: “Fisher’s LSD test”

L166: ANOVA P > 0.05?

L169: it seems that Rb1 also showed significant difference in Fig. 1A. Mention it here as well

L190: TC as well

L191: add “Fig. 3” here

L192: add “P > 0.05”

L195: “4,002,927”

L198: “OTUs”

L205: remove the extra “b”

L207: were the increase statistically significant?

L208: the decrease was too trivial to mention at all

L210-211: I think this is not these figures meant to show!

L214: support the statement with statistical test. Also, “richness and diversity”

L215: it is unclear which panel of Fig. S1 refers to which matrix

L219: “Rb1SalB”

L220: define LEfSe at first use (L156) instead

L224: “genera” instead of “genus”

L227: “Bacteroides”

L228: “Coriobacteriaceae” and “Deferribacteraceae”

L230: “Helicobacter pullorum and Mucispirillum schaedleri”. Also on L318

L267-268: provide a reference for the statement

L284-285: sentence of unclear meaning

L289: add “between the control and treatment groups”

L293: “Coriobacteriaceae”

L295: what kind of adjustment? increase or decrease?

L296: is the increase a good one or a bad one in this context?

L302: Helicobacter as well

L303: “enriched” instead of “enhanced”. Also on L313

L314: “depleted” instead of “down regulated”

L315: what do you mean by this sentence? The year information is missing here. Also, “H. pylori” instead of “HF”

Fig. 1 legend L4: remove “change”

Fig. 2 legend L5: why only 4 out of 8 mice were selected for this analysis?

Fig. 4 legend L9: does “RA” mean relative abundance?

Fig. 6 legend L1: “Linear”

Table 1: the number of decimal places used should be 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: GINSENOSIDE RB1, SALVIANOLIC ACID B AND THEIR COMBINATION MODULATE GUT MICROBIOTA AND IMPROVE GLUCOLIPID METABOLISM IN HIGH-FAT DIET INDUCED OBESE MICE
Review round: 1
Reviewer: 2

Output:
Basic reporting: The authors of the article titled ‘Ginsenoside Rb1, salvianolic acid B and their combination modulate gut microbiota and improve glucolipid metabolism in high-fat diet induced obese mice’ describes the effect of herbal extracts on metabolism through modulation of gut microbiota in obese mice model. The manuscript is well written in terms of understanding the finding, however it needs significant improvement before acceptance.
ALL the figure quality needs to be significantly improved in order to make the texts inside readable. In figure 1, reduce line thickness and clear indication of the comparing groups for statistical significance is lacking.
I have included the language edits as minor comments:

Line 49: the starting sentence in the 'introduction' needs to be changed or removed because it reads inappropriate.
Line 56: mention some of the ‘short and long term medical consequences’.
Line 59: change ‘emerged’ to ‘increased understanding’ because it was always there, we just identified its role in health and disease
Line 61: cite a reference discussing contribution of microbiota to human biology.
Line 62: change ‘closely related’ to ‘influenced by’
Line 63: change ‘dysbiosis is essential in’ to dysbiosis contributes to’
Line 67: it is confusing to understand here, ‘their’ means whose homeostasis, should be the host but it reads like it is of the microbiota.
Line 71: change ‘proportion’ to relative proportion
Line 72: whose metabolites? The authors can break the sentence here and explain the context of the metabolites from the microbiota or from the host itself.
Line 76: needs a reference to be cited, describing role of ginsenoside and salvianolic acid on various diseases.
Line 130: provide the kit details, e.g. catalogue number, vendor etc.
Line 189: cite reference for the statement that ‘long-term HFD feeding caused dyslipidemia’
Line 198: typo ‘OUTs’ should be OTUs
Line 293: reference for Coriobacteriaceae should be included. The references cited here only talks about Rikenellaceae
Line 325: remove ‘this inspired us that’
Lastly, in the discussion section there is inappropriate use of the word ‘meanwhile’ several times in the discussion. Authors should use their vocabulary to replace this word.
Experimental design: Major improvement in the text includes the following:

