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: XYLOSE FERMENTATION TO ETHANOL BY NEW GALACTOMYCES GEOTRICHUM AND CANDIDA AKABANENSIS STRAINS
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment.
Validity of the findings: No comments.
Additional comments: This manuscript describes the isolation, identification and characterization of new xylose-fermenting yeast. The work is interesting and significant amount of work has been done. However, the current version is not publishable if the following minor comments are addressed?
Minor comments
•	Besides ethanol production, naturally occurring xylose-fermenting normally produced significant amount of xylitol. Authors should include xylitol production in characterization as majority xylose-utilizing yeast primarily produce xylitol.
•	Yp/p = 0.29 – 0.35 g/g were quite significant. It is also interesting to know the yield of xylitol and cell biomass.
•	Glucose and xylose are most abundant monomer sugars in lignocellulosic biomass hydrolysate. Could authors also characterize the strains on glucose and xylose co-utilization and co-fermentation?
•	Currently there are many high-performance engineered xylose-fermenting S. cerevisiae. They not only could ferment xylose, but also able to co-ferment glucose xylose and produce high-concentration ethanol. In addition, they are also tolerant to the inhibitors derived from biomass hydrolysate. Are there any advantages in using the naturally occurring xylose-fermenting yeast over the metabolically engineered S. cerevisiae? Although discovery of new naturally occurring xylose-fermenting yeast is interesting, we cannot ignore the fact that plenty of recombinant xylose-fermenting yeast were developed. They are probably more suitable for industrial application. Author should include this in introduction and results/discussion.
•	Line 50, $ sign at the beginning sentence should be deleted.
•	Line 273-274: The ethanol production reached 5.12 g L-1. This was very low. What if sugar concentration is high? Can the selected strains tolerant ethanol?
•	Line 312: should be “was described”.
•


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: XYLOSE FERMENTATION TO ETHANOL BY NEW GALACTOMYCES GEOTRICHUM AND CANDIDA AKABANENSIS STRAINS
Review round: 1
Reviewer: 2

Output:
Basic reporting: I found a clear report of the data and supportive of the findings the authors commented on the text.
Some literature citations are not well cited (please check general comments to the authors).
And the discussion requires a better job to connect relevant previous findings of the bioethanol production field.
Experimental design: The experimental design is correct, but it needs some description about the calculation of some parameters (please check general comments).
Validity of the findings: The results provided are novel and interesting for further research.
Additional comments: The isolation of new strains with the capacity of ethanol production from xylose fermentation is an important source of new genetic traits for improving the ethanol yields of the bioethanol industry.
	Here, Valinhas et al. isolated and identified three of two hundred and two yeast strains from plant samples, which are able to produce ethanol from xylose consumption. They characterized the ethanol productivity in synthetic media with xylose supporting previous results where they demonstrated that the production of ethanol from sunflower hydrolysate, using 2 of the 3 strains explored in this paper, is due to xylose and not only from other sugar consumption (Matos et al 2018 Quim. Nova).
	I found an interesting piece of work, which might be published in PeerJ if some of the issues, I reported below, are fixed.

Major comments:
1. I missed in the discussion a comparison of the new strains with industrial xylose fermenting strains, and how the new strains identified by the authors might replace the industrial ones, which I consider from the data presented unlikely, or they must explain how the new strains might improve the existing ones.
2. Authors consider UFVJM-R10 and UFVJM-R150 to show the same growth profile a wrong conclusion based on the shape of the presented curves in Figure 4. While R10 showed two curves probably due to a shift in its metabolism before finishing the fermentation, R150 has not that behavior. Please correct that conclusion.
Minor comments:
1. Authors provided supplementary data that I do not see a citation in the text. In addition, the supplementary data must be translated to English.
2. How are the maximum growth rate calculated? There is not citation to the methodology nor explanatory text.
3. Check references. In many places, for example Matos et al in lane 171 and in the discussion lane 339, there are incorrect number correspondence to the citation.
4. In table 4 there are conditions with identical response in all three strains. It must be removed based on the table description title.
Minor corrections
1. A dollar symbol is found in lane 51.
2. Typo in lane 58: change ethano to ethanol
3. Typo in lane 73: change Methodes to methods.
4. Typo in lane 171: change comfirmed to confirmed.
5. Typo in lane 180 and in other parts of the text: change Two hundred two to Two hundred and two.
6. Through the text the authors wrote that one of the strains was isolated from taro, but in Table 2 it says yam.
7. Lane 312: Galactomyces must be in italics.


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

Input:
Title: XYLOSE FERMENTATION TO ETHANOL BY NEW GALACTOMYCES GEOTRICHUM AND CANDIDA AKABANENSIS STRAINS
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Authors have covered all my comments and with a minor correction, I consider the revised version of “Xylose fermentation to ethanol by new Galactomyces geotrichum and Candida akabanensis strains” by Valinhas et al able to be published by PeerJ.
Minor corrections
1. Authors indicated, “was describe”, line 331, as corrected but it is still showing the typo.


