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: BALEEN WHALE INHALATION VARIABILITY REVEALED USING ANIMAL-BORNE VIDEO TAGS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Overall this is a well written paper and a creative approach to addressing the methodical challenges of measuring energetic requirements of large whales.  The introduction is concise and clear, citing relevant literature and the authors clearly state the goals of the project.
There are a few sections in the methods and discussion that are unclear and the authors should make some revisions to help clarify their statistical analysis and discussion points. Details are provided below in other sections.
Experimental design: The authors clearly state the research questions and why the study is needed. This is a great use of data collected for other studies. They clearly defined what criteria the video must meet to be included and I appreciate how they performed tests on a subset of the data to defend their methods (only measuring one side, etc). One point I would like to see – is how hard is it to get this data – did they tag 100 whales with video tags and there were only 6 useable datasets? How many hours did they use for each whale (could be added to Table 1). That would allow the reader to assess if this is a method that can be relied on.  They also talk about how need more validation. How will you validate this for large whales?
The statistical analysis section is a little confusing and the authors should provide more information on model validation.
Line 218-236:  I am a little confused by this section.  I thought you normalized the data so you could compare the whales, but here you say you normalize and then test separately?  Then one sentence later you say given the individual variation you “also” plotted separately. You need to make it clearer why you normalized if you still analyzed separately. But when I look at your stats and results, you use GLMM with  whale ID as random effect – so then you did not analyze separately like you stated above?  I suggest revising this so it is a little easier to figure out what you did. Also – there seem to be some difference between species – I am not sure it is appropriate to keep them both in the same model? At a minimum you need to justify why you think this is okay.
Line 238 – 245:  I like that you linked behavior to inhalation duration, but I thought a part of this would be linking things like IA and max nares expansion. If you say those vary, especially for terminal breath, I thought you would want to link this to activity (at the end of the discussion I see you justified why you just did duration – but you might want to suggest that here because I felt like it was missing until the very end). Also if you are only including duration, would it be that different than if you just counted respirations? Could you also look at relationship between # of respirations to dive parameters and then maybe compare? – does duration give you better results than # of respirations?   The variation in inhalation duration is much smaller in the minkes between all breath types so maybe for this species respiration count would be just as useful as total surface inhalation duration.  The comparison might make a stronger argument for why this much more time consuming technique is a better method – at least in some species.
Validity of the findings: In general, I think the authors conclusions were appropriate for their findings. However, I would like them to provide more information about their models. This is important to include so the readers can assess their conclusions. While they provide p-values, they do not provide any information on effect size, confidence intervals or model fit which are all necessary to understand what the significant finding mean.
On line 368-375, when the authors discuss how measures of IA and maximum expansion may improve estimates of body density, drag, etc., I felt like they were overreaching a bit. They don’t make it clear how variation they found will help with these measurements. There are still many issues to overcome such as tag placement. In general, this superficial discussion point came off a bit like a tangent and overreach. I think the discussion would be stronger without it.
Paragraph 386- 412:  The authors comment on longer surface inhalation durations during intermediate dives. The explanation of this pattern is a bit weak. They talk about how may manage on level of a bout, but it is not clear why it would be shorter after long dives (they do a better job of clarifying in the conclusions).  It would also be helpful to see more model results.  I did not find Fig 4 very convincing –there is a lot of variation in the data both within and between individuals so would be good to have information on effect size and confidence intervals.
Line 424-425:  The authors mention species may differ – so is it appropriate to put both in the same models?  That is what your methods and  Table 3 results suggests you did.
Additional comments: I enjoyed reading this manuscript and appreciate the creative approach. Below are a few small suggestions to improve the readability.

Abstract
Line 28: Don’t need word historically.
Line 33: don’t need word additional (this would be the first free-ranging correct?)
Line 38:  Don’t you think you need the word physiological.
Introduction
Line  62: Pederson reference I think is spelled wrong and not in ref list.
Line 69: respiratory physiological does not make sense – do you mean respiratory physiology?

Methods:
109 – 113:  Awkward and a little repetitive first sentence.  Try to rewrite so more direct or break in two sentences - Maybe more like:  This study used data from video-recording motion sensory tags from Customized Animal Tracking Solutions (CATS) that were deployed for other projects (citations).
Line 113-115 – also a bit awkward.  You have a very complex subject – suggest revising so verb is earlier in the sentence.  Maybe something like: Whales were selected if the video tag was placed directly behind and facing the nares, the nares were clearly visible, and there was at least 2 hrs of video where the tag did not shift position.
Line 120:  Don’t think you need word “based”
Results:
Line 248 – 249:  I find this sentence a little vague – what do you mean by timing did not vary? – duration?  Because the next sentence highlights a difference in timing – when they reached max expansion. Be a little clearer about what you mean or state that they did vary slightly?
Discussion
Lines 319-325:  I don’t think the comparison to flow rate is as clear as the authors think – or I am missing it.  Flow rates are high very early, and nares are just starting to expand when flow rate is already high. Maybe explain a bit better so easier for reader to grasp.


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: ESCHERICHIA COLI TRANSCRIPTION FACTORS OF UNKNOWN FUNCTION: SEQUENCE FEATURES AND POSSIBLE EVOLUTIONARY RELATIONSHIPS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Duerte-Velazquez and colleagues have presents analysis of 58 putative transcription factors. These TFs are known to be expressed genes that have been previously experimentally reported to bind to DNA.
From a technical perspective, the manuscript is well understood, and professional English is used throughout. The literature references are mostly adequate. The figures and tables are well-structured and sufficient raw data is provided.
The key points of their analysis are:
1. These genes are located at genome regions containing significant number of mobile genetic elements (MGE) and have promoter characteristics of duplicated or horizontally transferred genes (HTG). They display highly variable sequence/genetic landscape, which is consistent with organism need for highly variable regulation factors.
2. Yet, despite high variation, these genes display adaptation to expression in E.coli indicated mainly by codon adaptation similar to other known E.coli TFs.
3. These putative TFs have divergent protein sequence retaining 3d structure, in respective structural groups, that goes beyond DNA binding structural elements.
4. Some of these putative TFs show a correlation with some stress expression profiles, hinting on single process regulation.
Altogether, Authors speculate that these might be the features of backup TFs, or silent genes with TF evolutionary potential.
Experimental design: Authors have used the wide variety of data sources and methods to describe this putative TFs. The methods are sound and have been (with minor notes) described with sufficient detail. The research was sufficiently defined and is within the aims and scope of the journal.
Validity of the findings: The arguments and analysis results are sound, definitely valuable and intriguing. Although, proving backup TFs function (knock-outs) in the most cases will be experimentally challenging.
Additional comments: The reviewer has only minor questions concerning the manuscript:

1. In the abstract and further in the manuscript, the authors refer to various TFs as bona fide TFs, core TF. Please, clarify which TFs are these? Experimentally confirmed, annotated, etc? Is there a list of this core TFs?
2. Line 67: provided link leads to page showing error: ":ALL-PROTEINS-5 not found". Please provide query to reproduce said 248 TFs list.
3. Line 121: Isihama and colleagues in paper from 201 refers to 285 DNA-binding TFs that are to be analysed via SELEX screening (page 2070, last paragraph). Have this progress been finally reported by Ishihama and has it been included in the analysis?
4. Line 132 vs Figure 2: Authors cite publication on AlphaFold2 while claiming to use AlphoFold. Please clarify which method was used. This typo is also present in later parts of the manuscript (i.e. line 555).
5. Line 141: Authors have used two services to produce structural alignments. Was there a step to agree results of these servers? How were the differences resolved? Also, Figure 2 description name only one structural analysis server.
6. Line 149: GLAM2 was used to discover sequence motifs. In general GLAM2 can generate own alignments, how did this differ to structure-derived ones?
7. Line 159: Again, authors have used to tools to predict promote regions. What was the final result, an interjection of these results, a sum?
8. Line 234: Authors have used different tool to again compare and overlap structures (mTM). Did this results agree with Raptor results.
9. Line 558: When reviewing structural models and model overlaps, reader should have known which regions do not overlap due to structure divergence but are of high confidence, versus parts of models that are of low confidence. So that false hypotheses are not formed on poorly modelled regions. Can model confidence be included into supplementary information (unless column 11 of raw data models in Raw Data\PDB files correspond to confidence scores)?
10. Line 106: Which 14 of 58 analysed TFs are located in prophages. Please provide database link or some sort of evidence.
11. Line 106: These 14 TFs display sequence/structure divergence conserved with other putative TFs. Which TFs are these, are they also in prophages, or the presence in such regions does not correlate with conservation?
12. Line 109: How does the third position variation look like in "bona fide" TFs in comparison to analysed 58 TFs. Can you comment on the possible difference?
13. Line 132-140: I am confused which structures were finally used for further structural overlap and analysis, these made with AlphaFold (if so which of the 5 generated model), these downloaded from EBI or these from PDB?
14. Line 136: What was the process of model confirmation? Did the authors compare their AlphaFold models against experimentally determined structures of the same TFs?
15. Paragraph Results/TF of unknown function sequence analysis: The reviewer would love to see a figure not only with structural overlap of structures in groups but also domain annotation on sequence level (at least similar to motif locations in Figure 4), that would help to identify common domain context within clusters and easily compare then between groups.
16. Paragraph Results/TF of unknown function sequence analysis: have authors tried to compare structures from different sequence-based groups (aside 1-3)? Were there any similarities beyond DNA binding domain?
17. Line 283: Have the motif been mapped to domain context and to the 3d structure? Does this motif correlate with genome location, putative horizontal transfer origin hypothesis?

