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: AUTONOMOUS SENSORY MERIDIAN RESPONSE SELF-REPORTERS SHOWED HIGHER SCORES FOR COGNITIVE REAPPRAISAL AS AN EMOTION REGULATION STRATEGY
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1.	Include citation for first sentence.
2.	Missing full stop at end of last sentence of first paragraph of introduction.
3.	Include citation for the creation of the term “ASMR”. I’ve included one that supports:

Morris, M. (2018). A simply marvellous reaction: Autonomous sensory meridian response and the desk tutorial. Architectural Design, 88, 50-57.

4.	Line 69 – 71 – While I agree that there have been few attempts at establishing the prevalence of ASMR in the general population, there have been some recent studies that have offered estimates. I’ve included them below for your reference. It may strengthen your point to note the range of estimates when commenting on how widespread the phenomenon appears to be. In addition, it may be good to include a note/statistics to support your statement about the popularity of ASMR on the internet (i.e. google search trends etc.).

Poerio, G. (2016). Could insomnia be relieved with a YouTube video? The relaxation and calm of ASMR. In F. Callard, K. Staines, & J. Wilkes (Eds.), The restless compendium (pp. 119–128). Cham, Switzerland: Palgrave Macmillan. http://dx .doi.org/10.1007/978-3-319-45264-7_15

		Roberts, N., Beath, A., & Boag, S. (2020). Autonomous sensory meridian response: sensitivity and personality correlates. Journal of Individual Differences. https://doi.org/10.1027/1614-0001/a000337

5.	Line 74 - Consider including some citations to support the increasing interest in ASMR research.
6.	Line 89 – Minor citation error re: et al.
7.	Line 92 – Similar to point above, there has been limited research on the emotional correlates of ASMR propensity, however there have been a few studies that have measured the role of empathy in ASMR experiences. While this doesn’t directly address the relationship between ASMR and emotion regulation, I think it would strengthen your introduction to note the mixed findings.

Poerio, G. L., Blakey, E., Hostler, T. J., & Veltri, T. (2018). More than a feeling: Autonomous sensory meridian response (ASMR) is characterized by reliable changes in affect and physiology, PLoS ONE, 13 (6), e0196645.

(See supporting information).

8.	Line 99 – I think the link between these two points could be improved with signposting.
9.	Line 100 – Consider (as you’ve done in line 117) including some of the specific regions involved (in brackets).
10.	Line 97-106 – I think the overall point of this paragraph (physiological and self-report evidence for emotional component of ASMR experiences) could be a bit clearer with some reorganisation.
11.	Line 107 – “On the other hand” – I think your point here is actually consistent with the previous paragraph (i.e. ASMR being a complex emotional experience with arousal components).
12.	Line 109 – Consider also including the following paper which assessed the relationships between self-reported misophonia and ASMR:

Roberts, N., Beath, A., & Boag, S. (2019). Autonomous sensory meridian response: Scale development and personality correlates. Psychology of Consciousness: Theory, Research, and Practice, 6(1), 22–39.

13.	Line 119 – Minor - change the capitalisation of “It”.
14.	Line 120 – I’m not quite sure what you mean by “such as the strategy deployed” – do you mean that ASMR may be a strategy/tool for emotion regulation?
15.	Line 130 – Great that you noted the role of neuroticism, however, it would be good to also include it in the personality traits associated with ASMR in paragraph 4.
16.	Line 147 – Consider rephrasing “ASMR self-reporters participants”.
17.	Line 149 – Consider rephrasing “this is the first study aimed to”.
18.	Line 156 – Consider opening the participants section with some more demographic information and details on where the participants were sourced.
19.	Line 158 – Consider rephrasing “we randomly subsample”.
20.	Line 165 – The ethics information may fit in better in the procedure section.
21.	Line 168 – Missing full stop.
22.	Just a suggestion, but it may be worthwhile moving some of the discussion of ASMR items (about videos etc.) to the materials section as a separate measure.
23.	Line 194 – “10-item”.
28.	Just a suggestion, but you may wish to move the first paragraph of the results (or just the reliability statistics) to the materials section (to compare prior and present reliability coefficients).
29.	Consider including a subsection under “normality” to help streamline your results section.
30.	Line 238 – Minor thing, just make sure you italicise the p and d statistics.
31.	Line 238 – Make sure you report the t statistic to two decimal places.
32.	Line 246 – “N” for total sample size, “n” for subsample.
41.	Line 327 – Last sentence could be expanded, or omitted.
42.	Line 338 – Consider including a citation here. I’ve included a reference for a study that assessed the effect of ASMR on sleep.

Lee, M., Song, C. B., Shin, G. H., & Lee, S. W. (2019). Possible effect of binaural beat combined with autonomous sensory meridian response for inducing sleep. Frontiers in Human Neuroscience, 13, 425. https://doi.org/10.3389/fnhum.2019.00425
Experimental design: 24.	Line 178 – Were all the participants Spanish speaking? Best to include this in the participants section, as well.
25.	Participants - Were participants rewarded in any way for participating?
26.	ERS - It would help the reader if you included the stem of the items, or some of the instructions that preceded the items, as well – i.e. how was the scale introduced?
27.	It would be good to also include some reliability statistics of prior studies (i.e. Cronbach’s alpha) when you mention how the translated scale has been validated.
33.	I understand that the ASMR qualitative responses were put into the supplementary materials, but I think it would be interesting to include a small discussion of the frequency that users engaged with ASMR media and the main reasons. Just a suggestion, but I think it may strengthen the story you are telling with regards to ASMR possibly being utilised as an emotion regulation strategy (as noted in the introduction).
Validity of the findings: 34.	Line 252 – What do these findings suggest? It may help the reader to briefly remind them what these two dimensions of emotion regulation refer to.
35.	Paragraphs 2 and 3 – I think these paragraphs (aside from discussion of present findings) may fit better in the introduction to support the strong affective dimension of the experience, and the rationale behind exploring the relationship between ASMR and emotion regulation.
36.	Paragraph 3 (Line 279) – This section may be better integrated into the first paragraph of the discussion, where you can first talk about the hypotheses and then what you found.
37.	Paragraph 4 – I think your argument could be more clearly signposted here. Some further explanation would help to communicate how these findings relate to misophonia. It may also be worth noting the overlap in the incidence of ASMR and misophonia, where the conditions/experiences may occur in the same people. See the following paper for some discussion of the overlaps of these experiences, and some methodological issues encountered when attempting to disentangle them with respect to trigger context and individual differences:

Roberts, N., Beath, A., & Boag, S. (2019). Autonomous sensory meridian response: Scale development and personality correlates. Psychology of Consciousness: Theory, Research, and Practice, 6(1), 22–39.

In addition, the Rouw and Erfanian (2018) paper you cited earlier found a 49% overlap in the incidence of ASMR in misophonic participants. They may very well exist as extremes on the same spectrum of sensory-affective experiences, but they also appear to commonly co-occur. It would be good to see some brief discussion of this.

38.	Line 303 – What does this suggest?
39.	Paragraph 7 – While there are definitely imbalanced samples in ASMR research, the effect may be partially attributable to utilising university samples. It may also be worthwhile to note whether these group differences (by gender) were significant in previous studies.
40.	Paragraph 8 – Good that you’ve noted some limitations, but it may also be relevant to note the potential limitations of the samples you drew from, confirmation bias, and limitations with self-report.
Additional comments: Thank you for inviting me to review your manuscript. The article expands on the current understanding of how ASMR relates to emotion regulation, and offers preliminary insight into the extent to which ASMR may reflect an emotion regulation strategy. The reviewer appreciates the breadth of related concepts the authors included in their discussion of ASMR experiences, particularly the clustering of both personality and physiological differences between ASMR-experiencers and controls. In summary, the paper appears to make an important contribution to our understanding of individual differences associated with ASMR propensity. However, I would advise that the authors refine sections of their manuscript to better emphasise this contribution.

I’ve made a number of suggestions and comments that I hope the authors will find helpful in refining the manuscript. Specifically, I would advise that the authors move some of the discussion material to the introduction. In particular, the prior research discussing the relationships between ASMR and other variables that weren’t included in the introduction. In addition, I appreciate the suggestion that ASMR may reflect an emotion regulation strategy, and I think it could be interwoven through the introduction a bit more explicitly.

Finally, while I understand that length may be an issue, it would be interesting to include some of the supplementary findings regarding motivation behind ASMR engagement to the results section. I think the results may provide support for the authors argument of the potential utility of ASMR as an emotion regulation tool.


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

Input:
Title: AUTONOMOUS SENSORY MERIDIAN RESPONSE SELF-REPORTERS SHOWED HIGHER SCORES FOR COGNITIVE REAPPRAISAL AS AN EMOTION REGULATION STRATEGY
Review round: 1
Reviewer: 2

Output:
Basic reporting: The writing in this manuscript is mostly clear and unambiguous (see relatively few comments in the accompanying annotated manuscript), with professional English used throughout. There are several typos, so the article requires a thorough copy-edit. The raw data was shared. A thorough overview of the background literature, complete with references, was also conducted, providing sufficient field background/context provided. I must commend the authors on an excellent job describing the current state of the literature on ASMR.

Although the article has a professional article structure, a formal discussion of the limitations of the study is lacking. Indeed, although the authors mention that their study cannot elucidate mechanisms and describe this as a limitation, they aren’t as forthcoming about the limitations of the study design (see section 2 on what I believe the limitations are).