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: COLLAGEN OVERLAYS CAN INHIBIT LEPTIN AND ADIPONECTIN SECRETION BUT NOT LIPID ACCUMULATION IN ADIPOCYTES
Review round: 1
Reviewer: 1

Output:
Basic reporting: The authors investigate the effect of collagen on the secretion of leptin and adiponectin in developed adipocytes. The paper is well written, succinct and objective. Structure appears appropriate. Raw data is included in supplemental file.

Intro provides context, with appropriately referenced literature. Authors should consider the paper by Q Li et al titled “The density of extracellular matrix proteins regulates inflammation and insulin signalling in adipocytes” (FEBS Letters 2010) that addresses a similar question, although in 2D, regarding collagen coating density on adiponectin secretion.

Figures are relevant, but some labels could be improved for clarity. For instance, what is the meaning of “Ins2d” “+I/D” and “+Ins” in Fig 1?

ELISA measurements are presented on a log-scale. This is likely done because in some cases, the differences do indeed span several orders of magnitude. However, the lower limit of detection is 62.5 pg/ml for the leptin (line 132; does the same limit apply for adiponectin?). Some of the plotted values may be near this cut-off (e.g., some values in Fig 2 B&C), and it’s difficult to tell on a log-scale how close these values are to the cut-off. As there is no reason to show values below the lower cut-off, the authors should consider plotting the data with the lower cut-off as the bottom.
Experimental design: This original primary research paper fits within the scope of the PeerJ journal. This research question is well defined and meaningful. The study aims to fill a gap in knowledge regarding how the 3D ECM environment affects adipocyte function. The gap exists because most studies of adipocytes use 2D surfaces.

Approximately 300 ul of collagen solution is added to the adipocytes (line 106). How thick of a layer does this produce (a rough estimate in my hands gives around 1.6 mm)? The motivation starting on line 76 gives the merits of a 3D environment, but the cells experience a rigid 2D surface surrounded by softer collagen. Did the authors visualise the cells to see if they migrated into the 3D collagen, or whether they remained attached to the plate?

Would the increased thickness of collagen, because it reduces advective mixing within the well, reduce oxygen or nutrient transport and thereby affect adipocyte function independent of collagen per se?

This reviewer has two questions regarding the ELISA measurements. First, the authors provide reasonable arguments against a deleterious effect of collagen on the ELISA measurements (lines 217-233). However, some of this concern (particularly regarding binding) could in principle be abated by performing a calibration curve in the presence of the collagen layer. Further, by the collagen occupying some physical volume (albeit small), there may be a steric hindrance effect that may influence the measured concentration. For these reasons, it would be useful to show a calibration curve both with and without the collagen. Second, what is the rationale for setting below detection limit ELISA values to 2 pg/ml, when presumably values up to 62.5 pg/ml would yield an undetectable result? Perhaps with a rank-sum analysis, this doesn’t make much difference in the statistics, but this seems arbitrary. Can this be clarified a bit more please?

Please state whether cells treated with PBS are deprived of glucose. Lines 102-103 do not mention glucose, which is typically absent from PBS, while the Hank’s buffer contains glucose (line 258; but at what concentration?). As the cells are cultured in high glucose medium (lines 91 and 94), glucose removal may therefore occur as a shock. Although I acknowledge that both PBS and PBS+Collagen groups would be similarly deprived, the absence of glucose seems peculiar. Lines 108-109 suggest that proper DMEM/FBS was returned at the “day indicated (Fig. 1)”. Perhaps this addresses the concern above, but if so, this is not entirely clear, in the text or in Figure 1.

It may have been useful to measure cell numbers. Cells in 3D environments tend not to divide as rapidly as those in 2D environments, and thus there could possibly have been a difference in cell numbers between buffer and collagen-treated samples, that could have contributed to the measured differences in TG or ELISA results.
Validity of the findings: Overall, the results appear reasonable sound subject to the questions or ambiguities raised above.

Is there an inconsistency between results shown in Figs 2B, 3B and 4B? In Fig 3B, cells cultured directly on either plate show strong leptin expression by 9 and 12 days. In Fig 2B, cells cultured directly on TC plates without collagen also show reasonable (but lower, relative to Fig 3B) leptin expression even with buffer disruption, while on the CellBind plate the leptin response is virtually eliminated. The explanation in the Discussion points to the disruption caused by buffer washes, but if this was the explanation, then why is response observed in Fig 2B and not Fig 4B?
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: COLLAGEN OVERLAYS CAN INHIBIT LEPTIN AND ADIPONECTIN SECRETION BUT NOT LIPID ACCUMULATION IN ADIPOCYTES
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript is clearly written using a professional English language style.
The Introduction is well referenced and the context providing a rationale for the work to be undertaken and the references used to support the work are appropriate.