As a general comment, this reviewer considers Verify3D as an obsolete MQAP tools. There are better tools, however in this case, AlphaFold2 provides some confidence scores itself.


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: ESCHERICHIA COLI TRANSCRIPTION FACTORS OF UNKNOWN FUNCTION: SEQUENCE FEATURES AND POSSIBLE EVOLUTIONARY RELATIONSHIPS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The topic addressed by the authors is very important and I see a lot of potential for the field. However, due to the complexity of the topic I recommend that authors consider rewriting some parts of the main text in order to make the reading more fluid.
English revision is mandatory.
Experimental design: The authors did not develop any new pipeline of platforms. Here the authors are exploring 58 unknown TF based on structural similarities, sequence homologies, and alignments. So, I am not convinced that this work is innovative but the description of available tools in the literature.

I struggle to understand what the goals were.

A Workflow for the work would be helpful.
Validity of the findings: As mentioned before I am not convinced that this work is innovative.
Before analyzing unknown TFs and their TF binding sites, the authors should validate the proposed method, for a group of experimentally validated TF in which the functions are well described.
Additional comments: The paper is too long and the main text is confusing and prolix.
The quality of the image resolution needs to be improved.


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

Input:
Title: ESCHERICHIA COLI TRANSCRIPTION FACTORS OF UNKNOWN FUNCTION: SEQUENCE FEATURES AND POSSIBLE EVOLUTIONARY RELATIONSHIPS
Review round: 2
Reviewer: 1

Output:
Basic reporting: All my concerns and question have been addressed. I find them quite satisfactory.

1. Minor numbering differences between responses and actual manuscript did not hamper the re-review.

2. The case of YgeK gene being in vicinity of prophage-derived genes is not obvious to reviewer. Reviewer has found in quoted source genes from type III secretion system (Pat.Isl.) and some genes/domains from  transposition process. The reviewer lacks the required expertise to validate prophage-derived origin of these genes.

I would recommend PeerJ to accept this article.
Experimental design: The experiment design did not change, and is OK. Even better, as some technical methods were additionally explained.
Validity of the findings: Authors have cross-checked tool results and formed hypotheses that require heinous experimental work to confirm. No additional comment on re-review in this section.
Additional comments: No additional comments


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

Input:
Title: KNOWLEDGE, ATTITUDE AND PRACTICE TOWARDS BIO-MEDICAL WASTE MANAGEMENT AMONG HEALTHCARE WORKERS: A NORTHERN SAUDI STUDY
Review round: 1
Reviewer: 1

Output:
Basic reporting: The researchers have used clear English throughout the manuscript; however, I suggest revising carefully for language and syntax errors. Below are a few suggested edits that could contribute toward more clarity and consistency.

Abstract:
Consider rephrasing the methodology section starting with referring to the current study (tool) then describing sampled population:
e.g: The present study assessed KAP through a self-administered and validated questionnaire. Using a multistage probability sampling method, 384 HCWs from different healthcare facilities participated in this survey. We cross-tabulated the knowledge, attitude, and practice category to find the associated factors with the sociodemographic variables. Spearman's correlation test was performed to find the strength and direction of correlation (rho) between KAP scores.

Minor edits throughout the manuscript:
Inconsistent spacing across the document:
e.g: Line 84: extra space “and / or”
line 107: add "the" before "failure"
Inconsistent hyphenation---- sociodemographic sometimes used and sometimes sociodemographics.
Experimental design: Health care workers are the first in line in managing the BMW. Therefore, assessing BMW's knowledge, attitude, and practices (KAP) is vital to ensure that BMW is properly managed and take the necessary actions if the inadequacies are identified, so the risk to healthcare worker and the general population is at a minimum. Accordingly, the research question of this study is well defined and relevant.
This paper contributes to that knowledge base; however, more detail is needed when describing the methodology of the research and more consideration of how the work links to the literature.

Below are some comments and suggestions for improvement :

Survey questionnaire:
 line 170: The researchers need to verify if the tool adapted from the previously published study is a validated tool, as the referenced study methodology does not indicate that their tool is validated. It was only tested for internal consistency (reliability).
The researchers need to provide more details about the final tool they used and the modifications they applied to the original instrument and explain why they were made. Explain the type of questions used to assess knowledge. It would be helpful to provide the survey as supplemental material.

Line 180: Please, provide literature that supports the information referenced. Please, describe the "other "category of healthcare workers.

Statistical analysis section:
Please, make sure you name the statistical test when testing the association of the subscales' categories and sociodemographic characteristics.
Validity of the findings: I want to thank the researchers for their valuable research and for making the data available, well-labeled, and organized. The statistical analysis looks sound; however, I would recommend further analysis that could put more insight into your study finding and how it can be used later in designing BMW educational programs (please, see additional notes). I would recommend sharing the data in an Excel file for easier accessibility.
Below are some comments on the results and the discussion section:

Results section:
Lines 219- 222 that explain table 4 could use more details on the significant findings.
Tables:
Table 1: add a footnote that describes other healthcare worker category
Table 2: Researchers should provide a  rationale for grouping low and medium score categories in the methods section. This should be noted in the methods section.
A footnote that describes the statistical test used and used statistical significance level (alpha) is missing.
Tables 3, 4, 5 I would recommend including subscale name (knowledge, attitude, or practice) in the table heading for more clarity.
Please. Ensure all tables include the (n) total number of participants; this can be done in the tile or within the table.

Discussion:
The researchers need to revise and rewrite the discussion. If possible,  incorporate the studies' findings, including numbers/statistics, while including your study's findings.
Additional comments: I would recommend a graph that plots KAP subscales’ categories to visualize differences better.
If at all possible, run a regression model to find significant predictors of the different subscales.
I would suggest testing the KAP correlations by work setting and work experiences and seeing if the same patterns will be observed.
I would recommend that the researchers provide some background information on the policies and regulations, for training healthcare workers about BMW  management and what educational programs are in place in the studied healthcare settings, its availability, and if mandatory or not, and so. That would be helpful to interpret the results, especially if a difference is noted with nationality type and duration of work (experience).

Also, it would be useful to provide information on how the study finding was communicated with concerned authorities and the organization involved and subsequent actions that were taken or considered to address the issue if any.

According to the study finding, marital status is significantly associated with the behavior score; could the researchers explain this finding? Is there literature available that supports testing this aspect? Support this finding?

Otherwise, well written.
Well done on your work in this important area. Your research covers some really important aspects of this topic and I hope that it will provoke further research in this area – from your team and from others.


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: KNOWLEDGE, ATTITUDE AND PRACTICE TOWARDS BIO-MEDICAL WASTE MANAGEMENT AMONG HEALTHCARE WORKERS: A NORTHERN SAUDI STUDY
Review round: 1
Reviewer: 2

Output:
Basic reporting: -Clear and unambiguous, professional English used throughout: please for article English drafting
-Literature references, sufficient field background/context provided: yes
-Professional article structure, figures, tables. Raw data shared: Methods section needs more depth
-Self-contained with relevant results to hypotheses: yes
Experimental design: -Original primary research within Aims and Scope of the journal: yes
-Research question well defined, relevant & meaningful. It is stated how research fills an identified knowledge gap: yes
-Rigorous investigation performed to a high technical & ethical standard: needs more explanation.
-Methods described with sufficient detail & information to replicate: inadequate
Validity of the findings: -Impact and novelty not assessed. Meaningful replication encouraged where rationale & benefit to literature is clearly stated: yes
-All underlying data have been provided; they are robust, statistically sound, & controlled: some results need more discussion
-Conclusions are well stated, linked to original research question & limited to supporting results: yes
Additional comments: Title: please remove "multicenter study"


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

Input:
Title: KNOWLEDGE, ATTITUDE AND PRACTICE TOWARDS BIO-MEDICAL WASTE MANAGEMENT AMONG HEALTHCARE WORKERS: A NORTHERN SAUDI STUDY
Review round: 2
Reviewer: 1

Output:
Basic reporting: Dear Editor,

Generally, the authors have responded appropriately to my comments. They addressed all my concerns and suggestions.

Again, this work is valuable and contributes to the literature.

I have just one comment on how the regression analysis results are presented. The authors did not clearly state which categories/groups of the predictor variables (e.g., age ) have significant differences from the reference group (higher odds of the outcome), especially for variables with three categories. I would recommend using more clear language in describing the results of the regression table, clearly stating what groups have higher odds than which group.
Experimental design: No comment
Validity of the findings: No comment
Additional comments: I want to thank the authors for responding to all my comments, considering my suggestions, and providing a clear explanation for my questions. I enjoyed reading this work, and I think it has added value to the literature and research community.