Line 51-54: discuss in brief about the hunter-gatherer groups where physical fitness was necessary to gather food and cite appropriate references. Review recent papers of Janelle Ayres.
The authors should either experimentally show that relative abundance of Firmicutes and Bacteriodetes controls obesity OR, discuss extensively several studies that supports this phenomenon as mentioned in section 4.2.
In the results section, a concluding statement should favorably be included.
In the discussion, authors should also include possibility of the direct effect of these products on host cell metabolism apart from changing the microbiota. If the receptors are identified or mention a speculated mode of entry into the cells. Moreover, the last paragraph has a lot of repetition and can be significantly reduced. Names like ‘Dorea’ has been mentioned several times which is unnecessary.
The authors should include a pathway/signaling description about how the glucolipid metabolism changes on administering these extracts to support their argument about reducing obesity.
I have no comments on the conclusion section.
Validity of the findings: The authors should mention the number of times they repeated the experiments to support their observation. Statistical significance should be included with +/- SD for all the experiments, as the statistical significance is described for the sequence analysis.
Additional comments: This study is important in terms of evaluating the potential of herbal compounds for treatment of ever-increasing metabolic disorders around the globe. It also highlights the role of gut microbiota composition and relative abundance in controlling metabolism in mammals. To increase visibility of this study and relate multiple aspects of biology, the manuscript needs to address most of the comments before being considered acceptable for publication. I appreciate the authors’ intention to evaluate therapeutic potential of herbal products for the treatment of metabolic disease e.g. obesity. However, I think addressing the above comments and including them into the text would significantly improve its validation, quality and would be appealing to a greater audience.


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: GINSENOSIDE RB1, SALVIANOLIC ACID B AND THEIR COMBINATION MODULATE GUT MICROBIOTA AND IMPROVE GLUCOLIPID METABOLISM IN HIGH-FAT DIET INDUCED OBESE MICE
Review round: 1
Reviewer: 3

Output:
Basic reporting: The authors show the role of ginsenoside and salvianolic acid in glucolipid metabolism. A big portion of the study is repetition of previous work. The figures are relevant but need some modifications. The experiments are mostly well executed with control; the exceptions are mentioned in general comments.
Experimental design: The study is of importance. A major portion is repetition of previous work. Since, PeerJ accepts replication experiments, this study comes within the scope of PeerJ. The research plan is well defined but further validation experiments need to be done to link the change of gut microbiota to glucolipid metabolism.
Validity of the findings: The study looks statistically sound. Conclusions are well supported by results except the ones that I have mentioned in the general comments.
Additional comments: Overall, the study is interesting. Although, there are certain topics that need attention as followed.

1. The authors wrote in the abstract (line 81-82) that the effect of Rb1 and SalB in regulating gut microbiota is not known but there are studies which looked at it previously. For example, check the review by X. An et al., 2019 in Biomedicine & Pharmacotherapy. I would suggest correcting that statement accordingly and cite proper references.
2. Fig 1, 2, 3 and to some extent Fig 4 is repetition (Ref. S. Lee et al.,Int J. Mol. Sci. 2012 and D. Zhao et al., JTCMS, 2017 etc.) of previous work and there is very less new information.
3. In the first half, for the body weight and blood glucose studies, an essential experiment is missing; the effect of these plant products (Rb1 and SalB) in food intake and activity of mice is not followed. Since weight gain and blood glucose can be affected by change in food habit or physical activity, these aspects cannot be overlooked.
The latter half of the study shows the effect of Rb1 and SalB on gut microbiota alteration and they relate this change to the metabolic effect they saw. Feeding mice with any plant product might change the gut microbiota but linking that change to some metabolic effect need further validation, which is missing in this study! In the current form, the authors only show change in the gut microbiota on feeding Rb1 and SalB and tried to draw a correlation with metabolism, but it is inconclusive and need further experimental results.
4. The writing in the ‘results’ section is weak and needs to be improved.
5. For Fig 1B, the Y-axis label could be replaced with ‘% body weight change’. The current label is confusing to a reader. Also, the ‘*’ used to denote level of significance are randomly placed; it should ideally be placed at the top of the data points.
6. For Fig 2: The Y- axis label for 2D, F is missing.
7. For Fig 4: Figure labels are small, unreadable, and pixelated. I would suggest replacing with better quality picture. Same is true for Fig 6 cladograms.


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: GINSENOSIDE RB1, SALVIANOLIC ACID B AND THEIR COMBINATION MODULATE GUT MICROBIOTA AND IMPROVE GLUCOLIPID METABOLISM IN HIGH-FAT DIET INDUCED OBESE MICE
Review round: 2
Reviewer: 1

Output:
Basic reporting: L98: somehow the given name “Xinyue” is incorrectly used as the surname in the citation of “Wu et al. (2018)” here. A quick look also identified some other similar mistakes, e.g. it should be “Pan et al. 2018” instead of “Yanyun et al. 2018” on L604. Please carefully check all of the citations again.