The figures produced are relevant and appropriate but there is ambiguity in understanding and interpreting the results due to a poor experimental design. More detail in Figure 1 outlining the design would be helpful and provide a more accurate reflection of the work carried out.
Experimental design: This is an area of real interest and raises a cautionary note when comparing results between different groups who may be using different methods.  However, the research question is not well defined and the experimental design reflects this.  The manuscript seeks to determine the effect of collagen overlay on the expression of leptin and adiponectin and triglycerides in 3T3 L1 adipocytes.

My comments below reflect my areas of concern and consequent ambiguity of findings:

1)	Apparent poor use of controls to reflect glucose concentration:
a)	Line 91: Cells maintained in high glucose DMEM – this is equivalent to 25mM glucose.  The collagen was prepared in 1X HANKS – I have been unable to access the precise details from the product number provided but standard HANKS would be 5.5 mM.   There is nothing in the manuscript that alludes to the rationale for undertaking this with high glucose and given previous work demonstrating that glucose controls adipogenesis in 3T3 cells (Jackson RM, Griesel BA, Gurley JM, Szweda LI, Olson AL Glucose availability controls adipogenesis in mouse 3T3-L1 adipocytes via up-regulation of nicotinamide metabolism. J Biol Chem. 2017 Nov 10;292(45):18556-18564. doi: 10.1074/jbc.M117.791970. Epub 2017 Sep 15.) it would seem appropriate to carefully control the concentration of glucose of each replicate.  While this may have been carried out, the description does not reflect it.  Line 101-109 outlines the preparation of the collagen to be in either HANKS or PBS.  Again there is a clear difference in the glucose concentration between the PBS and the HANKS and yet there appears to be no mention of this at any point during the manuscript.
b)	The samples that did not contain any collagen presumably were either the corresponding PBS or HANKS, did not polymerize (line 107).  It is not clear if the control solutions were added to cells containing media but if so this would have acted to dilute all the constituents in the media.  In addition the authors state that the additives appropriate to differentiation stage was added (line 109) as illustrated in Fig 1. One presumes this is the +I/D, +lns and Lns2d. However, the details of the meaning of the symbols was not evident in the figure legend.  Greater clarity in the presentation of the data may clarify current concerns regarding controls and glucose concentration.
c)	Lack of acknowledgement of gaseous perfusion: going hand in hand with the glucose concentration difference and the cells that have a collagen overlay will have less access to oxygen.  This will affect glucose metabolism and resulting changes may at least in part be reflected by this difference.  A concentration of 3.3% is still likely to allow for diffusion of the oxygen but it will be delayed when compared to those in the absence of an overlay and I wonder if the authors have determined the oxygen levels in the overlay?  This should be acknowledged in the interpretation of any results observed.
2)	Lack of documented morphology of cells in different treatment groups: the authors refer to the rounded nature of the cells on the CellBind plate (lines 168-170) and wonder if any of the other treatments caused morphological changes and if so, this may be worthwhile to include in the results rather than to refer to them as unpublished observations.
Validity of the findings: The manuscript reports some interesting findings but with cautionary note based on previous comments.
Results show that the timing of the addition of the matrix will affect production of TG, leptin and adiponectin.  The author’s comment that this is likely to be due to different stage of differentiation is supported by the data and the general increase in levels over time also supports this.  The effect on adiponectin were reported to be more dependent on collagen preparation “line 245-248.  This is possible, but the contribution from differences in glucose concentration, oxygen content and differentiation status is difficult to interpret without further details and will be a consequence of comparing PBS with HANKS.  Furthermore, the measurement of some of the down-stream regulators of these proteins would consolidate these conclusions.
Additional comments: The manuscript highlights some important aspects of cell culture with the environment having a profound effect on TG, AD and Leptin. This will clearly affect the cells and caution should be applied to articles where the response of cells is varied and where a different culture model is used.  Greater clarity of the research design would improve ability to interpret this data for example images of cells at different stages, more detailed figure 1 including acronyms used and comparison with controls.  To combine this with an extended section for methods on a) cells and reagents and b) collagen matrix to include how the collagen was used and applied to the cells to facilitate understanding of context.