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

Input:
Title: ENGINEERING A TUNABLE BICISTRONIC TETR AUTOREGULATION EXPRESSION SYSTEM IN GLUCONOBACTER OXYDANS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Overall, the manuscript is written in clear, unambiguous, professional English language. The introduction and background show the context. Relevant literature is well referenced. The figures are relevant, well labelled and described.
Experimental design: According to the AAB literature available, the experiments and data presented are original primary research and are within the scope of the journal. The research question is well defined, relevant and meaningful. The experiments have been performed according to technical standards. The methods are described with sufficient detail and information to replicate.
Validity of the findings: The data presented appear to be robust and statistically sound. The conclusions are stated, linked to the author’s original research question and mainly limited to the supporting results. The conclusion on the LuxR-Plux system seems to be too early without further results.
Additional comments: Comments on the manuscript “Engineering a tunable bicistronic TetR expression system in Gluconobacter oxydans“

The availability of genetic systems for tunable expression of target genes in acetic acid bacteria (AAB) is still very limited. One reason for this is the (high) leakiness of classical repressor-based expression systems often seen in AAB. Now, the manuscript presented adds an interesting aspect for engineering tunable expression systems for AAB by pointing to the E.coli study of Hensel (2017), in which stochastic simulation showed that negative autoregulation of bicistronic expression of the repressor gene and a gene of interest can be necessary to reduce noise and produce more gradual response to induction. This has not yet been tested in AAB. Consequently, the authors tested this design principle based on autoregulation in the AAB G. oxydans. For a TetR-Ptet system they found that the bicistronic tetR expression system constructed and tested was inducible up to more than 3000-fold and was highly tunable with almost no background expression when non-induced. This makes bicistronic tetR systems potentially useful for engineering G. oxydans and possibly other AAB.

The following comments are intended to improve the manuscript:

The aspect of “autoregulation“ is suggested to be included already in the title.

Lines 199-201 and Figure 2:
Why is non-induced UidA reporter activity of p452-luxR twice as high compared to the other constructs? Plux-uidA is always the same in all constructs and basal expression should always be the same? Could this be related to luxR expression levels and discussion lines 269-274?
Using the weak P452 for luxR expression is resulting in the highest induced UidA activities, while induced UidA activities were much lower when luxR was expressed with strong P0169 or P264.
How about inclusions bodies resulting in non-functional LuxR protein?
Furthermore, how about codon usage of the luxR sequence in G. oxydans? Are there some/many rare codons worth to be mentioned in text?

Line 210:
5-fold to 12-fold increase is good induction? How do the authors define good induction?

Line 212:
“... low background expression when not induced“? Wasn‘t it a higher non-induced background expression?

Lines 215-216:
"when not induced" or "in the absence of an TetR effector" should be added.

Line 237:
A sigmoidal response cannot be seen in Figure 4.

Lines 256-257:
Here, also name your system and refer to the system from Figures 3+4.

Line 263:
Contradiction: highly induced, yet only 5-fold? What is the author´s definition of highly induced?

Lines 269-270:
What is the expression strength of Plux in G.oxydans in the abscence of the luxR gene?

Lines 272-274:
What are the top 2 candidates in G.oxydans according to the BLAST searches?

Lines 275-277 and Line 318:
First, lower levels of AHL (gradually below 1 µM) should be tested and data should be shown, before this conclusion can be drawn.

Lines 278-279:
Confusing: the same data sometimes are considered as low levels of basal expression and sometimes as high levels of basal expression?

Line 297:
According to the author‘s discussion in the lines below, the point is not "directing the transcription" per se, rather it is "driving/controlling the transcription". I think "Directing" would be misleading here.

Line 299:
Not only sufficient expression, also not too high expression to avoid inclusion bodies / non-functional TetR protein. Also check wording (“the repression the“).

Lines 102-104 and elsewhere:
put always a space between number and unit

Lines 118-151 plasmid construction:
The spacings between the RBS and the start codons should be mentioned in the text.

Figure 3 legend:
- UidA should be uidA and italic
- tetR gene (not TetR) is terminated
- translation of tetR and uidA, not TetR nor UidA

Line 209:	check wording
Line 225:	check wording (“in when“)
Line 231:	check wording (“in in“)
Line 234:	check wording
Line 236:	check commas
Line 253:	check wording
Line 291:	check wording / grammar / typo
Line 315:	check wording

Please explain n.s. in Figure 6.


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: ENGINEERING A TUNABLE BICISTRONIC TETR AUTOREGULATION EXPRESSION SYSTEM IN GLUCONOBACTER OXYDANS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The paper is well written.
Experimental design: This work is well designed.
Validity of the findings: The findings are valuable in this field.
Additional comments: Tunable expression of desired protein is a key technology for protein engineering, metabolic engineering, studying physiology, biochemistry, and so on. Bertucci et al. developed such the expression systems in their manuscript entitled “Engineering a tunable bicistronic TetR expression system in Gluconobacter oxydans”. Honestly, I do not appreciate this paper as for novelty. Also, it is confusing that use of the reporter gene are different in the experiments. Even though the criticism is true, the biological experiments are well and convincing, and the paper is well written. Thus, I recommend this manuscript to accept for publication, if they consider the following suggestions.

Major concern:
1.	Interpretation on the results of LuxR is strange for me. I anticipate a simple possibility that the affinity of LuxR to AHL is higher than they expected. The dissociation constant of LuxR with AHL would be less than 1 micro M. Do the authors have any evidence to exclude this possibility?
2.	Do the authors have any experimental results to connect Figures 4 and 6? Because the reporter genes used in these experiments are different from each other. Therefore, it is difficult to compare two data. Alternatively, please explain a compatibility of two experiments, i.e. Figures 4 and 6.

Minor concern:
1.	What about growth behaviors of the recombinant G. oxydans strains constructed in this study?
2.	L250, What is “medium strength RBS”? I understand ribosome-binding sequence (site), but what is medium strength? One or more citations may helpful, if available.
3.	L253, “The maximal GFP induction ratio” or “The maximum GFP induction ratio”?
4.	L317, “Tn10-based”.
5.	Table 1, Please italicize genotype.
6.	Table 1, I recommend to insert a new line for pBBR1MCS-2.


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: ENGINEERING A TUNABLE BICISTRONIC TETR AUTOREGULATION EXPRESSION SYSTEM IN GLUCONOBACTER OXYDANS
Review round: 2
Reviewer: 1

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


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

Input:
Title: UBIQUITIN-PROTEASOME SYSTEM IN DIABETIC RETINOPATHY
Review round: 1
Reviewer: 1

Output:
Basic reporting: The English language could be improved to ensure maximum clarity. Sufficient background is provided. Figures need to be added for further clarity and ease of understanding.
Experimental design: The survey methodology is nice. PubMed and Medline databases are used in search of the scientific literature. It would be nice to use the Google Scholar search engine too for any missing literature in this scientific area.
Validity of the findings: 1.	In the abstract (line no. 29) of the manuscript, the authors stated that ‘In diabetes, the overall activity of the UPS is inhibited by the oxidative stress’. This is true in major conditions of diabetes, however, in diabetic myopathy, the over activation of UPS leads to muscle wasting. Hence, authors should consider this aspect too.
2.	The E3 ligases are the vital components of UPS that confers specificity to UPS. Hence, authors need to include the latest developments in identifying retinal E3s and their role in diabetes and diabetic retinopathy.
3.	PubMed and Medline databases are used in search of the scientific literature. It would be nice to use the Google Scholar search engine too for any missing literature in this scientific area.
4.	Authors need to follow the general rules of writing gene names (Line no. 179, 185, 186, 188, 189, 190 etc.).
5.	It is very informative that the authors depicted the role of UPS in DR as Figure 1. However, the manuscript has only one illustration and the addition of a few more adds to the ease of understanding.
6.	Conclusion is well written and It could be better if the authors add ‘Future Directions’ of this area
7.	Few typos are identified, for example:
Ubiquitin enzymes instead of ubiquitinating or ubiquitination enzymes in line no. 96.
Ubiquinate instead of ubiquitinate in line no. 135
Ubiquitine instead of ubiquitin in Figure 1 legend
Hiperglycaemia instead of hyperglycaemia in the Figure
8.	The authors abbreviated diabetic retinopathy as DR. However, it is not followed consistently.
9.	The English language could be improved to ensure maximum clarity. Some examples where the language could be improved include lines 36, 53, 141, 307, etc.
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: UBIQUITIN-PROTEASOME SYSTEM IN DIABETIC RETINOPATHY
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: Several sentence need to be reworded to make them valid. The specific examples are given in the additional comments.
Additional comments: The authors present introductory level review of the role of the UPS in DR. Mostly, the review was comprehensible, though the authors are strongly encouraged to revise selected lines (listed below). Additionally, the authors are also strongly encouraged to consult a colleague proficient in English, and familiar with the subject matter, or contact a professional editing service to make the article more readable and clear.
There are few factual errors that the authors should revise prior to acceptance (see below). Generally, the review is adequately written and cited, save the issues listed below. The references consulted were appropriate, indicating an appropriate methodology of survey, but organization of study comparisons can be better executed. In the end the review provides an introductory view of the UPS and its involvement in physiology (e.g. oxidative stress) that occurs in DR, that would be of interest to someone who is unfamiliar with these topics.