Moreover, the authors describe frisson occurring in the context of musical experiences; although this is often considered the “primary way” folks experience frisson, this is not the only way. This should be amended.

Finally, the authors once again mention misophonia at the end of their paper, though don’t examine misophonia in their paper. I think it makes sense in the introduction, but this discussion should be more limited in the discussion section; see validity of findings section below.
Experimental design: Although the manuscript consists of original primary research within the aims and scope of the journal, there are a few key issues with the design of this study that must be addressed:
1.	The authors only use one single question to determine the presence of ASMR. This is a major limitation of the study; the way the question is worded could be congruent with the experience of frisson/chills as well, and I imagine most lay folks won’t know the difference between ASMR and frisson. Indeed, I do not experience ASMR, but I experience frisson, and probably would have selected yes here. There are several measures of ASMR that have been used in the literature (e.g., the ASMR checklist by Fredborg et al., 2017, 2018) that could have been employed to ensure the ASMR-group truly experienced ASMR, and I wonder why the authors did not use such a measure. To put it another way, it is unclear whether the control/naïve participants are truly “naïve”. For instance, in an internet-based survey study of ASMR (Fredborg et al., 2017), potential control participants watched a 5-minute popular ASMR clip and completed a short survey to determine their eligibility in the control group. Although this method is not “perfect,” (i.e., perhaps some control participants truly experienced ASMR in other contexts), it supports the notion that this group is naïve to the ASMR experience.

I suggest that you run this analysis again with only the people who have ASMR and have actually sought out ASMR videos. That way, you have more evidence that these folks actually experience ASMR. You can add this as a follow-up analysis.

2.	I also wonder if this was a biased sample, given that it appears that recruitment primarily took place over ASMR-related forums: “Participants were recruited by an online invitation to participate in the present work. This invitation was spread through social media, specifically through Instagram and Facebook, posted in different groups, either ASMR and non-ASMR related.” I am wondering if the authors had a biased sample toward ASMR based on their recruitment strategy. That said, I am happy to see that they used identical sized groups for the analyses. I’d like to see the authors discuss the limitations of self-report over the internet.

3.	I am curious if the authors conducted any additional “validity checks” of the data. For instance, if questionnaires were checked over for folks who repeatedly answered the same response throughout, etc.

4.	I understand that the authors used a random sample of the ASMR participants to compare to the control group. Did they try this multiple times (e.g., came up with multiple ‘random samples’ of ASMR participants and compared them to the control group)? If so, that would speak to the “robustness” of their findings and add more credence.
Validity of the findings: I believe this article mostly meets all criteria outlined by PeerJ with regards to validity. One issue is the following statement: “283 This finding provides further evidence supporting the notion that misophonia and ASMR 284 represent two ends of the same spectrum of sound sensitivity.” I believe the authors cannot make this claim based on the findings (the study only looked at ASMR vs. non-ASMR, not misophonics) and this should be revised.
Additional comments: Excellent work!


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

Input:
Title: AUTONOMOUS SENSORY MERIDIAN RESPONSE SELF-REPORTERS SHOWED HIGHER SCORES FOR COGNITIVE REAPPRAISAL AS AN EMOTION REGULATION STRATEGY
Review round: 2
Reviewer: 1

Output:
Basic reporting: - Line 100: Minor error – Omit “Katherine” from citation
- Line 106: Minor suggestion – It might be useful for the reader if you include a couple of common misophonic triggers
- Line 122: Minor citation error with “J.”
- Discussion: Line 421: Missing brackets around “2018”
- Line 422-423: I think this sentence could be phrased a bit more clearly.
- Line 445-451: Minor, but be sure to use “and” for the out of brackets citations (as opposed to “&”)
- Line 459: Very minor, but instead of “proved”, it may be more accurate to say it has shown promise/demonstrated effectiveness, or something to that effect.
Experimental design: - Results: The discussion of the differences in ASMR groups by ASMR-15 scores was well done. However, was there an analysis of the total ASMR-15 score across groups, as well (i.e. sum of subscales)? If so, it would be great to include the results for the total score here, and the reliability of the total score measure in the materials section.
Validity of the findings: No additional comments here.
Additional comments: The reviewer commends the authors on their thorough and systematic integration of suggestions from the first review. Overall, aside from some minor suggestions, the reviewer believes that the paper has been substantially improved upon refinement. In particular, the emotion regulation focus has come through much more clearly in the introduction and discussion.


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: AUTONOMOUS SENSORY MERIDIAN RESPONSE SELF-REPORTERS SHOWED HIGHER SCORES FOR COGNITIVE REAPPRAISAL AS AN EMOTION REGULATION STRATEGY
Review round: 2
Reviewer: 2

Output:
Basic reporting: Please see previous review - excellent work! There are still several minor typos that should be addressed (e.g., line 126 - "J" in citation, line 230 - google is in lowercase), but overall, very lovely work addressing mine and Reviewer 1's comments : )
Experimental design: Now that you've added in ASMR-15, this study is so, so much stronger and quite excellent. Wonderful work! All of my concerns were addressed.
Validity of the findings: I have no concerns here, all of my comments were addressed.
Additional comments: I love this paper. Thank you for contributing in such a meaningful way to the field! I'd love to perhaps do a replication here in Canada, if you are open to chatting! bfredborg@ryerson.ca.


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: IDENTIFICATION AND ANALYSIS OF CHRYSANTHEMUM NANKINGENSE NAC TRANSCRIPTION FACTORS AND AN EXPRESSION ANALYSIS OF OSNAC7 SUBFAMILY MEMBERS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The general structure of the artical are intact and clear. Introduction is straight and narrow. The main text is generally easy to read, however, almost all the figures lack figure legend to aim reading. The data roughly support the main theme.
Experimental design: The experimental design is rational. Most of them are descriptive. However, more web-lab experiments are needed to support the linkage of CnNACs and stresses.
Validity of the findings: The descriptive part is fine. More web-lab experiments are needed to support the linkage of CnNACs and stresses.
Additional comments: In this manuscript by Wang et al., the NAC transcription factors of Chrysanthemum nankingense are systematically analyzed by bioinformatics tools. Those features of the CnNACs including phylogenetic relationship, conserved peptide motifs, putative subcellular localization, and rough expression patterns are summarized. In addition, some individual genes are selected to analyze the potential stress response. As mentioned by the authors, the Chrysanthemum nankingenseare shows a good performance of stress resistance; meanwhile, the NAC transcription factors are deeply involved in the stress response in many plant species. This is a good selling point to emphasize the significance of this work, while the support is relatively weak. A general description of CnNACs is good but not sufficient to profile the link between CnNACs and stresses. Since the functional analysis of several CnNACs might be hard to investigate due to lacking reliable transgenic system, ectopic while in planta analysis should be conducted at least in Arabidopsis. For example, based on the stress responses showed herein, some candidates can be transformed into Arabidopsis to see the in planta effects.
Some detailed comments are as following:
1. The manuscript has two title pages. However, the affiliations are inconsistent.
2. Although I can understand the figures, legends are always required for any figures.
3. The reference citations are not well-formatted in the text. For example, in line 38, the “[4]” should be superscript as other citations. Please carefully check and fix those errors one by one.
4. Line 65, ABA is not abiotic stress, though it is important for the response of abiotic stress. Line 66, the virus resistance is related to biotic stress rather than abiotic stress. The sentence in the line 64-66 is too wordy with redundant information.
5.The third paragraph in the introduction section (line 64-81) is describing the link between NACs and stresses. The sentence in the line 67-68 is not suitable to be put here.
6. In the methods & materials section, it is usually not required to cite figures. Please refer to line 133.
7. The scale bar of Fig.1 is strange. Since the authors do not change the pot for the plant during the growing, the scale bar for the middle and right panel could be mispresented. Please carefully check the raw data.
8. For Fig.2, the bootstrap of the tree is always required. At the lowest level, only the node with the bootstrap higher than 50% is reliable and could be presented. In addition, the multi-alignment data should be provided accordingly.
9. Also, there might be something wrong with the “Rootless phylogenetic trees” as shown in the ANAC063” clade. A root appears there. Please check the raw data.
10. Since the authors have involved rice NACs in the tree, why only 6 rice NACs are analyzed? It should be unbiased to generate the tree, therefore, if the authors want to involve rice NACs, all the rice NACs should be put in the tree. So do the Arabidopsis NACs.
11. More information are needed to support why c3ulxA and d1ut7a are proper templates for modeling.
12. The individual panels in Fig.3 are not easy to read details. The authors may consider arranging the data as a multi-panel figure.
13.  Only the prediction of subcellular localization is presented. Since the NAC proteins are transcription factors, it is easy to understand their nuclear localization. However, extracellular localization and membrane localization are also predicted. It is interesting, while the subcellular localization of several typical NAC candidates should be validated by wet-lab experiments. At least, transient expression analysis should be used.
14. Statistical analysis should be applied for Fig. 6,7, and 8.
15.  In the discussion section, the authors should not present results in detail.


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: IDENTIFICATION AND ANALYSIS OF CHRYSANTHEMUM NANKINGENSE NAC TRANSCRIPTION FACTORS AND AN EXPRESSION ANALYSIS OF OSNAC7 SUBFAMILY MEMBERS
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The manuscript by Hai et al. uses bioinformatic, genetic, and molecular approaches to identify NAC transcription factors and determine expression levels of OsNAC7 subfamily members in C. nankingense. The authors have generated a solid amount of data and the conclusions are justified by the results. Besides that, the details in some experiments are missing or need to be clarified in the text and legends.