L185: the correct citation for QIIME should be Caporaso et al. (2010, nature methods)

L381: “H. pylori” in italics

Figure 1: a legend for the newly added panel A is missing

Figure 3: mention the panel numbers in suitable places of the legend

Figure 4: the legend needs to be updated with the newly named panels. Also for Figure 6.

Figure 6: “Linear” is still misspelt in the legend
Experimental design: L161: is it the “TIANamp Stool DNA Kit”? Name it explicitly

L163: provide proper citation(s) for the primers

L167: it is still unclear what purification kit was used along with the beads

L171: was the “2 x 100 bp” or “2 x 125 bp” chemistry used?

L177-178: it is still unclear which “reference database” was used in assigning taxonomy to the reads
Validity of the findings: L47-48: According to results of Figure 1B and 2A, I am still not convinced by the claim that “the combination of ginsenoside Rb1 and salvianolic acid B showed a better effect on decreasing body weight and improving glucose tolerance”. Also for L313-314.

L264-265: given that statistically significant grouping is now detected, this sentence should be removed

L268: is this analysis based on the weighted or unweighted unifrac distance?
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: GINSENOSIDE RB1, SALVIANOLIC ACID B AND THEIR COMBINATION MODULATE GUT MICROBIOTA AND IMPROVE GLUCOLIPID METABOLISM IN HIGH-FAT DIET INDUCED OBESE MICE
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors have addressed all the comments on a point-by-pont manner which has significantly improved the manuscript quality.
Experimental design: Experimental observations are aligned with evaluation and discussion of the finding.
Validity of the findings: The authors must include the limitations of using these drugs, if being readily brought into applications and describe the potential side effects as a possibility.
Additional comments: I believe the minor comments are addressed meticulously by the authors including grammatical errors and typos which I overlooked.


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: GINSENOSIDE RB1, SALVIANOLIC ACID B AND THEIR COMBINATION MODULATE GUT MICROBIOTA AND IMPROVE GLUCOLIPID METABOLISM IN HIGH-FAT DIET INDUCED OBESE MICE
Review round: 2
Reviewer: 3

Output:
Basic reporting: Several improvements are made in the revised manuscript.
Experimental design: Well done.
Validity of the findings: Acceptable
Additional comments: Should go through and correct the typos and other mistakes, such as in Line 297- ‘that have results in…’- should be - have resulted in.


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: GINSENOSIDE RB1, SALVIANOLIC ACID B AND THEIR COMBINATION MODULATE GUT MICROBIOTA AND IMPROVE GLUCOLIPID METABOLISM IN HIGH-FAT DIET INDUCED OBESE MICE
Review round: 3
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Thank you for the revisions.
However, there is one more point to fix:

Figure 6: The panel numbers in the legend do not align with those shown on the figure.


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

Input:
Title: GINSENOSIDE RB1, SALVIANOLIC ACID B AND THEIR COMBINATION MODULATE GUT MICROBIOTA AND IMPROVE GLUCOLIPID METABOLISM IN HIGH-FAT DIET INDUCED OBESE MICE
Review round: 3
Reviewer: 2

Output:
Basic reporting: I reviewed the answer to the comments and they are acceptable.
Experimental design: Experimental design is aligned to the question addressed.
Validity of the findings: Based on evidence provided the authors claim seems to be justified.
Additional comments: The authors have addressed all comments and the manuscript is suitable for acceptance.


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

Input:
Title: LIVER CANCER MORTALITY OVER SIX DECADES IN AN EPIDEMIC AREA: WHAT WE HAVE LEARNED
Review round: 1
Reviewer: 1

Output:
Basic reporting: English language is clear and well-written throughout the manuscript. However, there are some minor points that need to be revised:

Line 57: Add reference
Line 261: Revise ‘these different patterns’
Experimental design: Line 152: Why did you select 35-64 as the threshold?
Line 153: What about people that are elder than 74-year-old?
Validity of the findings: Line 226: Please briefly explain the population structure changes over the decades
Additional comments: This study summarized the changes in liver cancer mortality rates over the decades in Qidong. The authors also highlighted the possibility that the change in aflatoxin intake may contribute to the decrease of liver cancer mortality rate.