Line 29 – 31: The two sentences do not seem to flow together, possibly due to the usage of “however”.
Line 58: “microangiopathy” should be defined
Line 111: not clear what is meant by “density “ of ub substrate? Authors should clarify.
Line 111-112: The fate of ubiquitinated substrates does not only depend on the number of ubiquitin, but also the geometry of ubiquitin chains. (French et. al, 2021, Cell Discovery)
Line 112 should be e.g. instead of i.e. as many different chain lengths can target for degradation as well, even multiple monoubiquitinations.
Line 115 – 117: There is one constitutive proteasome, which is the catalytic core also referred to as the 20S. The others such as the 19S regulator (which associates with the 20S to form the 26S; the 19S is not a proteasome itself) or the 11S regulators (PA28αβ, PA26) are collectively referred to as Proteasome Activators, regulatory complexes of the proteasome. (Stadtmueller & Hill, 2012, Molecular Cell)
Line 118: The appropriate abbreviation for the immunoproteasome is 20Si, as referred to throughout the proteasome field.
Line 122 – 124: The immunoproteasome is assembled using a slightly different group of subunits from the constitutive proteasome, hence the difference in proteolysis activity (ie. the reduction of caspase-like activity), this should be clarified. e.g. “misses caspase activity” is not clear.
Line 125 “compensate the ubiquitin process” is not clear.
134 is wrong, ERAD proteins are NOT degraded in the ER lumen. Instead proteins are extracted from the ER and degraded in the cytosol. This could be an English problem but must be fixed, because it is false as stated.
Line 144, use of the word “derangements” is awkward and non-tradition and unclear what is meant.
Line 170, Protein degradation of what?
Line 203, deletion of pa28 may not result in a decrease in proteasome activity, this should be worded more carefully. E.g. PA28 binding to the 20S could prevent 26S formation decreasing other types of proteasome activity. Also the proteasome “activity” can be assessed via many methods, the type of activity that is being referred to should be made clear.
Line 205 – 208: The 26S proteasome is known to disassemble in the state of hypoxia. It is not that the 26S is ineffective at degrading oxidized proteins, but that the hypoxic environment simply decreases the ratio of 26S to 20S. The references utilized for this point are appropriate though the interpretation is not accurate.
Line 203 & 224: As noted by the authors, several studies contradict as to whether the increase or decrease in proteasome activity is involved in the progression of diabetic retinopathy. The review can be improved by discussing substrate-specificity of the 20S when associated with different regulatory complexes. Perhaps the point is not to simply stimulate or inhibit proteasome activity but to target specific proteasome substrates.


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: UBIQUITIN-PROTEASOME SYSTEM IN DIABETIC RETINOPATHY
Review round: 1
Reviewer: 3

Output:
Basic reporting: In this manuscript, Svikle et al. tried to summarize the biology of UPS associated to DR. The UPS biology has not reviewed recently in the context of DR and it could be interest for the journal’s readers. Unfortunately, the manuscript is not well-written and a high % of citations are other reviews (not original papers). Regarding DR, it not covered if UPS activity is different in the different retinal cellular components or how diabetes leads to UPS dysregulation in retina. The manuscript seems to be an immature draft (see below) and it should be re-written from the scratch. It is absolutely incredible that 7 authors did not detect so many errors in the text. It does not fulfil minimal requirement to be published in the journal.

1.	The abstract of the manuscript is chaotic. it should provide an overview to the readers without making them read the entire paper.
2.	The most important issue is that it is not clear if the overall activity of UPS is increased or decreased in DR. Would enhancers (or inhibitors) of UPS have benefits to combat DR? How is UPS activity by oxidative stress in DR? Once the reader finishes the reading….. there is not a clear message.
3.	Why were the terms “telomeres” and “telomere length” included in the literature search?? There is nothing about telomeres in the manuscript at all.
4.	References 15-24 are missing. No text in the manuscript include those citations. It sounds that a section was completely erased.
5.	The sentence “accumulating evidence demonstrates the potential role of UPS in diabetes” is repeated several times (abstract line 2, line 71, line 146). What does it exactly mean?? Do the authors want to say that UPS dysfunction play a major role in pathogenesis of DR??
6.	Reference 12 do not support that “regulation (of protein quality) is mainly mediated by the UPS in the retina.
7.	The different molecular mechanisms associated with DR are not mentioned (polyol pathway flux, activation of protein kinase C isoforms, increased hexosamine pathway flux, and increased advanced glycation end-product formation (doi: 10.1155/2014/801269). Are those metabolic abnormalities impacting the UPS activity in diabetic retina?? For example, it has been reported that hyperglycemia covalently modifies the 20S proteasome DOI: 10.2337/db08-1565
8.	Most of the information in the text is not about diabetic retinopathy. It should be clear when the information is related to retinal biology.

Minor comments:

1.	Why is term “novel biomarkers” italicized? (line 67)
2.	The first time you use an abbreviation in the text, both the spelled-out version and the short form should be presented. Then, the spelled-out version should not show up anymore (e.g. diabetic retinopathy (line 66, line 248, line 265, etc) . Also, “Unfolded protein response (UPR)” appears twice (line 138 and 153)
3.	It's not clear what the authors wanted to say with the sentence “the destiny of ubiquitinated substrates relies on the amount of ubiquitin added” (Line 111).
4.	Different font size in line 162.
5.	symbols for genes should be italicized whereas symbols for proteins are not italicized and in upper-case.
Experimental design: not applicable
Validity of the findings: not applicable
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: UBIQUITIN-PROTEASOME SYSTEM IN DIABETIC RETINOPATHY
Review round: 2
Reviewer: 1

Output:
Basic reporting: The revised manuscript is much better in the language. Sufficient background with references provided.
Experimental design: The sources are adequately cited and the review is organized logically into subsections.
Validity of the findings: no comment
Additional comments: The authors have responded reasonably well to all of my queries.


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: UBIQUITIN-PROTEASOME SYSTEM IN DIABETIC RETINOPATHY
Review round: 2
Reviewer: 2

Output:
Basic reporting: This reviewer appreciates the effort the authors made to improve the manuscript.  The manuscript was refocused and rewritten and, now, the text covers properly the information available about the role of the UPS system in diabetic retinopathy.
Some comments:
•	In lines 368-371, the authors include the paragraph “Additionally, a variety of novel preparations targeting different components of UPS are under development and testing in neurodegenerative diseases [19]. As neurodegeneration is a crucial mechanism of diabetic eye disease, we are looking forward to preclinical studies in DR.” in the subsection “UPS targeted therapies in diabetic microangiopathy” This paragraph should be included in the “UPS and neurodegeneration in DR” subsection.

•	In lines 104-106, it is not clear what the authors mean by “The fate of ubiquitinated substrate depends on the amount of ubiquitin added A protein with a single ubiquitin molecule attached continues to function in an altered way”. In addition, it should be discussed how UPS degradation is dependent on Lysine chain-specific polyubiquitination (Lys48-linked polyubiquitin chains compared to Lys63 or Lys27, for example).

•	It is not clear what the authors mean to say in line 118, in line 214-215, in line 347, lines 211-215, in line 286.

•	A reference should be included to support the sentence in lines 172-173.

•	The “numerous downstream autophagosome-related proteins” should be listed.

Minor comments:

•	The manuscript contains multiple typographic mistakes and some grammatical errors:

o	Every time the authors use a term composed of two words (e.g. diabetes-induced), the term appears with hyphens first (–diabetes-induced). The first hyphen must not be included (lines 163, 165, 202, 208, 244, 296, 319,
o	A blank space is missing in lines 51, 68, 114, 124, 130, 197, 202, 204 (PA700), 207, 209, 263, 363)
o	A blank space should be removed in lines 87, 271, 316, 333.
o	A period is missing in line 107 and 205.
o	In line 133, “Endoplasmic” should be change to “endoplasmic”. In line 135, “endoplasmic reticulum” should be change to “ER
o	In line 138, UPR should be spelled out. The authors use the term UPR throughout the text but it is not defined.
o	In line 214, “chymotrysin” should be change to “chymotrypsin activity”.
o	In Table1 , “Gene name” should be change to “Gene description” and “Gene Symbol” should be change to “Gene Name”.

o	In line 202, the authors use “-hyperglycema-induced”. The term is not correct
o	Some words are written without a blank space in between: “neurodegenerationetc” (line40), “assessablemarkers” “severityof” (line 407) “glycotoxicity[116]” (line 263), “action[88]” line (256), “Siah[92, 93]” (line 263),  “PSMD9SNP”  (line 181), “immunoproteasomeis” (line 120), “subtypesdiffering” (line 115), “PSMB9Cfol” (line189), etc.
o	The authors must check the names of all genes and molecules: “pten” (line 347) should be written in capital letters, parkina (Parkin?) and Parkin (lines 257 and 270). Also NRF2 and KEAP1.
o	In line 233 the authors use the word “nocive”. This reviewer would suggest the use of “harmful”
o	In line 288, the authors include information about REDD1 (regulated in development and DNA damage responses 1), but they refer to the protein as  “stress response protein regulated in development and DNA damage 1 (REDD1). It must be corrected
o	In line 183 the authors use the term “MODY3”, but it is not defined.
o	The authors must check typographic mistakes as the one that appears in line 53: (- .)
o	In lines 214 and 215, the authors mention chymotrypsin and atorvastatin, respectively, but the function of these substances is not explained.
o	This reviewer suggests to change “In a cultured human retinal pericytes that is exposed to” to “In cultured human retinal pericytes that are exposed to” (line 300)
o	in vivo,  in vitro and et.al should be written in italics and without a hyphen
Experimental design: N/A
Validity of the findings: N/A
Additional comments: N/A


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

Input:
Title: WHEN DOES SELF-REPORT OF PAIN OCCUR?: A STUDY OF OLDER ADULTS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Thank you for the opportunity to review the manuscript entitled “When does self-report of pain occur?: A study of older adults”. The manuscript deals with a topical concern, i.e., self-reporting technologies among older adults. The authors test a simple tangible interface to self-report pain levels among 24 older adults.

Abstract: The abstract is clear and comprehensive. The authors introduce the background, describe the methodology, clarify the aims and scope of the research and highlight the main results and outcomes, as well as some limitations (e.g., limited time frame of the experiment).