Line 119: italic “A. thaliana” and “O. sativa”.

Figure 2: It would be better to differentiate which are from A. thaliana, O. sativa, and C. nankingense in legends.

Figure 4: brief explain the meaning of E-value, sites, and width.

Figure 6: Explain relative expression to what? And add statistical analysis.

Figure 7 and 8: include the meaning 20% PEG6000 and 200 mM NaCl for osmotic and salt stress, respectively.

Supplementary data of Figure6_7_8 is not cited 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: IDENTIFICATION AND ANALYSIS OF CHRYSANTHEMUM NANKINGENSE NAC TRANSCRIPTION FACTORS AND AN EXPRESSION ANALYSIS OF OSNAC7 SUBFAMILY MEMBERS
Review round: 1
Reviewer: 3

Output:
Basic reporting: Abbreviations are not properly defined. For example, NAM, ATAF, and CUC (line 36-37); CnNAC (line 98)

Statements lack citations. For example, line 69-71; line 92-93; line 260; line 298-303

Figure 1 does not provide any meaningful information.
Experimental design: The authors should state clearly what is used as the control sample for RT-qPCR experiments related to Figure 6-8.
Validity of the findings: Change in gene expression during stress treatment does not necessarily mean the genes are important regulators of stress response. The authors should not over interpret the results.
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: IDENTIFICATION AND ANALYSIS OF CHRYSANTHEMUM NANKINGENSE NAC TRANSCRIPTION FACTORS AND AN EXPRESSION ANALYSIS OF OSNAC7 SUBFAMILY MEMBERS
Review round: 2
Reviewer: 1

Output:
Basic reporting: NIL.
Experimental design: NIL.
Validity of the findings: NIL.
Additional comments: I appreciate the efforts of the authors to improve the manuscript. The previous concerns are addressed accordingly. The statistical analysis had been added in Fig. 6,7, and 8, but they need to be described in the legends.
However, the revised text, including that in the main text and methods, MUST be edited by a native speaker. A lot of grammatical errors are there, and the sentences are not readable. In addition, the presentation of the legends is really bad, especially for the multi-panel figures. A key point is that, NEVER to use “A”, “B”, “C”, etc as a subject in a descriptive sentence. “A”, “B”, “C”, etc, are only the labels of the panels.
For example:
>> “A means 28 days after sowing, which is the seedling stage.”
--- “A” does not mean that at all.
Do revise it like that, “A. The picture showing the seedling 28 days after sowing.”

>> “A, B, C, D is a subcellular observation of 1300: : GFP. A is the root of 1300::GFP, B is the leaf of 1300::GFP, C is the root tip of 1300::GFP, D is the stem of 1300::GFP.”
---“A” is not the observation, thus the whole sentence is nonsense.
Do revise it like that, “A-D. Subcellular localization of GFP in root (A), leaf (B), root tip (C), and stem (D).”

I can’t point them one by one, but DO please revise all such sentences entirely.


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: IDENTIFICATION AND ANALYSIS OF CHRYSANTHEMUM NANKINGENSE NAC TRANSCRIPTION FACTORS AND AN EXPRESSION ANALYSIS OF OSNAC7 SUBFAMILY MEMBERS
Review round: 2
Reviewer: 2

Output:
Basic reporting: The structure of revised manuscript is clear with all the required sections.
Experimental design: The experimental design is well defined to answer questions.
Validity of the findings: The findings are well described and discussed.
Additional comments: The revised manuscript addresses the reviewers' comments with detailed descriptions and additional analyses/experiments. I do not have 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: IDENTIFICATION AND ANALYSIS OF CHRYSANTHEMUM NANKINGENSE NAC TRANSCRIPTION FACTORS AND AN EXPRESSION ANALYSIS OF OSNAC7 SUBFAMILY MEMBERS
Review round: 2
Reviewer: 3

Output:
Basic reporting: The English in the present manuscript is not of publication quality and requires major improvement.

The figure legends are particularly confusing. I highly recommend the authors to follow standard figure legends formats of scientific papers. Also, in Figure 2, there are no individual descriptions of the three panels; in Figure 6 and Figure 7, there is no description of what the small letters (a, b, and c) above the bars represent.
Experimental design: It's nice to add the CHR00069684::GFP experiment. However, important details of this experiment are missing. For example:

1) information and references of pSuper1300

2) DNA sequence or the genomic coordinates of the ORF of CHR00069684 inserted into the vector.

3) promoter driving the expression of CHR00069684::GFP or GFP

4) meaning of CHR00069684::GFP. Is it a fusion protein?
Validity of the findings: Since only the ORF of CHR00069684 was cloned into pSuper1300 vector, I'm assuming the vector contains some ubiquitously expressed promoters to drive transgene expression. If so, why is pSuper1300::GFP expressed in leaves, while pSuper1300::CHR00069684::GFP is NOT?

I cannot tell whether the fluorescence is in cytoplasm or on cell membrane in Figure 9. The authors should provide images with higher magnifications and better qualities. Staining the tissues with cell membrane, cytoplasm, and nucleus markers would also help. Also, the authors should show images of the leaves of the pSuper1300::CHR00069684::GFP line to support their claim that there is no expression of CHR00069684::GFP in leaves.
Additional comments: No additional comments


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

Input:
Title: TAXONOMIC DESCRIPTION AND PHYLOGENETIC PLACEMENT OF TWO NEW SPECIES OF SPALANGIOPELTA (HYMENOPTERA: PTEROMALIDAE: CEINAE) FROM EOCENE BALTIC AMBER
Review round: 1
Reviewer: 1

Output:
Basic reporting: all ok !!

I do see clearly the impact of the ct scans, they are not very precise, can you explain
Experimental design: all ok
Validity of the findings: all ok, nice finding for a rather poorly known group
Additional comments: whereas the other six species are Nearctic or Neotropic species
remove last 'species'

Character 1: Hyaline break in parastigma. 0: absent, 1: present. The third character
143 state (“partial”) in Darling (1996) was removed
please, precise to which remaining state absent vs present they are transferred, and why ?

What mean Presence of short-winged females.
short compared to what, can you precise ? please

Character 17: Shape of forewing. 0: narrow, length greater than 3.5 times width, 1:
199 wide, length less than 3.5 times width
what about if it very close to 3.5 ?

Fore wing with transparent region (“hyaline break”) in parastigma ......................3
271 — Fore wing with large admarginal setae below marginal vein..................................4
the two states are not opposed ?

the diptera in syninclusion is a female Ceratopogonidae


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

Input:
Title: TAXONOMIC DESCRIPTION AND PHYLOGENETIC PLACEMENT OF TWO NEW SPECIES OF SPALANGIOPELTA (HYMENOPTERA: PTEROMALIDAE: CEINAE) FROM EOCENE BALTIC AMBER
Review round: 1
Reviewer: 2

Output:
Basic reporting: Clear and unambiguous, professional English used throughout - for the most part.  Problem areas highlight in the pdf returned with this review.  The literature citations are complete as far as I can tell.
Experimental design: The 3D models constructed based on the CT data add nothing to the paper.  They do not clarify any aspects of the morphology as presented and if anything detract from the other figures in the manuscript.  The habitus drawing (Fig. 1) is minimally useful but should show the admarginal setae present in Fig. 2.
Validity of the findings: I am not convinced that two species of Spalangiopleta are represented in this study.  S. darlingi is well supported by Figure 2 but the status of the specimen described as S. semialba is less clear -- the white genae are certainly not prominent in Figure 4.  And the distinctive mesopleuron of the genus is not apparent in Figure 4.  The Diagnosis of S. semialba suggest to me the possibility that this specimen is not referable to Spalangiopelta.
Additional comments: See annotated pdf for suggestions on wording and other comments.

Figure 1.  The habitus drawing is minimally useful but should show the admarginal setae present in Fig. 2.
Figure 6.  This should be deleted.  The "syninclusions" add nothing to the paper.
Caption, figure 8.  Add (above) and (below) to the inset text.
Table head.  What are Amira measurements.  Explain here or in the text.


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

Input:
Title: TAXONOMIC DESCRIPTION AND PHYLOGENETIC PLACEMENT OF TWO NEW SPECIES OF SPALANGIOPELTA (HYMENOPTERA: PTEROMALIDAE: CEINAE) FROM EOCENE BALTIC AMBER
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript is an important addition to the knowledge of a rather poorly studied group of chalcid wasps. It adds two new fossil species, keys all known species of Spalangiopelta, and provides a cladistic analysis of the genus. The most important area where the authors should pay attention is the key to species, where in several couplets the two parts do not match entirely, i.e. some characters are mentioned in the first part of the couplet, but are not repeated in the second part, or vice versa. The authors should also clarify the use of the terms 'absent' and 'indistinct' as different, which can be confusing. Regarding the phylogenetic analysis, although it is not very informative regarding the basal part of the tree, it provides some interesting correlations between some morphological characters.
Experimental design: No comment
Validity of the findings: The two new species are well described, allowing comparisons with previously described species. They are well illustrated and benefit from the cutting edge technology of micro CT.
Additional comments: Please see and address all suggestions and comments 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: TAXONOMIC DESCRIPTION AND PHYLOGENETIC PLACEMENT OF TWO NEW SPECIES OF SPALANGIOPELTA (HYMENOPTERA: PTEROMALIDAE: CEINAE) FROM EOCENE BALTIC AMBER
Review round: 2
Reviewer: 1

Output:
Basic reporting: All indicated changes/comments have been addressed.
Experimental design: All indicated changes/comments have been addressed.
Validity of the findings: All indicated changes/comments have been addressed.
Additional comments: All indicated changes/comments have been addressed, thank you.