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: LIVER CANCER MORTALITY OVER SIX DECADES IN AN EPIDEMIC AREA: WHAT WE HAVE LEARNED
Review round: 1
Reviewer: 2

Output:
Basic reporting: Professional English throughout
Sufficient literature references
There are many mortality terms in the manuscript and they can be defined more clearly
Experimental design: It is a really original primary research. To tell you the truth, I haven't read an article that investigated such long-term cancer mortality in an epidemic area.
However research question in not well defined and meaningful. Because the result are predictable.
Validity of the findings: Conclusions are well stated and linked to original research question.
However, there is no impact and novelty in the manuscript.
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: LIVER CANCER MORTALITY OVER SIX DECADES IN AN EPIDEMIC AREA: WHAT WE HAVE LEARNED
Review round: 1
Reviewer: 3

Output:
Basic reporting: I appreciate authors for putting great deal of effort into this study that details of Liver Cancer Mortality trends. This study details analyzing data coming from two different sources- one being retrospective death survey data and another being cancer registry data.  Study data spans over few decades. Authors have used various metrics such as natural death rate, cancer mortality rate, liver cancer mortality rate in this study to look at trends over many years.

This is well done study with many data points and have certain limitations in terms of presentation.

•	As a reviewer, I suggest re writing and rephrasing entire abstract to make more sense. In current structure it is tough to comprehend. Since this manuscript aimed at reaching to wide range of international audience it would be better to have abstract with better sentences.
•	As a reviewer when I looked at the abstract and at the rest of the manuscript, there is a clear difference of English between both.  I suggest reframing and restructuring sentences in abstract.
•	Abstract is repeated twice this manuscript.

•	In methods of abstract it should be age standardized rate instead of age stand ardized rate

•	In results section of the abstract, the natural death rate percentage symbol should be "%"

•	Authors might want to think if this is appropriate way of representing cancer mortality rate in results section of the abstract. I feel it is not in sync with what is detailed in manuscript. In the body of the manuscript it is represented as 10 to the power of 5 but here in abstract it is represented as 105 at five instances.

•	At line 99- I understand that ICD-10 system is the latest. Appropriate references to showcase ICD-10 implementation date in China would be helpful. In United States ICD 10 system has not been implemented until later of 2010.
Experimental design: •	From line 77-93 in methods I would also suggest how details to include if the data analyzed was deidentified or identified or how the deidentification was done.

•	Authors might also add few details about which software was used for data analysis and running the statistics in methods section.
Validity of the findings: no comments
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: LIVER CANCER MORTALITY OVER SIX DECADES IN AN EPIDEMIC AREA: WHAT WE HAVE LEARNED
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The authors have addressed the questions that I raised in the first edition. The quality of the manuscript has been improved in the current version.


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: LIVER CANCER MORTALITY OVER SIX DECADES IN AN EPIDEMIC AREA: WHAT WE HAVE LEARNED
Review round: 2
Reviewer: 2

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


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

Input:
Title: COVID-19: EXPLORING IMPACTS OF THE PANDEMIC AND LOCKDOWN ON MENTAL HEALTH OF PAKISTANI STUDENTS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Though the article was written clearly and was lucid, there were instances where English could have been improved. However, instances of such mistakes were not too many. Consulting a language editor would really improve the quality of manuscript. Sufficient background information was provided. All the relevant references were mentioned. Article structure was fine. However, tables could have been presented in a more professional manner. They were too clumsy to look at. There is no need to display each and every grid of the table. The article was self-contained with relevant results to hypotheses.
Experimental design: Authors had well defined questions and pursued them rigorously. Methods were defined in sufficient details.
Validity of the findings: . It is well known that women are more prone to anxiety and depression compared to men. Given the poverty prevalent in Pakistan, not everyone can switch to online mode of education. Given the extreme patriarchal nature of Pakistani society, women, including students, do bear most of the brunt of household chores. This may reflect in poor academic performance. Hence, female students being significantly anxious does not surprise me.The findings of the study not very surprising.
Additional comments: It would have been great if authors mentioned that Covid-19 spreads through droplet infection, instead of giving analogy to a virus spreading through internet, and viruses not needing visa ( See lines 78-79).