Introduction: The section “Introduction” is clear. The authors state the general problem and introduce some relevant aspects of the issue. Further, the authors propose a literature review of the topic, introducing in a clear and comprehensive way the difference between tangible user interfaces and digital technologies.  However, I suggest the authors including more recent studies, since several of them date back to the early 2000s or even before. For instance, is there the chance to quote more recent articles related to: “this process can be affected by memory bias” (line 30) or “pain can vary on a daily basis” (line 33)?
Although significant, I would avoid saying: “The short duration of the study due to nursing home regulations - means that we were not able to study long term adoption or patterns” (lines 80-81). I would include them in the sub-section “Limitations” at the end of the manuscript.
Experimental design: Materials and methods: The section “Materials and methods” describes the experiment and the prototype. The authors include references to justify the adoption of some features (e.g., buttons, sound effects). Is there the chance to define what “LilyPad Arduino” is?

As regards the study context, the authors describe the sampling techniques: first, the snowballing sampling technique; second, the list of people in the nursing home. However, the authors should better define how the sampling technique has been implemented, and how the nursing home has been selected. Further, they refer to “a list of people who met the inclusion criteria”. Could the authors define these “inclusion criteria”?

Further, as concerns the “oral interview”, how was it structured? Which questions have been asked to the participants? Please clarify. If this oral interview is the same described at lines 194-199, please try to make it clearer.

In the field of “description of study participants”, I would move this paragraph to the beginning or the section “Results”.

The description of the experiment is clear.

As regards the description of the “quantitative analysis” (lines 201-205), the authors should refer to successful studies applying the same methodology. It helps in strengthening the research.
Validity of the findings: Results: The research questions presented in the section “Introduction” are not the same presented in the section “Results”. Please, use the same research questions, in order not to create confusion among readers.

Results are presented in a comprehensive and clear manner. Descriptive data are supplemented by figures and tables. Further, I agree with the inclusion of participants quotations.
Discussion: The section “Discussion” and the sub-section “Design Insights” are clear. The authors present novel issues and refer to several previous studies, highlighting the transversality and the utility of the present research. The authors refer to the above-mentioned research questions and follow a coherent approach along the entire manuscript.

Conclusions: Conclusions are adequate and tie with the entire research.
Additional comments: The authors should respect the “guide for authors” as regards in-text references. References should be included within brackets in the manuscript.


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

Input:
Title: WHEN DOES SELF-REPORT OF PAIN OCCUR?: A STUDY OF OLDER ADULTS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Self-reporting of pain with the use of technology is a relevant and promising area of work for improving the quality of life of older adults. This is important work, however, there are few limitations, which I would like to highlight and would want to see how authors are addressing them in their research.
In text referencing style is not appropriate throughout. At least, I have not seen that style of referencing. It is certainly limiting my ability to read.
English should be proofread throughout. There were grammatical errors.
Regarding introduction: Focus on older population for pain self-reporting is relevant and important. Authors have mentioned key challenges for older adults or elderly population in self-reporting pain using digital technology. However, cognitive impairment and dexterity are other two important aspects, which I would suggest authors to consider or include. Also, there is a general critique around single rating scales for capturing complexity in pain. Also, measurement errors and validation issues have been reported previously. Given that, please justify, why it was essential to use single rating scale, and how it is more beneficial than other pain assessment tools?
Experimental design: Experiment
“Each experiment had a duration of between 230 and 245 minutes per participant”, reading it feels like it was done in one session. Please rephrase and clarify. If it is a longitudinal study?
Factors that trigger people’s pain reporting behaviour is very interesting aspect of pain self-reporting, however, that is limited only to emotional state, patient activity and heart rate. Could participant submit any pain diary to mention those aspects, which above stated pain triggers could not capture?  Also, patient activity and heart rate were measured in real-time using wristband, how did you capture emotional state in real-time?
Validity of the findings: Results
Not clear, how data on emotional state was collected and analysed.
Participant could report their pain, whenever they had a pain sensation. It means, no pain intensity score will be assumed as such, without considering a design flaw. For example, a participant may forget, or is in too much pain that limited his ability to report pain. If this is not considered then I would say caution should be considered in interpreting the results.
I can understand that there is a methodological challenge of summarising multiple data points across the population, but I would like to see how this method of data collection can be used for population health research.
Additional comments: My biggest concern is use of single pain rating scale. A clear justification for it might be helpful in addressing my concern


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: WHEN DOES SELF-REPORT OF PAIN OCCUR?: A STUDY OF OLDER ADULTS
Review round: 2
Reviewer: 1

Output:
Basic reporting: Thank you for the opportunity to read and review the revised version of the manuscript entitled "When does self-report of pain occur?: A study of older adults". I have carefully read the responses of the authors to my comments/suggestions, as well as the entire revised manuscript, which seems to me suitable for publication in the Journal. The authors have substantially revised their research and added several novel (and more recent) details, reaching a good quality standards and an adequate scientific soundness. I am convinced of the importance of the research.
Experimental design: N/A
Validity of the findings: N/A
Additional comments: N/A


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

Input:
Title: WHEN DOES SELF-REPORT OF PAIN OCCUR?: A STUDY OF OLDER ADULTS
Review round: 2
Reviewer: 2

Output:
Basic reporting: Authors have considered all my previously made suggestions, and responded each of them appropriately. I have no further comment or concern
Experimental design: Authors have considered all my previously made suggestions, and responded each of them appropriately. I have no further comment or concern
Validity of the findings: Authors have considered all my previously made suggestions, and responded each of them appropriately. I have no further comment or concern
Additional comments: Authors have considered all my previously made suggestions, and responded each of them appropriately. I have no further comment or concern


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

Input:
Title: DIVERSITY AND DISTRIBUTION OF AIR-BREATHING SEA SLUG GENUS PERONIA FLEMING, 1822 (GASTROPODA: ONCHIDIIDAE) IN SOUTHERN JAPANESE WATERS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The language is ok, though reads clunky in some parts.
The flow is ok.
I have checked and confirm that GenBank accession numbers are live.

Can I check if the specimens used in their study have been or will be deposited at any natural history collections? If so, please state, and if not, perhaps include contact information to someone responsible for these specimens? Should another researcher wish to review them at a later date.

Line 180–183: Sentences could do with a bit of re-ordering; the present flow is confusing. Suggest to (1) mention that you did ABGD first, then (2) mention you used the online portal, and the settings used. Finally, (3) mention that you used the same COI dataset. To add on, authors could also clarify why it was written as “COI trees from ML analyses”. As far as I recall, ABGD does not require an input tree (vis-à-vis Poisson Tree Process, see Zhang et al., 2013). I am unable to confirm on my end, as the link provided in the manuscript does not work at time of writing this review.

Fig. 4A: Missing a label for P. setoensis.

Figs. 5–7: Figs. 6 & 7 were mentioned first before Fig. 5 in the manuscript (Line 250 vs. Line 258), so there is some ordering issue here. I think Figs. 5 & 6 can be combined into one figure tile since they both convey the same message on size differences between species (the boxplots are quantitative while the dorsal images are more qualitative), so one can become tile A and the other tile B of the newly combined figure.

Fig 7. Suggest that authors include significant pairwise comparisons mentioned in Lines 260–263 into the respective boxplots for better clarity.
Experimental design: I have one issue regarding their analysis. May I know why the authors chose to perform ABGD analysis with K2P model? But calculate uncorrected p-distances for COI (mentioned Line 223)? Most studies I have seen generally do one or the other. Please also see the critique by Srivathsan & Meier (2012) on the inappropriate use of the K2P model in DNA barcoding literature. I suggest sticking to the use of uncorrected p-distances instead. Link: https://onlinelibrary.wiley.com/doi/full/10.1111/j.1096-0031.2011.00370.x

Lines 178–179: Was there a specific package in R that you used to construct a pairwise distance matrix? Do include it here. Relatedly, authors also mentioned later on having calculated uncorrected p-distances for COI. How was that calculated? I find no mention of that in the Methods, please add it in.
Validity of the findings: Given that Onchidiidae is a poorly-studied family, the authors study of Peronia here will be useful in adding to the extant knowledge of sea slugs.

I have some comments on some things mentioned by the authors:

Line 30: Authors mentioned that they “identified a potential species complex within P. setoensis” in the abstract. This claim needs to be tempered, as they do not provide justifications why this is so. It was mentioned that genetic variation was large (Line 229) but it would be good to give some sort of yardstick for which they base their judgements on. Are they applying the 3% threshold here to delimit species? In addition, a 3.6% variation is within range of known intraspecific distances in P. verruculata, and below interspecific ranges of 4.3–5.4% between P. williani and P. sydneyensis. Both examples they described (Lines 432–435) do not provide compelling evidence to suggest potential cryptic speciation in P. setoensis; they just point to a need for more careful interpretation of mitochondrial genetic variation results in the genus.

Line 488: “…could be a different species”? You cautioned careful interpretation of results earlier for P. setoensis. Please exercise the same caution here! Your sequence GB18 originated from Dayrat et al. (2020). Their study combined morphology and genetic data (both mitochondrial and nuclear) and yet, they did not erect the Mauritius clade as a new species. Furthermore, your node in Fig. 4 is not even well-supported to begin with. I cannot stress this enough—genetically different does not always mean a different species!
Additional comments: Title: Perhaps consider removing “of species” from the title, to become “Diversity and distribution of the air-breathing sea slug genus Peronia Fleming, 1822 in southern Japanese waters”. Authors could also perhaps include some of the higher taxon classifications here to provide more context, e.g., (Gastropoda: Onchidiidae).