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: PSYCHOMETRIC PROPERTIES OF THE ATTITUDES TOWARDS MEDICAL COMMUNICATION SCALE IN NURSING STUDENTS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The authors have been very conscientious and systematic in their preparation, writing and reporting the results of the cross-cultural validation and adaptation of a scale.
 The introduction is well founded. However, in the bacground section, you should have contemplated and cited other validated scales in Spanish in your immediate area. For example The Communication Skills Scale (CSS) (Leal-Costa et al., 2019; Leal-Costa et al., 2016).
Experimental design: The method is well explained and contains all the sections that a validation research and adaptation of a questionnaire must have.
The results have been flawlessly displayed. Finally, there are 11 items in the Spanish version and I sincerely believe that your great work is credited.
Validity of the findings: I only must make a final reflection on the meaning of your work. The Attitudes Towards Medical Communication Scale was initially validated in medical students. Later it has been used in health professionals in various fields and in students as well.
I think, to be honest, the external validity of your validation should be improved. For this, it would be necessary to use a representative sample of the Spanish population to which the scale is targeted. Not only fourth year nursing students in the southeast of Spain. I mean that validation should be considered in a multiprofessional sample (mainly postgraduate doctors and nurses). I believe that students have limited access to communication with patients. Communication is reduced to their limited clinical practices and their clinical skills are not sufficiently developed, leaving non-clinical skills in the background. But this is just a reflection that should be considered when naming the scale in Spanish (indicating that its validity includes only nursing students or that the population it is addressed to are students). However, I think this would be a target for further research, so I only suggest that you include these arguments in the limitations section.
Additional comments: Thank you very much for giving me the opportunity to review this excellent work. It is a very professionally written manuscript with an impeccable scientific structure.
The authors have been very conscientious and systematic in their preparation, writing and reporting the results of the cross-cultural validation and adaptation of a scale.
 The introduction is well founded. However, in the bacground section, you should have contemplated and cited other validated scales in Spanish in your immediate area. For example The Communication Skills Scale (CSS) (Leal-Costa et al., 2019; Leal-Costa et al., 2016).
The method is well explained and contains all the sections that a validation research and adaptation of a questionnaire must have.
The results have been flawlessly displayed. Finally, there are 11 items in the Spanish version and I sincerely believe that your great work is credited.
I only must make a final reflection on the meaning of your work. The Attitudes Towards Medical Communication Scale was initially validated in medical students. Later it has been used in health professionals in various fields and in students as well.
I think, to be honest, the external validity of your validation should be improved. For this, it would be necessary to use a representative sample of the Spanish population to which the scale is targeted. Not only fourth year nursing students in the southeast of Spain. I mean that validation should be considered in a multiprofessional sample (mainly postgraduate doctors and nurses). I believe that students have limited access to communication with patients. Communication is reduced to their limited clinical practices and their clinical skills are not sufficiently developed, leaving non-clinical skills in the background. But this is just a reflection that should be considered when naming the scale in Spanish (indicating that its validity includes only nursing students or that the population it is addressed to are students).
However, I think this would be a target for further research, so I only suggest that you include these arguments in the limitations section.


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

Input:
Title: PSYCHOMETRIC PROPERTIES OF THE ATTITUDES TOWARDS MEDICAL COMMUNICATION SCALE IN NURSING STUDENTS
Review round: 1
Reviewer: 2

Output:
Basic reporting: See general comments
Experimental design: See general comments
Validity of the findings: See general comments
Additional comments: The manuscript by Escribano et al describes the authors’ attempt to translate the Attitude Toward Communication Scale for use among nursing students in Spain. The authors seem to have a good level of sophistication about the requirements of creating a translation and demonstrating its psychometric characteristics, and the paper therefore contributes to the literature in disseminating a tool for use in a culture and in a health profession for which it was not previously available. Although generally favorable toward this paper, I came across several instances of sentences that did not make sense, and it appeared that they more related to proofreading problems than English-Spanish translation. This should not have happened.
There were several issues, some larger, some smaller, where I thought the paper could be improved:
•	The authors refer to the fact that there seems to be little skills training in communication, referring to nursing, for which I agree. However, their references largely relate to medicine. They should be more careful about being sure to cite nursing literature if they want to make a case about nursing.
•	As noted above, there are some issues about meaning. Line 75: In “This evidence-based practice approach…” it’s not clear what this refers to. Lines 129-130, something is missing. Line 152—what additional study questions? Line 228: the sentence is missing something.
•	The authors used two study sites, one with a very high response rate the other very low. Nowhere did I see any explanation for this or a concern for combining the two sites’ data together. In fact, given the very small n in the second site, I would recommend that the authors demonstrate that the data were comparable across sites in order to combine the two, or even better, that they simply delete the small number from the second site in their analyses.
•	Why do the authors never show us he items? Referring to them by number when the reader is totally in the dark as to their actual content is a disservice. Especially when we come to the cognitive interviews, we are lost without knowing what the items were.
•	Does the fact that there were ceiling effects for many of the items not concern the authors? This needs further discussion.
•	In terms of validity evidence, the authors rush through the fact that their scale was moderately correlated with the Self-Efficacy scale. This seems like very limited validity evidence, very weakly explained. If the authors want to spend so much time providing information on their fancier statistics, why do they not spend time talking about types of validity and what their moderate correlation with another validated scale means.
•	On lines 296-297, the authors make reference to the Theory of Planned Behavior in a way that is both loose and inaccurate. If they want to give their paper some conceptual/theoretical backing, they should do so with far more care. Otherwise, it is simply window dressing for those who will be impressed by the simple mention of the theory.
•	The authors report gender differences. This is good and these findings are consistent with lots of other findings in the literature. It’s surprising that they don’t tell us about the scale scores in relationship to training in communications within their sample, nor do they tell is anything about their scores in relationship to those found in other cultures and in other health professions. This would make the paper far richer.
•	The authors mention notable limitations in a couple of sentences and then simply move on, as if mentioning these was sufficient. Once we are aware of the limitations, the authors are obligated to inform readers what to make of these limitations and/or how they might be addressed or overcome in the future.
In general, this is a solid paper, done by people who clearly know their way around translation and back-translation issues and who hove good statistical expertise among their team and consultants. It is my feeling that the improvements suggested here could be made without a major re-write, and that these improvements would increase its contribution significantly.


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:  PARTR2: PARTITIONING R2 IN GENERALIZED LINEAR MIXED MODELS
Review round: 1
Reviewer: 1

Output:
Basic reporting: This manuscript is well-written in a professional way.
Experimental design: The presentation is well-thought and comprehensive.
Validity of the findings: This manuscript presents a useful R package to the research community.
Additional comments: The authors introduce partR2, a versatile package for estimating part R2, inclusive R2, structure coefficients and beta weights from mixed-effects models. They also show how to use partR2 with real example datasets for Gaussian and binomial GLMMs. The manuscript is well-written and the presentation is well-thought and comprehensive.

My only comment is that partR2 can be explained in more detail in the text. It is an important and subtle concept. It would be nice if it can be defined in the text using the notations in Figure 1 so the reader does not need to read Nimon et al. (2008).


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

Input:
Title:  PARTR2: PARTITIONING R2 IN GENERALIZED LINEAR MIXED MODELS
Review round: 1
Reviewer: 2

Output:
Basic reporting: In their paper Stoffel et al. describe a potential framework for calculating semi-partial R^2 for GLMMs.  Such a contribution would be ground-breaking and very exciting.  Some features of the author's function partR2 appear useful.  However, I am not convinced that the author's overall approach, in the context of GLMMs, is valid.  I have five main concerns:

1) The definition given for R^2 is confusing and non-rigorous. The coefficient of determination is the proportion of the variance in the dependent variable that is predictable from the independent variable(s).

2) The paper introduces an algorithm for calculating semi-partial R^2, however most scientists are much more familiar with partial R^2.  Only a brief mention of partial R^2 is given on lines 90-93.  A concise comparison of these methods, along with a motivational paragraph concerning the undervalued importance semi-partial R^2 would be useful.

3) The paper lacks mathematical rigor throughout.  Only one inline formula is given.  Specifically, on line 66 the authors confusingly give the equation for the mean function of a linear model in the context of a sentence describing structure coefficients.  Lacking and badly needed are explicit formulae for semi-partial coefficients of determination and structure coefficients, along with mathematical frameworks for general linear mixed models, generalized linear models and generalized linear mixed models.

4) Importantly, it is unclear how isolated correlation coefficients (structure coefficients) squared or otherwise would provide insight concerning partial and semi-partial R^2 in a GLMM.