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: COVID-19: EXPLORING IMPACTS OF THE PANDEMIC AND LOCKDOWN ON MENTAL HEALTH OF PAKISTANI STUDENTS
Review round: 1
Reviewer: 2

Output:
Basic reporting: SAS items tap both affective and somatic symptoms; there is no discussion of such symptoms.
Experimental design: Anxiety diagnosis is a clinical process and structured interviews need to be utilized apart from using scales. Moreover, recent research (Dunstan DA and Scott N, Depress Res Treat 2018:9250972, 2018) has questioned whether the existing cut-off for identifying the presence of a disorder might be lower than ideal. Mathematical formulas, including Youden’s Index and the Receiver Operating Characteristics Curve can be applied to positive diagnoses (presence of a disorder) from the clinical sample and negative diagnoses (absence of a disorder) from the community sample.
Validity of the findings: Findings need to be substantiated with more evidence/data by using multiple scales to analyze the effect confidently.
Additional comments: 1) Section 1.3 is not needed in Introduction section.
2) Scientific deductions can only be made with valid citations. Citations in line 109, 126 do not seem appropriate.
3) Citations for many sections not included. (one example: line 170-173)\
4) Discussion is not cited at many places


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: COVID-19: EXPLORING IMPACTS OF THE PANDEMIC AND LOCKDOWN ON MENTAL HEALTH OF PAKISTANI STUDENTS
Review round: 1
Reviewer: 3

Output:
Basic reporting: Baloch et al. have studied the impact of the pandemic and lockdown amongst the Pakistani students. They have found that anxiety levels among women students are significantly more compared to male students. The manuscript is well written; however, it is still missing a few key points which are enlisted below. Therefore, at the current stage, this manuscript cannot be accepted, however, if authors can convince with a major revision and rewriting this manuscript explaining their results and subsequently providing their rationales, the decision can be reversed.
Experimental design: The authors should include the name of the cities and expand the area to comment on the whole country.

Research questions are very much important for the society and designing government plans to fight with the ongoing pandemic. However, the details of the statistical methods used in the study should be properly mentioned.
Validity of the findings: It lacks complete novelty, as because similar studies with COVID-19 and other pandemics are already available. However, earlier studies used a very limited sample size, so it would be better if the authors can increase the sample size significantly.
Additional comments: Major comments:
# No of samples (494) are severely low to comment on the anxiety level for the entire country. I recommend increasing the sample size significantly.
# Origin of the students and their locality (city wise) should be properly documented.
# It would be better to include students whose family or relatives are directly affected.
# previous studies, Azad et al. (2017) and Mirza & Jenkins (2004), had already shown similar findings with similar demographics, so proper justification and relevance of this study are completely lacking.
# Compare this study with previously reported the national anxiety level before this pandemic.
# Details of Statistical analyses are missing.
# I recommend including a section discussing the drawbacks of this study and future perspectives.

Minor comments:
# Data (the anxiety level among male and female students) should be shown as histograms and significance levels should be labeled and discussed properly in the text.
# Zung self-rating anxiety scale (SAS), the original paper should be properly cited.
# Azad et al. (2017) and Mirza & Jenkins (2004), both papers are missing in the reference section.
# Software used for Statistical analysis should be properly mentioned.


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: COVID-19: EXPLORING IMPACTS OF THE PANDEMIC AND LOCKDOWN ON MENTAL HEALTH OF PAKISTANI STUDENTS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The manuscript reads much better now. The English language editing has made the manuscript easier to read.
Experimental design: After the revision, the experimental design is easy to understand.
Validity of the findings: The major revisions of manuscript have made the findings more valid.
Additional comments: I appreciate the efforts authors have put in to revise the manuscript thoroughly.


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: COVID-19: EXPLORING IMPACTS OF THE PANDEMIC AND LOCKDOWN ON MENTAL HEALTH OF PAKISTANI STUDENTS
Review round: 2
Reviewer: 2

Output:
Basic reporting: Raw data for Zung's classifications not shared.
Experimental design: As mentioned in the drawbacks, this study, being a cross-sectional design, could not provide causal relationships. The resultant participants may not reflect the bigger community of university students in Pakistan. The various data points of field of study and variate analyses does not provide any substantial result. Rigorous investigations does not seem to be performed.
Validity of the findings: No comment
Additional comments: Statements like:  "Although both male and female students encountered stress and anxiety, females suffered higher levels of anxiety. This is likely the result of the patriarchal edifice of Pakistani society, where females are the perpetual sufferers of pre-existing inequalities" is not substantiated by results. The data is not robust