Lines 72: Is there a citation for the phrase “fisheries may not completely understand which species they are harvesting”? Or do the authors mean that it is actually researchers who do not have data/information on what species are harvested in the Peronia trade?

Line 95: Change “a DNA barcoding technique” to “DNA barcoding”.

Line 171: repetitions

Line 213: P. verruculata unit #1 (sensu Dayrat et al., 2020).

Line 295: Can emphasise that P. setoensis was previously only known from Wakayama, Honshu, Japan.

Line 318: Change “(e.g., larger individuals being targeted)” to “(i.e., larger sizes preferred)”.

Line 353: 0 to 1.7%. Standardize with Lines 228–229.

Line 301: Authors should avoid speculating which fisheries capture which Peronia spp based species distributions. Being in the same locality as a naturally-occurring Peronia population does not necessarily mean those are the species that will be harvested. Supply-chains? Fisheries could be importing their onchs from elsewhere. Does size really matter? What about taste? I think there is just not enough information on the Peronia trade, or context provided, to make this link.

Line 358: The haplotype pattern is referred to as “star-like”

Line 439 or 469: Authors could mention DNA barcoding of nuclear genes, or genome sub-sampling as potential ways to better understand genetic variation in P. setoensis.

Line 483: See comment on Line 353.


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

Input:
Title: DIVERSITY AND DISTRIBUTION OF AIR-BREATHING SEA SLUG GENUS PERONIA FLEMING, 1822 (GASTROPODA: ONCHIDIIDAE) IN SOUTHERN JAPANESE WATERS
Review round: 1
Reviewer: 2

Output:
Basic reporting: In this manuscript, the authors sampled Peronia species in the Ryukyu Islands to better understand the geographic distributions of these intertidal slugs in the waters of southern Japan, and investigate possible external morphological differences. This study was based on a DNA barcoding approach and using phylogenetic analyses to cluster new DNA sequences with identified DNA sequences from a previous taxonomic study by Dayrat et al. (2020). The authors expanded the known distributions of multiple species, and provided photographs of live specimens in situ for the recently described species P. setoensis. This is a well-written paper that I enjoyed reading, that includes relevant references to recent literature on onchidiid diversity and previous studies on Peronia species in Japan. There were a few references missing from the Literature Cited, the authors should add the details for their references to Katagiri 1998 (Line 64), Johnson & Gosliner (Line 147), and Edgar 2004 (Line 152). Also note that on line 180, the Puillandre reference is listed as published in 2011 while in Literature Cited it is listed as 2012. The manuscript also includes background information on the fishery of Peronia species in Japan and how these organisms are prepared, which I found quite interesting and is relevant to future work to understand how harvesting may impact the relative abundance of these species locally.

In the results section, the ML analysis of the concatenated dataset is not described (lines 207-208). How did the topology of that analysis differ?

All of the figures are relevant, and overall there are high-quality. I especially like Figure 6 with the photos of the 4 species of Peronia that approximate the average size of each species, and the plates with in situ photographs as well as the high quality images of the ventral coloration. I also appreciate that a map was included (Fig. 12B) showing the locations of Peronia fisheries. There are some minor corrections of the figures or captions needed that I have noted here:
In Figure 4A, the haplotypes belonging to P. setoensis are not labeled with the species name.
For Figures 8 & 9, the captions indicate that the ventral view of the animal is labeled A-D, but in the figure they are labeled G-J. Similarly, for Figures 10 & 11, the captions indicate that the ventral view of the animal is labeled A, B, and C but in the figure they are labeled G, H, and I.
In Figure 12A, it is confusing to have the map of the islands repeated 4 times very closely together. I strongly recommend revising Figure 12A to place the island groups for each species distribution map further apart so that the map for each species can be clearly differentiated.

I recommend adding a brief figure caption to the supplementary tables to clarify the content to the readers (particularly to label which Table gives GenBank accession numbers for COI sequences, and which is for the 16S sequences). Also note that some of the supplementary tables are referred to with numbers (S Table 1) and others with S preceding the number (S Tables S2 & S3).
Some of the figure numbers need to be corrected in the text. Throughout the morphology section (including headings on line 331 for P. verruculata, lines 412, 474, 528) the figure numbers are incorrectly reported. For P. verruculata the figures are Fig. 6A and Fig 8, not 7A & Fig. 9. Please review if the figure references in the text are correct.
One last suggestion on the figures, in Figure 1, the most northern locality is labeled Nishidomari, but in the charts in Figure 7 this locality is referred to by the Prefecture name Kochi. It would be easier for the reader if the same name was used consistently for this locality in all figures and the main text (Kochi is used in the text, so I suggest updating the name in Figure 1 to Kochi or “Nishidomari, Kochi”).
Experimental design: The research question in this study is well-defined and provides data to improve the understanding of species distributions of these marine slugs in Japan. It expands on previous research on Peronia species with expanded sampling and DNA barcoding efforts. The methods are described in sufficient detail. The specific primer sequences provided in Table 1 are not strictly necessary as these primers are previously published and widely used and cited, and are cited in the Materials and Methods section. Table 1 could be omitted without negatively impacting the paper.
Validity of the findings: The phylogenetic analyses and haplotype network analyses are appropriate and well-described. There is some inconsistency between the haplotype network illustrated in Figure 4 and the description in the results. Based on Figure 4, it looks like the text in Line 243-244 should read 6 to 14 base pairs changes in COI and 1 to 5 base pair changes in 16S (or if the text is correct, the figure needs to be corrected).

There are some issues in reporting of the morphological data:
Lines 247-250: the smallest spm of P. verruculata is listed as 16/11mm but in the Supp. Table 1, the specimen BAB0 is listed as being 7/4 mm. Also note that the average length given here (38± 12.94 mm) is not the same as listed in the morphology section of P. verruculata (37.56 ± 13.69 mm). I observed the same inconsistency in averages reported in P. setoensis between the Results and Morphology sections, so these numbers need to be checked for all species to ensure the correct values are being reported. Similarly, the largest specimen of P. setoensisis is listed as 28/16 but I only see a specimen listed as 30/16. I request that the authors review their data and correct the parts of the text that are not consistent with their measurements.

The statistical analyses are appropriate.
Additional comments: The paper is well-organized and provides observations from the field on the abundance and ecology of each species. The species P. setoensis had previously only been described from a single site, so the additional data on its distribution in southern Japan is fascinating. At the beginning of the Discussion, the authors mention that this is the only region with four Peronia species found. This is very interesting and makes the Peronia in Japan a very interesting group of onchidiids for further study! I think it would be worth mentioning in the abstract that this is the only island group where four Peronia species have been found to co-occur. Readers will naturally wonder how these Peronia species differ from closely related species in other regions, where only one or two species are found. I suggest to the authors to indicate in the Discussion that there is limited data on the ecology and developmental biology of Peronia species globally, but this study aimed to provide data on the distribution of co-occurring species in Japan with notes on their abundance and habitats. Below I detail a few areas that I think need clarification, a few minor edits and some suggestions. Overall, I think this paper contributes to our knowledge of onchidiid geographic distributions, and I recommend it for publication with minor revisions.

Line 56- I haven’t seen the word tetrapods used before as a substrate, I have only seen this word to refer to a clade of animals. I see later in the manuscript that you are using this term to refer to artificial structures. If this is the correct terminology, I would suggest adding “artificial structures” to this line to clarify its meaning, as you have done on line 390.
Lines 202-203: Add to the end of this sentence that the new DNA sequences generated for this study were deposited in GenBank:  “From a total of 87 specimens, 81 COI and 75 16S sequences were successfully sequenced, and in total 84 specimens were identified to species level. All new COI and 16S sequences generated for this study are deposited in GenBank (Table S1).”
Line 230- change “I examined” to “examined” since this study has multiple co-authors
Line 270, elsewhere: if the scientific name is at the beginning of a sentence, instead of abbreviating the genus name as P. it should be written out as Peronia.
For line 322- This sentence is confusing to me. It says that Takagi et al. 2019 mistakenly identified specimens of P. verruculata as P. setoensis, but P. setoensis had not yet named and formally described in 2019 and was not mentioned in Takagi’s paper. In the Takagi paper, the majority of Peronia specimens sequenced were correctly identified as P. cf. verruculata and the specimens identified as Peronia sp. (Grp 2) belong to P. peronii. So, I don’t see that any specimens of Peronia in the paper were misidentified. If you disagree, be specific about which specimens were misidentified and the evidence that they are misidentified. In the introduction to that paper, Takagi referenced previous work that suggested a small hidden species of Peronia that they referred to as Peronia sp. (= P. setoensis) but it was not sampled in Takagi’s study.
Line 400: Dayrat et al. 2020 indicated that the larvae of onchidiids are released in sea water, but this does not necessarily mean that all Peronia species have pelagic larvae, some species may crawl away. I recommend qualifying this statement at the beginning of the sentence. For example, it could be edited to read: “At least some Peronia species have a pelagic larval form, but the reproductive mode differs between some Peronia species (Ueshima, 2007) and needs to be investigated further.”
In the section on the Molecular phylogeny for P. setoensis it is mentioned that there are genetic differences between specimens collected from different localities. I think this is an interesting finding. It is up to the authors, but they could add a statement in this section to highlight that this species was previously only known from a single locality, and that the expanded sampling in this study is the first to reveal genetic structure within the species. As you noted, the clear correlation of the genetic structure observed with the sampling localities would be expected with direct larval development. It would be interesting if future research investigated differences in larval development and ecology further between these Peronia species.
Line 431: Is it Katagiri & Katagiri, 2007 that indicated developmental differences between the Peronia species or Ueshima, 2007?
Line 566-567: There are many statements throughout the manuscript of things that need to be studied in the future, but this is normal in scientific research. Perhaps his text could be combined with the previous sentence to make it more concise. Something like “These differences in external morphology might be due to animal sizes and their developmental stages, but this would need to be investigated further."
Line 575-576: Interesting observations!