5) The background and literature cited is insufficient. For instance, missing from the list of R functions and packages that calculate partial and semi-partial R^2 is the library ppcor which has functions that calculate both of these entities, along with performing null hypothesis tests.  More troubling, the authors claim that their framework for semi-partial R^2 for GLMMs is based on R^2.  However quantifying explained variability in generalized linear models, including those with binomial errors, is a difficult and contentious subject with a large body of literature.  None of this work is considered by the authors.
Experimental design: Not applicable here
Validity of the findings: Insufficient background is provided to allow confirmation of the validity of the author's approach.
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:  PARTR2: PARTITIONING R2 IN GENERALIZED LINEAR MIXED MODELS
Review round: 1
Reviewer: 3

Output:
Basic reporting: No comment
Experimental design: Not applicable
Validity of the findings: No comment
Additional comments: I have reviewed the referenced manuscript and find it provides a nice addition to the literature. The manuscript followed the R code well and the contained data files made it easy to replicate the analyses presented. Please find below opportunities to improve the accessibility of the material presented:
1.	I think more explanation needs to be provided including formulas for each statistic calculated. I did find some information by using help(partR2) and in the vignettes, but it was not complete. In particular, what is the formula for R2 of which part R2 is based?
2.	I am unfamiliar with the term inclusive R2 and wonder if more information could be provided to support how and why it is being calculated.
3.	Communality should be commonality on line 92.
4.	Line 80 - I disagree that the analysis presented is similar to commonality analysis.


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:  PARTR2: PARTITIONING R2 IN GENERALIZED LINEAR MIXED MODELS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The paper is dramatically improved with the inclusion of clarifying equations and responses to reviewers.  However, a few issues still exist that I have described in an attachment.
Experimental design: Not applicable
Validity of the findings: These seem reasonable now given additional background.
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:  PARTR2: PARTITIONING R2 IN GENERALIZED LINEAR MIXED MODELS
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: partR2: Partitioning R2 in generalized linear mixed models
I have reviewed the revised version of the referenced manuscript and appreciate how the authors attended to the reviewer comments.
1.	Thank you for the definition of inclusive R2 as presented in the manuscript. I am not clear however why a new term is needed is explain the relationship between a criterion and a predictor as multiplying the squared structure coefficient by Multiple R2 always results in the squared validity coefficient or zero-order correlation between the predictor and criterion. This can be seen by examining the formula for structure coefficients (see for example Nathans et al., 2012).
2.	Communality should be commonality.
3.	It is not clear how the analyses is conceptually related to commonality analysis.
4.	Line 80 - I disagree that the analysis presented is similar to commonality analysis.


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:  PARTR2: PARTITIONING R2 IN GENERALIZED LINEAR MIXED MODELS
Review round: 3
Reviewer: 1

Output:
Basic reporting: The authors have addressed my comments adequately.
Experimental design: Not applicable
Validity of the findings: The findings seem reasonable now given additional background.
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:  PARTR2: PARTITIONING R2 IN GENERALIZED LINEAR MIXED MODELS
Review round: 3
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The authors have addressed all previous comments. I appreciate very much the information provided in Figure 1.


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: RUN FOR YOUR LIFE: CAN EXERCISE BE USED TO EFFECTIVELY TARGET GLUT4 IN DIABETIC CARDIAC DISEASE?
Review round: 1
Reviewer: 1

Output:
Basic reporting: Bowman et al provide a comprehensive review of cardiac metabolism and how it related to diabetic cardiomyopathy. This review provides insight into the links between insulin action, metabolism and cardiomyopathy. The Authors also provide an opinion on how exercise may overcome diabetic heart complications. This is an interesting topic and interesting article.

Language – Overall the article is well written.

The review generally covers field with required references. However, the Authors do not mention the recent publications linking heart pyruvate transport into mitochondria with cardiac hypertrophy. Is this relevant to their argument lining glucose metabolism with diabetic heart? https://www.nature.com/articles/s42255-020-00276-5

The review is comprehensive and covers a lot of ground. However, it is also quite long. It would be useful to edit for conciseness if possible.
Experimental design: Survey methodology is clear and appropriate.
Validity of the findings: The Authors clearly outline to context and scope of the review.
Additional comments: 1.	The Authors make a put together an argument that altered fuel utilisation (lower glc) leads to impaired ATP or CrP for contraction– “Excessive FFA uptake and therefore accumulation may lead to insulin resistance and starve the heart of the glucose that it needs to rapidly and efficiently generate the ATP that powers excitation contraction coupling. The relatively smaller contribution of glucose to total cardiac metabolism under normal conditions does not diminish its functional importance.” This appears to be a key argument, although as the Authors state, the evidence is limited. Should this be include in figure 3?
a.	However, the authors also state “that “T2D has been linked to cardiac mitochondrial dysfunction that manifests as lowered intracellular CrP levels in T2D hearts (Bashir, Coggan & Gropler, 100 2015). This adaptation does not appear to reduce glycolytic flux” – these data appear to this point to there being no change in glucose metabolism. Is there any evidence for functional coupling between glucose metabolism (and not FA metabolism) and CrP?
b.	If this is not linked directly to glucose metabolism, what are other possible mechanisms by which insulin resistance lowers the CrP concentrations?
2.	Line 246 onwards:
a.	Increased UCP3 expression is proposed to link FFAs to cardiomyopathy. But the Authors do not directly relate this change back to ATP and CrP – its implied, but a direct statement would be helpful.
b.	It might be useful to include a statement that the idea that the ATP/CrP ratio can be linked to GLUT4 has not been tested, but could be tested in KO or TG models.
3.	"Can we specifically target GLUT4 with exercise" – Is there any data on how exercise increases GLUT4 expression in heart? How might GLUT4 be stabilised at the protein level in response to exercise? Is this a reasonable target for therapy?
4.	The section on exercise is lengthy and might benefit from editing to make sure it’s a concise as possible. In addition, this section is about 50% of the review, but has no summary figure. It would help the reader to have a figure for reference.
5.	Exercise interventions are notoriously difficult to maintain – how do the authors propose to overcome this limitation?
6.	The discussion of the use of IPCS cells is excellent. Perhaps the Authors could extend very slightly to talk about organoids - https://www.cell.com/cell-stem-cell/pdfExtended/S1934-5909(19)30108-0. Do these 3D cultures have a better GLUT4/GLUT1 expression profile?

Figures:
Figure 2 – the figure suggests that contraction and insulin both increase glucose and fatty acid uptake.  How does the heart balance the use of each substrate (text or legend). Is it purely what is available in the circulation? Does the Randle cycle dictate use of FAs over glucose, but both CD36 and GLUT4 are mobilised so that any available substrate is taken up?
Figure 3 – describes the links between altered metabolism and diastolic dysfunction. Changes in ceramide are linked to changes in insulin signalling, so should there be a link between ceramides and insulin signalling or even directly to GLUT4? In addition, while this figure highlights the changes in metabolism that may link to diastolic dysfunction, there is no information on how these link to contraction. Could this be included?


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: RUN FOR YOUR LIFE: CAN EXERCISE BE USED TO EFFECTIVELY TARGET GLUT4 IN DIABETIC CARDIAC DISEASE?
Review round: 1
Reviewer: 2

Output:
Basic reporting: See general comments.
Experimental design: NA
Validity of the findings: NA
Additional comments: Many thanks for your review. My bias is that all pharmacological interventions should be judged versus exercise, so I enjoyed reading this review.
Line 46: Slightly awkward sentence structure.
Line 59-62: As above. Considering separating into 2 sentences?
Line 73: I find the logic in this subsection a little difficult to follow. You’re saying that IR precedes deterioration of cardiac function, therefore DCM is a consequence of diabetes?
Line 140: Period after the reference.
Line 156: Sentence structure is off. Consider commas either side of therefore?
Line 163: needs a comma after exercise
Line 187: missing a reference
Line 198: Period after the reference
Line 220: missing some commas
Line 246: I wonder if you could comment on glucose-fatty acid cycle and how this may affect glucose uptake in the heart. I think this model has been larhgely disregarded by the work of Shulan and others, but I believe it is still a widely held belief.
Line 356: Anaerobic threshold, rather than lactate?
Line 412: Perhaps some comment about the athletes paradox is warranted here. Exercise may not decrease TGs, but it may decrease the lipotoxic intermediates.
Line 416: You draw parallels to insulin signalling in cardiac and skeletal muscle at the beginning of the review. You make the important point that it is difficult to asses the effects of exercise on cardiac tissue. Do you think you can infer this from the effects on skeletal muscle, which have been well investigated?
Line 442: I’m unconvinced re: the argument that there are responders and non-responders to exercise training. And I disagree with the statement that this is a widely-held belief. I would also question the statement that all “individuals can adapt to exercise training, however the required load, optimal programme and degree of adaptation achieved will show inter-individual variability”. In the context of type 2 diabetics who, I do not think it would be unfair to say, are likely well-below average ‘fitness’. For these individuals, any structured exercise is likely to induce favourable, potentially disease modifying adaptations.
Line 461: Again, I think it would be useful to draw parallels with SM here. Presumably, the type of exercise best suited to increase SM GLUT4 translocation would also apply to cardiac muscle?
Line 513: It might be useful to review how drugs may interact with exercise to modulate adaptations. In particular, the effects of metformin may be interesting.
Line 521: 1:40,000? rather than 1:40,00
Line 522: My understanding is this J shaped relationship is only observed when weekly duration exceeds ~>10 hours, so may not be a factor here?
Line 630: gap after reference
Line 598: I find the section on iPSC-CM a little too speculative to warrant inclusion, but it’s not a deal breaker.
Once again, thank you for your efforts in bringing this review together. I look forward to hearing your responses to my recommendations in due course.


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: RUN FOR YOUR LIFE: CAN EXERCISE BE USED TO EFFECTIVELY TARGET GLUT4 IN DIABETIC CARDIAC DISEASE?
Review round: 2
Reviewer: 1

Output:
Basic reporting: See below
Experimental design: See below
Validity of the findings: See below
Additional comments: Many thanks to the authors for their revisions and consideration of the points I raised.