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: COVID-19: EXPLORING IMPACTS OF THE PANDEMIC AND LOCKDOWN ON MENTAL HEALTH OF PAKISTANI STUDENTS
Review round: 2
Reviewer: 3

Output:
Basic reporting: Now, the manuscript looks much better. Authors have successfully addressed all the major concerns.
Experimental design: All the relevant pieces of information regarding the statistics are now included.
Validity of the findings: No comments
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: EFFECTS OF SELF-ISOLATION AND QUARANTINE ON LOOT BOX SPENDING AND EXCESSIVE GAMING—RESULTS OF A NATURAL EXPERIMENT
Review round: 1
Reviewer: 1

Output:
Basic reporting: Tables and graphics are good, but the writing and argumentative flow is a bit clunky in places, e.g., in the Abstract: “many have turned to digital social activities...’ (established?);  the sudden jump to loot boxes seemed odd; the statement that one might find that isolation leads to excessive gaming seems to almost pre-empt the findings. Again, why was the main hypothesis all about the PGSI and loot boxes? The final two sentences in the Abstract also felt a bit unclear. P. 3 ‘In
fact,’  (the flow of writing could be better here).

p. 4 I struggled with the logic that set up the study. If lots of people start increasing their gambling why would you expect relationships to get stronger? Couldn’t you also get floor effects and less variability as the majority of gamers spend more on gaming?

p. 4 ‘suggesting that they were’ (try to avoid this construction)
Experimental design: See below. A few concerns about the design.
Validity of the findings: I had a few concerns about the paper.

First, the definition of isolation and quarantine is ambiguous. This has a specific meaning in disease management contexts. Isolation usually refers to situations where people who are sick are kept apart from other people, whereas quarantine usually refers to people who are kept apart from others because they may have been exposed, i.e., they may eventually show signs of the disease. The definitions here appear to mix up people who: (a) may be fearful and who are isolating because they may feel that they are vulnerable to COVID; (b) have just had a test and are told to stay home or have been put into self-isolation, but not necessarily in quarantine. These people might be getting police checks, but they are not really in quarantine; (c) People in actual quarantine, e.g., hotels, in hospital or other places. Lockdown could be Stage 4 or Stage 2. Big difference. For these reasons, I have concerns about the grouping that forms the basis of the analyses.
I also wondered- if you were in hotel quarantine – would you get to do as much gaming as when you were at home?

Second, the PGSI is described as classifying people as moderate risk based on scores of 5-7, but it’s actually 3-7? Low risk = scores of 1-2. Is the scoring in the study in accordance with the regular scoring?

Third, I found the hypotheses a bit odd. I was surprised that the main hypothesis wasn’t about increases in gaming or the relationship between gaming and loot box use. Problem gambling is most likely related to loot box use because people who enjoy gambling tend to gravitate towards gambling content in games. However, the main thrust of this paper is that the stress of COVID drives people towards greater gaming (as per the WHO recommendation- yes, ironic given the IGD classification, but sadly it doesn’t surprise me after their performance earlier this year!).  In other words, wouldn’t one expect to see people’s gaming and loot box spending increase when they are really locked down and on mandatory stay-at-home orders? But it’s not so obvious that the cross over from gambling to gaming would occur.

Fourth, the study is all about moderation, but they haven’t really tested for moderation. It’s based on correlation difference tests. That’s OK, but it’s unusual. Maybe avoid referring to moderation and have a hypothesis that specifies that the simple first order effects will be different for the two groups.

Fifth, I didn’t feel that the paper provides enough detail about the demographic composition, gaming habits of the two groups; country etc..
Additional comments: Other general comments

p. 4 I struggled with the logic that set up the study. If lots of people start increasing their gambling why would you expect relationships to get stronger? Couldn’t you also get floor effects and less variability as the majority of gamers spend more on gaming?
p. 4 ‘suggesting that they were’ (try to avoid this construction)
p. 10 Another general point that needs to be made is that many people spent more on other things too: alcohol, take-away food. Netflix, etc. During the COVID period. It seems unlikely that a lack of access of gambling would increase loot box expenditure. There are many other reasons and people spend more on a range of things. People could have also increased their online gambling if they wanted to.