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: PLASTIC DEBRIS IN PLASTIC-MULCHED SOIL—A SCREENING STUDY FROM WESTERN GERMANY
Review round: 1
Reviewer: 1

Output:
Basic reporting: The submitted article is very interesting and suitable for their publication, although I'm not sure about a multidisciplinary journal such as PeerJ. The paper has several new information and potential interest due to the topic's novelty. I like the paper!

However, the paper needs to improve its discussion with other papers because this question is missing in the present version. Ok, the authors compare their data with german fields, but comparison with other countries is scarce. See some examples: https://doi.org/10.5194/soil-6-649-2020 https://doi.org/10.1016/j.scitotenv.2020.141917 or https://doi.org/10.1016/j.scitotenv.2019.03.368 During the reviewing process, i didn't see that this manuscript is a short communication or a technical report. For these reasons, I think that a good discussion is needed. Linked to this question, the authors refer several times to the previous paper, but i think more information can be added here.

L15 (abstract). I suggest to add "Three fields without any visual plastic" We can not talk about without any plastic, considering the multiple plastic sources in the
environment.

L47. "Crop rotations and shorter plastic applications of less than three years appear" Even less time (sometimes 6 monhts). Three years is the expected life-time for plastic greenhouse not for a mulch that usually is applied for each cultivation period (around 3-6 months). I suggest to read (EIP-AGRI Focus Group Reducing the plastic footprint of agriculture)  https://ec.europa.eu/eip/agriculture/sites/default/files/eip-agri_fg_plastic_footprint_minipaper_a_final.pdf

L170. "So far, only few studies have investigated macroplastics on arable land." In Germany? I suggest to see https://doi.org/10.5194/soil-6-649-2020 https://doi.org/10.1016/j.scitotenv.2020.141917 or https://doi.org/10.1016/j.scitotenv.2019.03.368 Besides, i suggest to add a comparison table with studies from all countries, and not only Germany. This section needs to be improved—a lot.
Experimental design: L70-79. Please, add species names as in the previous study https://doi.org/10.5194/soil-8-31-2022

L77-79. "None of the six sites has received any compost or sewage sludge applications in recent years. While this explicitly excluded some other potential sources of plastic inputs, littering and wind drift could not be controlled for." I agree with the authors on this question because plastics have been converted into an environmental contaminants such as Pb. However, can you add a bit more information about the study area? Potential contamination from industrial or urban sources?

L80. Only a minor comment: Can you add the study area's location on a Germany map?

L90-94.
- Why only these soil properties and not also pH or other properties? In the previous study, more properties were measured https://doi.org/10.5194/soil-8-31-2022
- "The soil texture on-site was determined in accordance with ASTM D422-63 (2007)." Hydrometer?

L96-100.
- Some researchers are indicating that a sieve tower should be used for soil sieving and plastic separation (5 mm, 2 mm and 1 mm). Why were separated directly by 2mm and not previously by 5 mm?

Why NaCl solution and not other solutions (H2O, CaCl2, ZnCl2, olive oil, etc)? NaCl could be good for LDPE plastics, but in an agricultural field, you could find other plastic items (PVC, PP, PET, etc) that could don't have a result with NaCl. Besides, other authors suggest the use of sequential extractions with different solutions at a different density ranges (e,g, H2O+NaCl, or NaCl+ZnCl2) (e.g. https://doi.org/10.1016/j.scitotenv.2017.10.213 or https://doi.org/10.1016/j.scitotenv.2020.141917) I agree with the authors on the use of NaCl (cheap, environmentally friendly...), but this question should be improved.

L101-112. Usually, organic matter digestion is an issue in agricultural soils, and for these reasons is, used Fenton or H202 extraction to remove organic matter https://doi.org/10.1021/acs.est.8b01517. However, why this approach was not carried out for microplastics? The sodium hexametaphosphate solution can be used as a substitute for Fenton/H2O2 reagent? I'm interested too because I have similar samples.

L107-112. The solutions and reagents were used for which interest? Sample purification? Py-GC/MS analysis? Please, add more information. Although this information could be previously explained (https://doi.org/10.5194/soil-8-31-2022), i suggest improving this information also here. All readers want to read the information in the paper and not read the previous paper ;)

L113. Please, add information about quality controls and measures to avoid plastic contamination in the lab. Steel or glass equipment? A positive control was carried out? This information was indicated in the previous work but not here https://doi.org/10.5194/soil-8-31-2022

L136. No information about statistical analysis.
Validity of the findings: Please, try to compare your data with previous data from other countries (not only Germany).
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: PLASTIC DEBRIS IN PLASTIC-MULCHED SOIL—A SCREENING STUDY FROM WESTERN GERMANY
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript reports a well-defined objective, that is to determine the presence of plastic micro-, meso-, and macroparticles in plastic-mulched soils from agricultural lands. A major shortcoming in the reporting of data is the definitions used for these three types of particles. Throughout the literature there are various ranges used to define macro-, meso-, micro-, and nanoplastics based on the physical dimensions of the particles, which creates a major barrier for comparing results from different studies. A publication by Hartmann et al. in 2019 (Are We Speaking the Same Language? Recommendations for a Definition and Categorization Framework for Plastic Debris https://doi.org/10.1021/acs.est.8b05297) had pointed out the need to use a common terminology in order to address this issue. I believe it is imperative to start using a common framework and suggest that the authors re-group their results using the limits suggested in this publication, i.e. microplastics: 1 - 1,000 µm, mesoplastics: 1 - 10 mm, and macroplastics: larger than 1 cm.
In addition, the concentration of macroplastics is reported as number of particles per area of field, mesoplastics as number of particles per mass of soil, and microplastics as mass of particles per mass of soil. Although it is perfectly understandable that the choice of units derives from the quantification methods used, I suggest that the authors go one step further and make appropriate assumptions in order to provide a qualitative comparison between the three types of particles, using the same units; preferably as number of particles per volume of soil. If the authors feel that other units are more appropriate, please feel free to use them.
Experimental design: The experimental design is very well planned and executed. The choice of reference fields provides the means to take into account the effect of factors other than mulching on the presence of plastics in soil. I would like to see a discussion about the possibilities of sample contamination during handling in order to further strengthen the quality of the experimental work presented.
Line 82: please elaborate on the four-eye principle, either in the main text or as supporting information.
Line 102: please report the concentration and pH of the sodium hexametaphosphate solution used.
Validity of the findings: no comment
Additional comments: This is an excellent field study on a subject with scientific and socioeconomic interest, which I recommend to be published once the issues mentioned above are addressed.


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

Input:
Title: PLASTIC DEBRIS IN PLASTIC-MULCHED SOIL—A SCREENING STUDY FROM WESTERN GERMANY
Review round: 1
Reviewer: 3

Output:
Basic reporting: I would like to thank the authors for developing comprehensive but not easy to apply (because of the necessity of a sophisticated instrument) methods for microplastic in soil matrices. The chemical used in the analysis is not also environmentally friendly.
The methodology was already applied in another previously published study by the same authors. However, I have a few concerns about the density separation and the chemicals that they used.

- NaCl based density separation is not suitable for PET or other high-density plastics. They should mention this limitation somewhere in the discussion section.
- Xylene has melting effects on especially polystyrene plastics. How did they tackle this issue? This part needs clarification. Also similar concerns for 1,2,4-trichlorobenzene.
- Please be consistence about units. fragments or particles or pieces or MPs.
- Where do plastic size categories come from? Please be consistent with the literature about size classification.
- As they mentioned that only PE and PP type mulches are used for mulching and then why did they consider PS? This part needs to be clarified.
- The term soil-associated debris is not actually the correct term for soil plastic pollution. If they used this term then we need to talk about non-soil-associated debris as well? Since the authors work on soil plastic pollution then it is not necessary to mention soil-associated debris in the text. Please fix this issue.
- How did they measure the contamination issue? Any blank?
- The plastic analysis part of the paper is not reader-friendly. Please provide a flow chart for explaining each step.
Experimental design: A couple of issues need to be clarified.