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: BIOTIN-STREPTAVIDIN-GUIDED TWO-STEP PRETARGETING APPROACH USING PLGA FOR MOLECULAR ULTRASOUND IMAGING AND CHEMOTHERAPY FOR OVARIAN CANCER
Review round: 1
Reviewer: 1

Output:
Basic reporting: The authors developed Biotin-streptavidin-guided two-step pretargeting approach using PLGA for molecular ultrasound imaging and chemotherapy for ovarian cancer. The major concern needs to be addressed before the paper is accepted for publication. Followings are recommended for the manuscript.
What is the novelty of work?
Purpose of study in the abstract is not clear. Therefore, the author needs to a breakdown in two sentences and make it clear.
The objective of the study should be incorporated at the end of the introduction.
If authors have used reported HPLC method for drug analysis then it should be incorporated in the manuscript such as the composition of mobile phase and instrument specification.

The vocabularies, grammar and minor writing style in the manuscript must be revised and improved
Experimental design: Please indicate both the manufacturer’s name and location (including city, state, and country) for all specialized equipment, kits, software, incubators, instruments, and reagents used in the experiment wherever required.

Authors should provide the process parameters, e.g. how much volume of the organic phase, temperature, stirring speed, the power capacity of sonicator and so on.

How is the stability of formulations physiological condition? How is the biosafety of formulations?

Authors did not show any experiments in which they used blank nanoparticles. Nanoparticles itself could induce an unwanted cytotoxic effect on cells. MTT assay should be performed also with blank nanoparticles.

The author needs to justify why the cytotoxic effects of formulations on at least one normal cell line were not assessed.
Validity of the findings: No comment
Additional comments: Discussion of the results especially formulations is laconic, the author needs to improve the discussion with compare to previous literature.

The quality of the Figures is poor. Please provide high resolution (300 dpi) images.


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: BIOTIN-STREPTAVIDIN-GUIDED TWO-STEP PRETARGETING APPROACH USING PLGA FOR MOLECULAR ULTRASOUND IMAGING AND CHEMOTHERAPY FOR OVARIAN CANCER
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: This article is entitled " Biotin-streptavidin-guided two-step pretargeting approach using PLGA for molecular ultrasound imaging and chemotherapy for ovarian cancer " by Zhou, et al. They report a pre-targeting strategy using streptavidin (SA) and paclitaxel (PTX)-loaded phase-shifting poly lactic-co-glycolic acid (PLGA) nanoparticles with perfluoro-n-pentane (PTX-PLGA-SA/PFPs) on the treatment and ultrasound imaging of ovarian cancer. The concept of pre-targeting strategy using streptavidin (SA) was quite interesting. Overall, I find this design and the scope of this paper is suitable for the journal after a major revision.
1.	There are no animal experiments to verify the results of in vitro experiments, I advise the author should supply the in vivo experiments to verify the effects of PTX-PLGA-SA/PFPs on the treatment and ultrasound imaging of ovarian cancer.
2.	The author reported that PTX-PLGA/PFPs were prepared with a single emulsion (O/W) solvent evaporation method, as far as I know, the PLGA nanoparticles were usually prepared with a double emulsion(O/W/O), why you choose a single emulsion (O/W) solvent evaporation method? Please explain this.
3.	The annotation of Figures cannot be seen clearly (Fig 1e, 1f, Fig 1g,1h, ect). The author should enlarge font.
4.	The SEM and TEM in figure 1c,1d lacks bar, and some other figures.
5.	Flow cytometry analysis of the connection efficiency of both SA and Ab bound to PTX-PLGA/PFPs in Figure, What is the sample size?
6.	The annotation of CLSM pictures in Figure 4b is wrong, please revise.


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: BIOTIN-STREPTAVIDIN-GUIDED TWO-STEP PRETARGETING APPROACH USING PLGA FOR MOLECULAR ULTRASOUND IMAGING AND CHEMOTHERAPY FOR OVARIAN CANCER
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have addressed all the concerns, improving the manuscript.  In my opinion, the manuscript is now acceptable for publication
Experimental design: The authors have addressed all the concerns, improving the manuscript.  In my opinion, the manuscript is now acceptable for publication
Validity of the findings: The authors have addressed all the concerns, improving the manuscript.  In my opinion, the manuscript is now acceptable for publication
Additional comments: The authors have addressed all the concerns, improving the manuscript.  In my opinion, the manuscript is now acceptable for publication


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

Input:
Title: WHOLE GENOME SEQUENCE ANALYSIS OF MULTI DRUG RESISTANT COMMUNITY ASSOCIATED METHICILLIN RESISTANT STAPHYLOCOCCUS AUREUS FROM FOOD FISH: DETECTION OF CLONAL LINEAGE ST 28 AND ITS ANTIMICROBIAL RESISTANCE AND VIRULENCE GENES
Review round: 1
Reviewer: 1

Output:
Basic reporting: This airticle describe the unique ST28/ t021 CA-MRSA in fish from India which is the first report, and it is co-harbouring the antibiotic resistance and virulence genes.
This article is well written in English and conform to professional standards of courtesy and expression. However, there is a minor mistake in spelling and grammar.
Literature references, sufficient field background/context provided. Data are showed clearly,if possible, it's better to use three-line tables and adjust font size to suit tables.
Line 49-50.  “three decade” or “three decades”？
Line 64 and 239. “CA-MRA” or “CA-MRSA”？
Line 73-74 and line163,171. “mepR, mgrA, arlR, LmrS, FosB, norA and GlpT and murA” are genes，so the first letter should be lowercase according to the standard.
Line 97. Please correct “16SrRNA”.
Line 115. Please correct “10-5”.
Line 131-132. “CDs” or “CDS”?
Line 145. Please delete “ :”.
Line 193 and 195. The phrase “community acquired CA-MRSA”, “community acquired” is synonymous with “ CA”. Please rewrite this phrase.
Line 258 and 261. Please confirm the phrase “CA-MRSAclones” and “CA- MRSA”.
Experimental design: The MRSA strain7 isolated from fish samples, AST was carried out and mecA , nuc gene  and 16S rRNA were confirmed by Sanger PCR. The authors analysis ARGs and virulence factors by WGS. This study contributes to filling the gap of the first reported CA-MRSA from the fish based on WGS in India. Methods described with sufficient detail. However, there are several problems need to solve.

1.In method, you have done the Antimicrobial susceptibility testing (AST), but this result have not been shown. Please add it.
2. If possible, it’s better to add more experiment such as animal experiment to prove the high virulence of this strains, because high virulence is an important point of this paper.
Validity of the findings: All underlying data have been provided. Conclusions are well stated, linked to original research.
Additional comments: No, thanks.


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: WHOLE GENOME SEQUENCE ANALYSIS OF MULTI DRUG RESISTANT COMMUNITY ASSOCIATED METHICILLIN RESISTANT STAPHYLOCOCCUS AUREUS FROM FOOD FISH: DETECTION OF CLONAL LINEAGE ST 28 AND ITS ANTIMICROBIAL RESISTANCE AND VIRULENCE GENES
Review round: 1
Reviewer: 2

Output:
Basic reporting: The text needs rephrasing since it is unclear in some parts of the manuscript. The figure quality is low, hence needs editing or regeneration.
Experimental design: The experimental design is well thought of. However, the genome assembly workflow is missing from the manuscript as well as the amount of throughout in the sequencing information. A novel genome sequence of MRSA strain from India is presented. Some methods are missing (such as assembly) and need to be added to the manuscript with recommended additional analyses. Comparison to other available strain genomes is highly recommended for novelty.
Validity of the findings: Distinction needs to be clear between results of the study and existing literature where ever required. Additional analyses are required for more concrete conclusions.
Additional comments: In the study “Whole genome sequence analysis of multi drug resistant community associated methicillin resistant Staphylococcus aureus from food fish: Detection of clonal lineage ST 243 and its antimicrobial resistance and virulence genes”, GK Sivaraman et. al. report the whole genome sequence of Staphylococcus aureus ST 243, the first of its kind from India. The authors performed analysis of the virulent genes present in this strain and its virulence factors. The study is novel, however the manuscript would greatly benefit from changes in the text and a few additional analyses

Minor corrections :
There are some abbreviations which occur only once in the text, hence short forms seem unwarranted.

Abstract : The abstract though informative is very wordy, and the text is a little redundant in the upcoming sections. I would recommend shortening it - something as in the lines of “Methicillin Resistant Staphylococcus aureus (MRSA) sequence type 243 (ST 243) and spa type t021 is a widely disseminated CC30, prototype of ST-30, Community Associated-MRSA (CA-MRSA) (lukS- lukF +). It is a multi- drug resistant strain harbouring staphylococcal endotoxins, haemolysins, ureolysin, serine protease and antimicrobial resistance genes.”
Abstract : The NCBI accession is not require din the abstract.
Abstract : Please modify as “The unique ST28/ t021 CA- MRSA in fish is the first report from India, and in addition to antibiotic resistance is known to  co-harbor virulence genes, haemolysins, aureolysins and endotoxins.”
Abstract : Please modify as “The detailed analysis of the whole genome sequence of CA-MRSA strains7…” -. “Comprehensive comparative genomic analysis of CA-MRSA strain7 can help further further understand their diversity, genetic structure, diversity and high degree of virulence to aid in fisheries management”

Introduction:
52 : Please capitalize staphylococcal, since SCCmec is not used again abbreviation doesn’t seem necessary
56-59 :  Please rephrase - “Its virulence is controversial based on the presence or lack of gene deletions in PVL in different animal models and human but the exact role of PBL in the CA-MRSA epidemic is debatable”
59 : Please remove “Morevoer”
60 :  Please remove and before skin
61 :  are responsible for its virulence
64 : severity of CA-MRA virulence
65 : indicated -> indicate
71 : which can possess highly virulent genes and multi drug …
73 : have not yet been reported based on whole genome sequencing from fish in India
76 : “MRSA” is redundant; highlighting its
82 :  Please move the project number to Funding and Acknowledgements
105 : Figure S1 is missing in the attachments
90-93 :  AST section can be merged into the Isolation and Identification section
95-99 : Primer sequences could be provided
109 : Please mention the read lengths and fragment lengths or insert sizes of the libraries
119 : Please rephrase since VirulenceFinder2.0 is the software used, add a sentence on how it works
126 : 7,731,542 bases assembled into 120 contains
128 : 66022 bp
142-145 : The sentence is unclear
146-148 : Please clarify if the antibiotic resistance is based on computational analysis in this study and add required references
154-159 : Please rephrase that the your statement of resistance is based on gene similarity
171 : 100% coverage of reference sequence
172-173 : Please add needed citation showing this point mutation leads to higher resistance to fosfomycin and add the amino acid or codon change in the mutation
174 : Correlation needs a statistical test, please mention if it was performed and the value of significance
177-178 : In silicons analysis revealed high level of conservation in the AMR genes
181 : The ending of the sentence is unclear
189-190 : Please remove sentence after its genes since the lengths and accessions are mentioned in Table 2
210 : Please mention the strain (aureus 55/2053) with accession in brackets
246 : Please rephrase the sentence, the drug users is unclear
279 : co-harbors antibiotic resistance to
282 : Further comparative analysis of CA-MRSA strain7 genomes
284 : The environment isn’t being studied here, I would recommend removing such references throughout the text

Major corrections:
1. Rephrasing of text throughout is recommended and removal of redundancies
2. The genome assembly workflow is missing, additionally information on throughput (number of reads, read size)
3. The clarity of Figure 1 needs to be improved, font needs to be of uniform type and size
4. Though Gene ontology (GO) analysis was performed and provided as Figure 1, the results were barely discussed excepting in context of very broad classifications such as biological process, molecular function and cellular processes. I would recommend the authors to add more info on this for discussion, and if needed look for GOs in only the virulence function or antibiotic resistance genes. It would be interesting to see how these genes are represented in pathways (KEGG, BioCyc or MetaCyc provide this information)
4. Additional analysis comparing the ARG genes in other virulent S. aureus genomes would be more informative, especially conservation or sites of evolution in these sequences. Phylogenetic trees of these genes would be very useful to understand their evolution
5. If possible, it would be greatly beneficial to compare this genome with genomes of other available MRSA strains in order to understand the virulence (presence or absence of particular genes, or higher dissimilarity in a handful of genes) or propose it as a future 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: WHOLE GENOME SEQUENCE ANALYSIS OF MULTI DRUG RESISTANT COMMUNITY ASSOCIATED METHICILLIN RESISTANT STAPHYLOCOCCUS AUREUS FROM FOOD FISH: DETECTION OF CLONAL LINEAGE ST 28 AND ITS ANTIMICROBIAL RESISTANCE AND VIRULENCE GENES
Review round: 2
Reviewer: 1

Output:
Basic reporting: Please recheck the references and add details when necessary during citations. There seems to be inconsistency in the data reported in terms of reads & total bases and softwares used
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Minor corrections:
Abstract : “The present study reported the” -> In this study we report the
98: Supplementary Figure S1 is missing in the documents I have access to. 103 : “WGS data were analyzed as” -> “WGS data was analyzed as”
104 : Please mention what’s the process in at least one to two lines, add more info if there were changes to the protocol.
104:  Sivaraman et al., 2017 says that MaSuRCA was used, but in supplement table 1 it is SPAdesV.3.20.0
109 : “raw” misspelt
110: “will be used for” -> “is used for”
116: “7,731,542 bases” in abstract and supplementary table 1, it is written as 7.73M reads, please check it thoroughly and add the number of bases & reads to the methods section rather than in the results
118: “N50 value” -> “N50 estimate”
126 : The average read length is 100 and there are 7.73M reads, the number of bases is written as 731.27M - please mention if these are based after QC or pre-QC
157: remove “the”
158: Start new sentence for “further revealed…”, instead the new sentence can start with “ARGs showed”
166: Formatting error near references - I assume “2020)” instead of “2020;”
171: lower case for “World”
172-173: “The ARO genes of mepR, mgrA, and arlR matched with 100% identity of the region and also with 100% coverage of reference sequence” -> “The three ARO genes mepR, mgrA, and arlR were 100% identical to reference and aligned with 100% coverage to the reference sequence”
174: remove “for”
178 : “and is highly resistant to”
179 : “In silicons” -> “In silico”
184 : For the phylogenies, highlight your strain(s)
189: Please start a new sentence, and separate sentences for each GO test
202 : “revealed the”
213 : “pathogens”
227: “harbors”
229 : Accession numbers in brackets
274-275 : Please add a reference, this is a major claim
278 : “The WGS study” -> “Further comparative genomic analysis ”
278 : “give insight” -> “provide insights”
280: “and further helpful” -> “This would further help”
289-291: The names of the antibiotics is redundant, would advise shortening in the lines of “co-harbors multi-antibiotic resistance and contains virulence genes, …”
292: “The detailed analysis of the whole genome sequence could reveal further comparative analysis of CA-MRSA strain7 genomes, diversity, and its high virulent and staphylococcus endotoxins genes from fish” -> “Future studies involving the comparative genomic analysis of CA-MRSA strain7 will help further understand the diversity, virulence and endotoxicity of Staphylococcus in fish”
294-296 : It is unclear whether you are proposing it as a future study or you are already working on this. This sentence can be removed from the conclusion and moved to discussion
299 : It would be advised to specifically name the staff and scientists involved in the work, since the authors are from multiple labs
209-314 : Use initials for author names, please write as a single paragraph rather than bullet points

Major corrections:
184: Methods missing for phylogenetic analysis
Figure 1 : The function categories are incomplete in some labels on the y-axis, please make sure that this is complete, the GO ID can be removed if required
Table1 : Reformat the table such that the words are not separated by lines, would recommend decreasing font size
I would recommend including all supplementary figures into one single document, likewise for supplementary tables since they are not large excel sheets


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: PROKEVO: AN AUTOMATED, REPRODUCIBLE, AND SCALABLE FRAMEWORK FOR HIGH-THROUGHPUT BACTERIAL POPULATION GENOMICS ANALYSES
Review round: 1
Reviewer: 1

Output:
Basic reporting: The language used in this manuscript is clear, except that some sentences are too long to read. Also, all species names need to be italicised.

The authors failed to provide adequate background of the field. In particular, they ignored all the widely accepted core genome MLST schemes for Salmonella, Campylobacter and Staphylococcus, which are hosted in EnteroBase and pubmlst. They also falsely stated in line 560 that cgMLST is not scalable, ignoring the fact that both cgMLST platforms can easily handle genetic relationships of 1000's or 10,000's of genomes. The workaround proposed by the authors is, actually, not scalable, as they have to divide datasets into subsets of <2000 genomes each.
Experimental design: Overall speaking, ProkEvo is a well designed automated framework that helps people. The authors proven that, with the help of Pegasus and large clusters, ProkEvo can be used to handle 1,000's or 10,000's of genomes in a reasonable time.

I, however, have doubts on the reproducibility (or future-proof) of the framework. The authors assumed that all the programs implemented in ProkEov are eternal, and have not implemented (or described) a version system for these programs. Different versions of the same program can perform very differently. For example, the assemblies generated by SPAdes 3.6 is likely different from those by SPAdes 3.10. Without a careful, yet powerful version system in hand, scientists 5-year later will not be able to reproduce what they get today.

Secondly, ProkEvo is also not so "versatile". For example, ProkEvo by default accepts only assemblies with <=300 contigs and N50 >= 25Kb. However, if the same criteria are applied on Y. pestis or Shigella, many high quality genomes will fail because their chromosomes carry too many transposons. Authors will need to describe how these parameters can be adjusted. Do the users need to build up a new workflow with different settings, or can they be changed on the fly?

Finally, while most of the analyses in ProkEvo look like a cascading, automated workflow, the roary->fastbaps sub-pipeline requires some sort of human interactions. For example, the authors have manually selected subsets of <2000 genomes for this sub-pipeline. How does this interaction work in practice?
Validity of the findings: The authors described in detail about how the parallelization has improved the performance. However, it is still unclear to me that how long does every module in the workflow run in average and which program is the bottleneck. It will be great if the authors can list things out in a supplementary table.

It is also interesting that, the authors mentioned ABRicate as the program in ProkEov, but later referred the AMR results to "Resfinder". What exactly has been used?