1) The corrosive effects issues of chemicals used for py-GC/MS have to be mentioned.
2) The limitation of the NaCl separation method needs to be mentioned?
3) The blank procedure is missing.
4) What about contamination control?
Validity of the findings: Already mentioned in previous section.
Additional comments: No additional comments


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

Input:
Title: PLASTIC DEBRIS IN PLASTIC-MULCHED SOIL—A SCREENING STUDY FROM WESTERN GERMANY
Review round: 2
Reviewer: 1

Output:
Basic reporting: All comments have been addressed. The manuscript quaility has been improved since last submission. New figures and new text in the discussion section improved the manuscript. It can be accepted.
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: PLASTIC DEBRIS IN PLASTIC-MULCHED SOIL—A SCREENING STUDY FROM WESTERN GERMANY
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors have adequately addressed all comments, except the reporting of the sodium hexametaphosphate solution pH. Even though the pH was not adjusted, the value needs to be reported, because it indicates the mechanism of stabilization of the aqueous suspension, i.e. highly negatively charged particle surfaces inducing electrostatic stabilization. Please report the pH value. No further action is required.
Experimental design: N/A
Validity of the findings: N/A
Additional comments: N/A


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

Input:
Title: CORAL MICRO-FRAGMENTATION ASSAYS FOR OPTIMIZING ACTIVE REEF RESTORATION EFFORTS
Review round: 1
Reviewer: 1

Output:
Basic reporting: This article is very good and this type of information is needed in the coral restoration realm. Reporting on the differences in survival and growth for two types of nurseries and for two species is commendable and will be of great interest to the rest of the restoration world. Literature could be more up to date in this industry as items are changing rabidly. There is great backround literature up thru 2018, but there is more relevant literature 2020-2022 available on this subject, including ones from some of the authors.
Experimental design: Experimental design is good, however 3 species and 3 types of substrates would have been better and a longer experimental period for monitoring would have been nice. All in all a good study for getting some results to present
Validity of the findings: All finding are valid, with the exception of one..
 In
423 contrast to the ex-situ outplanting site, there were no significant effects of nursery time, size
424 class, or the interaction of these effects for fragment survivorship at the in-situ outplanting site
425 for either coral species

How does that statement compare to this:  There was significant variation among the species across sites for both
480 survivorship and growth. M. capitata had the lowest survivorship (28%) at site 6 and the highest
481 survivorship (72%) at sites 2 and 10. P. compressa had only 42% survivorship at sites 5 and 8 to
482 a maximum of 72% at site 7 (Figure 4). There was approximately twice as much variation in
483 survivorship across individual colonies in M. capitata compared to P. compressa (36.4% vs
484 12.4%, respectively).
Additional comments: I would re work this statement.
Thus, for a project in which the objective is to outplant in the
650 least expensive and most cost-effective manner with the greatest coral survival outcome, for
651 these species at these sites we would recommend direct outplanting of medium-sized fragments
652 (~3cm2
) without any nursery care. In contrast, if the objective were to maximize growth for a
653 high value species (such as rare or ESA-listed corals) in which starting material was limited or
654 outplanting of large sexually reproducing colonies was the objective, then targeting ~1cm2
655 fragments raised in the ex-situ nursery would best achieve this goal

Line 650 makes it sound like the line 651 is the recommendation for least expensive, cost effective, survival, etc.
Line 653-655 makes i sound like this goal is only reached for the exception of lines 653/4

the option of all nurseries outplanting the  optimal size (medium, etc) should all look at starting with 1cm2 or less as a suggestion, not as a limited exception.

Also recommend these recent publications;
Land and Field Nurseries: D.Vaughan, 2021, In Active Coral Restoration, JJRoss Pub
Coral Fusion: Harnessing Coral Clonality for Reef Restoration: Forsman, Page and Vaughan 2021 in
Active Coral Restoration, JJRoss pub
Growth and Cyclin-E Expression in the Stony Coral Species Orbicella faveolata Post- Microfragmentation, Deanna Sopher, et.al 2022 Biological Bulletin
Vaughan, D., Tiecher, S., Halpern. G. and Oliver, J.  2019. Building more resilient coral reefs through new marine technologies, Science, and Models. Marine Technology Society Journal Vol 53:5


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: CORAL MICRO-FRAGMENTATION ASSAYS FOR OPTIMIZING ACTIVE REEF RESTORATION EFFORTS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Figure 6.

1. Using black dots and lines on a dark background makes this figure nearly impossible to see. Either lighten the background color or change the black dots and lines to white or yellow.

2. Since 62.5% of the population that has color blindness has a form of red-green colorblindness, I would suggest replacing the use of green blocks in the bottom graph of figure 6 with another color such as yellow.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: 1. I would like to see data on water quality and flow characteristics of the two out planting sites so that a data driven comparison of the two locales could be made. There are references given that mention that that there are differences but there is not data presented to show how the two sites differ. The difference in growth form for instance is related to water flow and light penetration since the HIMB site is more turbid and lagoon like compare to the more wave exposed site off of Sand Island but there is no data provided to backup this assertion.


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: ACTIVE INGREDIENTS AND MOLECULAR TARGETS OF TARAXACUM MONGOLICUM AGAINST HEPATOCELLULAR CARCINOMA: NETWORK PHARMACOLOGY, MOLECULAR DOCKING, AND MOLECULAR DYNAMICS SIMULATION ANALYSIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Few figures need to be improved. Such as Figure 2 and 4.
1.	If possible, the figure 4 could be edited for better view with clear content of labels.
Experimental design: 2.	In line no. 133, the authors have mentioned “A binding free energy less than zero indicates that the ligand binds to the receptor spontaneously”. Do the ligands bind repeatedly with the receptor or any spontaneous process of the thermodynamics occurs? Revise the statement accordingly. Please refer the below article for detailed information
        a.	Insights into Protein-Ligand Interactions: Mechanisms, Models, and Methods. International journal of molecular sciences, 17(2), 144. (2016) https://doi.org/10.3390/ijms17020144
Validity of the findings: 4.	The authors have selected only five best complex for molecular dynamics studies based on their binding energies. If possible display the remaining other targets with ligands not included in further analysis with binding energies in supplementary. It could be helpful for the follow-up studies
Additional comments: 3.	In line 174, “After the duplicates were removed, one thousand two hundred and twenty-three targets were obtained”, the authors could mention the same in numbers/integers and maintain uniformity.


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: ACTIVE INGREDIENTS AND MOLECULAR TARGETS OF TARAXACUM MONGOLICUM AGAINST HEPATOCELLULAR CARCINOMA: NETWORK PHARMACOLOGY, MOLECULAR DOCKING, AND MOLECULAR DYNAMICS SIMULATION ANALYSIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: .
Experimental design: .
Validity of the findings: .
Additional comments: The manuscript is novel, well written and the results are interesting, however, the major point that I have a concern regard are;
1- Experimental in vitro enzyme assay for the best selected target such as Hsp90 , PI3K , ....
one enzyme assay for one or two representative component of the plant will validate the in silico results.
2- Cell line inhibition measurement of the most representative component of the plant.
3- correlation of enzyme assay inhibition with cell line inhibition for both pure compounds and herbal extract will validate the finding more.
4- it is not clear if the anti-tumor effect of the herb extract is due to a principal compound or due to cumulative effect of the herb components, so I think it is required from the authors to clarify this point.
5- Statistical validation of the method used, use a reference or standard to compare with is essential.
6- I think addition of the scheme or flowchart for the sequence of the work will help understanding the logic of the manuscript, specially the idea seems novel.
7-Validation of the docking process by redocking of cocrystallized ligand and find RMSD values.


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: ACTIVE INGREDIENTS AND MOLECULAR TARGETS OF TARAXACUM MONGOLICUM AGAINST HEPATOCELLULAR CARCINOMA: NETWORK PHARMACOLOGY, MOLECULAR DOCKING, AND MOLECULAR DYNAMICS SIMULATION ANALYSIS
Review round: 1
Reviewer: 3

Output:
Basic reporting: This paper addresses an interesting topic: to shed some light on the mechanisms of action of the herb  Taraxacum mongolicum.  The authors use the principal databases and software for data analysis and predictions to their scope and they find two possible active ingredients (Austricin and Quercetin) that probably interact with the protein encoded by the gene HSP90AA1 (Heat shock protein HSP 90-alpha).
The paper is well organized, includes sufficient introduction and background and the methods are described precisely.
Experimental design: The research question is well-defined and meaningful.
The investigation is all in silico, but it is well performed.
The methods are described precisely.
Validity of the findings: All the data have been provided.
The results are quite well presented, however they could be better described and discussed. In particular, it should be shown or described where the molecules interact with HSP60 with respect i.e. to ATP binding site in order also to speculate on possible consequences of the binding. Moreover, at least in the discussion section, the gene symbols should not be used to identify the proteins. Since there are no lab experiments confirming the in silico results, It is also necessary to maintain doubt sentences.
The figures need captions and some of them do not appear of sufficient resolution.
Additional comments: The English language should be improved. Some examples where the language could be improved are included in the PDF revised file.
I have some minor suggestions to make, therefore, please, check the PDF file.


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

Input:
Title: ACTIVE INGREDIENTS AND MOLECULAR TARGETS OF TARAXACUM MONGOLICUM AGAINST HEPATOCELLULAR CARCINOMA: NETWORK PHARMACOLOGY, MOLECULAR DOCKING, AND MOLECULAR DYNAMICS SIMULATION ANALYSIS
Review round: 2
Reviewer: 1

Output:
Basic reporting: perfect
Experimental design: excellent
Validity of the findings: almost valid
Additional comments: no further comments


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

Input:
Title: ACTIVE INGREDIENTS AND MOLECULAR TARGETS OF TARAXACUM MONGOLICUM AGAINST HEPATOCELLULAR CARCINOMA: NETWORK PHARMACOLOGY, MOLECULAR DOCKING, AND MOLECULAR DYNAMICS SIMULATION ANALYSIS
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors have carefully revised the original manuscript. Moreover, they have performed lab experiments in order to support their findings. I have no other comments.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: No additional comments