The case studies are quite hard to follow, especially because the authors described populations using many different designations, but have not offered a figure to show the relationships between these population designations. There may be two trees in the middle of the rings in Figure 7? They, however, are not clear and large enough to show the branches. And why are there two figure legends for each of the ring?
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: PROKEVO: AN AUTOMATED, REPRODUCIBLE, AND SCALABLE FRAMEWORK FOR HIGH-THROUGHPUT BACTERIAL POPULATION GENOMICS ANALYSES
Review round: 1
Reviewer: 2

Output:
Basic reporting: In this manuscript, authors report the development of a framework for the large scale analysis of bacterial whole genome sequence data. The pipeline is split in two parts. The first part downloads raw sequence data from NCBI SRA and performs quality control of the data. The second part runs the actual population genomics analysis. The strength of the paper is that it appears to be designed work with very large datasets of over 20K genomes. However, there is limited innovation in the design. This seems to be another WGS pipeline that wraps several programs. They also dont compare this program with other recently published pipelines such as Bactopia. In fact these two looks very similar. Bactopia probably have more options such as the ability to run MASH.
Experimental design: From the methods section it is not clear how much flexibility this program has for input data. The default is to provide a list of SRA accession numbers as a file. Is there any option to load the data directly if the user has that in the local system? The  other main problem with this paper is extremely poor documentation. methods section gives three different links to the GitHub pages for the different versions of the program. However, clicking on that takes to the same page. Additionally, the installation seems to be quite complicated with the need of manually specifying paths. Unless these are fixed, nobody other than the authors may be able to use the pipeline.
Validity of the findings: I could not find the conda ad docker packages in the Github page. So there is noway to run the pipeline to see whether it works
Additional comments: If the authors  wish to see wide usage of their program, a much better documentation for the installation and use of the software needs to be provided. I spent quite a bit of time looking for the conda and docker versions mentioned in the paper. However, the links in the paper took me to the same page. The current documentation is poor and it was frustrating for me.


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: PROKEVO: AN AUTOMATED, REPRODUCIBLE, AND SCALABLE FRAMEWORK FOR HIGH-THROUGHPUT BACTERIAL POPULATION GENOMICS ANALYSES
Review round: 2
Reviewer: 1

Output:
Basic reporting: The revised manuscript describes ProkEvol, which is a useful automated framework for large scale genomic analysis. The authors have addressed most of the issues from previous review, except that they still have not italicized many of genus names. The authors listed https://peerj.com/about/author-instructions/#species-formatting, which describes: "The names of higher taxonomic levels (family, order, class, phylum or division, and kingdom) should be capitalized but not italicized. "

The genus names need to be italicized because they are not "higher taxonomic levels"
Experimental design: There is no remaining issue for the experimental design
Validity of the findings: The evaluations and examples are now well described.
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: EVALUATING THE CLEAVAGE EFFICACY OF CRISPR-CAS9 SGRNAS TARGETING INEFFECTIVE REGIONS OF ARABIDOPSIS THALIANA GENOME
Review round: 1
Reviewer: 1

Output:
Basic reporting: With the exception of minor changes to the text (listed in General Comments), Malik and colleagues have clearly and professionally written this manuscript and provided sufficient figures and data. The introduction and references are appropriate for this study.
Experimental design: Statistical analyses in this study are appropriate and accurately assess some of the features affecting sgRNA cleavage efficiency. This work is relevant as many other studies on sgRNA efficiency determinants focus on mammalian and/or coding regions. As interest in targeting ineffective regions grows, resources such as this manuscript for designing efficient gRNAs will be useful and interesting to the plant science community.
Validity of the findings: A significant concern I have with this study is the interpretation of the data used. A dataset is chosen from Wu et al 2018 in which several pairs of gRNAs are used to remove coding sequences of various genes in Arabidopsis thaliana. Malik and colleagues then remove gRNAs targeting coding regions and split the remaining gRNAs into two categories based on their ability to remove the coding sequence in Arabidopsis, as reported by Wu et al 2018. This binary functional/non-functional split is then used for the remainder of the analysis. Interpreting the data in this way results in two issues. First, Wu and colleagues report various gene deletion efficiencies for the gRNA pair. While it’s true that any deletion is an indication that the gRNA is functional, the relative efficiency of the gRNA pair could also be considered in the analysis. Second, and of major concern, is assigning gRNAs to the non-functional group assumes that both gRNAs must be ineffective at cleaving DNA. This is untrue as deletion events, as reported in Wu et al 2018, can only occur when both gRNAs are functional and have relatively the same cleavage efficiency. It’s possible that in many cases, for each gRNA pair in which a deletion is not recovered (and thus assigned to the non-functional gRNA group in this study), one of the two gRNAs is indeed functional. This results in an unknown number of gRNAs being incorrectly assigned to the non-functional category and impacting downstream statistical analysis used for the rest of the study.

Other considerations:

As this study examines sgRNA features affecting efficiency when specifically targeting ineffective regions, the study would be strengthened if analysis were also performed in parallel on sgRNAs targeting coding regions to compare differences in features for targeting each of these regions.

The study would be greatly strengthened if the authors also include experimental validation of their findings. The authors acknowledge this.
Additional comments: I found the research question relevant and an often-undervalued component of CRISPR/Cas9 gene editing. The interpretation of the initial dataset is incorrect, but I would encourage the authors to examine the same research question with an appropriate dataset. Data in which each gRNA cleavage efficiency is reported individually rather than as a pair should allow effective statistical analysis of the sgRNA features affecting cleavage efficiency.

Line 101- either remove “much” or replace with “very”

Line 139-140- wording is confusing, in particular “potentially” and “inactive”

Line 154- “sites” to “site”

Line 157- remove “and”, not the end of the list

Line 205- replace “either” with “whether”

Line 211- Include p-value of significance in main text

Line 213- replace “high” with “higher”, comparing two groups

Line 220- Include p-value of significance in main text

Line 252- replace “out” with “any”

Line 287- remove “Though”


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: EVALUATING THE CLEAVAGE EFFICACY OF CRISPR-CAS9 SGRNAS TARGETING INEFFECTIVE REGIONS OF ARABIDOPSIS THALIANA GENOME
Review round: 1
Reviewer: 2

Output:
Basic reporting: I believe. that there is data which contributes CRISPR community. I do have two issues with the MS:
1. Please remove too old references unless they are not pioneer,

2. English editing is a must and there is typos and should be checked.

3. The following recent review/research article would be good to add/discuss and compare with this research
- CRISPR/Cas9 in plants: at play in the genome and at work for crop improvement. Briefings in functional genomics 17 (5), 319-328	16	2018
-CRISPR/Cas9 genome editing in wheat. Functional & integrative genomics 18 (1), 31-41	86	2018

4. Figure 2 can be replaced with a different one. Is it possible show it differently?
Experimental design: This is good to show in Arabidopsis.
Validity of the findings: It is done.
Additional comments: I believe. that there is data which contributes CRISPR community. I do have two issues with the MS and listed above.


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

Input:
Title: EVALUATING THE CLEAVAGE EFFICACY OF CRISPR-CAS9 SGRNAS TARGETING INEFFECTIVE REGIONS OF ARABIDOPSIS THALIANA GENOME
Review round: 2
Reviewer: 1

Output:
Basic reporting: The text is now clear and well written.
Experimental design: My position has not changed. The statistical analysis is appropriate for the original question and work is relevant.
Validity of the findings: I agree with the authors rational for the study, that targeting ineffective regions may produce lower editing efficiencies.
I also agree with the author’s rebuttal that dual gRNAs may be recommended to enhance targeting efficiency, but this study is not examining pairs of gRNAs.
The dataset and conclusions would be appropriate if the statistical analysis considered sequence features of pairs of gRNAs. As is stated in their rebuttal letter, “for evaluating their features that collectively are responsible”. It is indeed possible that there are sequence features within a gRNA pair that impact editing efficiency but the authors are examining sequence features of individual gRNAs. The statistical analysis treats gRNAs as individuals once they are assigned to “functional” or “non-functional” groups.
Due to this fact, I still have issue with the dataset used in this study. In the authors rebuttal letter, they mention contacting the corresponding author of Wu et al., 2018, from which the data set was obtained, and received the response “they were functional but not always highly efficient” regarding gRNAs that did not produce deletions. This suggests some gRNAs that did not produce deletions were indeed highly efficient. Inappropriately assigning these individual gRNAs to the “nonfunctional” group would then affect the statistical analysis.
I believe it’s likely that many of the gRNAs are correctly assigned to functional and non-functional groups but even if a few “non-functional” gRNAs could produce high individual targeting efficiencies, the final conclusions of the statistical analysis could be compromised.
The authors have adequately addressed all of my other comments.
Additional comments: None


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

Input:
Title: EVALUATING THE CLEAVAGE EFFICACY OF CRISPR-CAS9 SGRNAS TARGETING INEFFECTIVE REGIONS OF ARABIDOPSIS THALIANA GENOME
Review round: 2
Reviewer: 2

Output:
Basic reporting: There revised version seems better.
Experimental design: Looks good
Validity of the findings: There would be Moree validation however this is good enough for the paper.
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: IDENTIFICATION OF KIF4A AS A PAN-CANCER DIAGNOSTIC AND PROGNOSTIC BIOMARKER VIA BIOINFORMATICS ANALYSIS AND VALIDATION IN OSTEOSARCOMA CELL LINES
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: Line 128: Please mention how the crystal violet dye was dissolved before measuring the absorbance
Validity of the findings: Line 196: Fig 5C does indicate statistical significance. Please run stats on multiple blots ran on different days and provide statistics. This also applies to Fig 6 (line 202)
Additional comments: Line (220-222): Please silence KIF4A in the non-cancer cell line and compare the effects with the current OS cell-lines.
Also, it is important to exogenously overexpress KIF4A of a constitutively active promoter in the KIF4A  silenced OS cell-lines to see if it rescues inhibition of cell viability, migration and reverses apoptosis. This will significantly improve the quality of the manuscript.