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: COLLAGEN OVERLAYS CAN INHIBIT LEPTIN AND ADIPONECTIN SECRETION BUT NOT LIPID ACCUMULATION IN ADIPOCYTES
Review round: 1
Reviewer: 3

Output:
Basic reporting: 1.	BASIC REPORTING
In general this is a well written manuscript, with an appropriate introduction and discussion. There is a good literature review.
Experimental design: 2.	EXPERIMENTAL DESIGN
The article is in scope and looks rigorous. Good detailed methods. The authors used three-way ANOVA in Figs 2 & 4 but two-way ANOVA was sufficient in Fig 3 for (what looks like) datasets with a similar complexity. More justification for the use of the different statistical methods would be desirable in the Methods.
Validity of the findings: 3.	VALIDITY OF THE FINDINGS
In general, the data is robust and the discussion sound
Additional comments: 4.   GENERAL 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: SELECTION OF QUALITY INDICATORS FOR NUTRITIONAL THERAPY IN PEDIATRICS: A CROSS-SECTIONAL STUDY CONDUCTED IN BRAZIL
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: As for the rigorous performance of the methodological course I have some observations.
I understand as a fragility of the study the participants' choice for vocational training, different places of work (private and public system) and not considering the disparate number among them.

Just as a suggestion, I believe that if it were a comparative cross-sectional study between different populations,  the results would be less questionable.

However, the choice of multiplicity of actors and sites does not invalidate the research.

"When you put different professions and different places of action you can say that we can be faced with multiple views and different angles", that is, I believe that in the continuity of this study we must stratify the qualification of the indicators according to place of performance and type of professional activity.
Validity of the findings: It`s OK
Only, the conclusion can be improved by identifying the need for further study..
Additional comments: Congratulations!
I believe that this study could be further developed so that it becomes a reference for other services to point out their best quality indicators in Pediatric Nutritional Therapy.
The experience in this area brings us multiple glances and we certainly need these to better serve those who so badly need this type of therapy performed effectively, efficiently and quality.


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: SELECTION OF QUALITY INDICATORS FOR NUTRITIONAL THERAPY IN PEDIATRICS: A CROSS-SECTIONAL STUDY CONDUCTED IN BRAZIL
Review round: 1
Reviewer: 2

Output:
Basic reporting: English: Recommend the english be revised by a professional native english scientific editor
Literature - There is a publication on the topic of quality control in clinical nutrition in pediatrics, from the ILSI force task on pediatric nutrition, from March 2017, available on line at http://ilsibrasil.org/publication/indicadores-de-qualidade-em-terapia-nutricional-pediatrica/  which should be quoted and discussed by the authors
Article structure - Pertinent to the area
Results and hypotheses - no comment
Experimental design: Aims and scope - no comments
Knowledge gap - At ILSI - Brasil , it is available a document written by the pediatric clinical nutrition task force, under the chairman of Dr Rubens Feferbaum, that suggest the pediatric IQTN
rigor and ethics - no comments
Methods - no comments
Validity of the findings: Replication - The authors may want this study to be replicated by all the pediatric nutrition support groups in Brazil, in order to validate their preliminary findings
Data - Suffers from the bias of being done only in Rio de Janeiro state, and by invitation, what is a selection bias of participants
Conclusions: The authors may want to rephrase the conclusions that would be applicable for Rio de Janeiro state, and invite validation
Additional comments: The authors should be commented by trying to stablish the top 10 quality control indicators for pediatric clinical nutrition
Some modifications are important to be included , in order to improve the understanding and scope of 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: SELECTION OF QUALITY INDICATORS FOR NUTRITIONAL THERAPY IN PEDIATRICS: A CROSS-SECTIONAL STUDY CONDUCTED IN BRAZIL
Review round: 1
Reviewer: 3

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: 283/5000
The following correction is suggested for the present study: In Table 1, if categories of Qualitative Indicators in Nutritional Therapy refer to the same ones mentioned in the introduction and published by Waitzberg DL, for better understanding they should be described in methods.


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: SELECTION OF QUALITY INDICATORS FOR NUTRITIONAL THERAPY IN PEDIATRICS: A CROSS-SECTIONAL STUDY CONDUCTED IN BRAZIL
Review round: 2
Reviewer: 1

Output:
Basic reporting: ok
Experimental design: ok
Validity of the findings: The authors have answered adequately all my questions, no further comments
Additional comments: The authors have answered adequately all my questions, no further comments


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

Input:
Title: IMPROVING PHYSICAL ACTIVITY, SEDENTARY BEHAVIOUR AND SLEEP IN COPD: PERSPECTIVES OF PEOPLE WITH COPD AND EXPERTS VIA A DELPHI APPROACH
Review round: 1
Reviewer: 1

Output:
Basic reporting: The quality of the writing in this manuscript was generally very good.  However, several important details of the study were not well described (explained in detail below), making it difficult to assess/understand the methodology.

The introduction gave good context for the study, and cited references were relevant to the topic. Data was provided as supplemental files.
Experimental design: This manuscript falls within the scope of the journal.  The research question is well defined and meaningful. As noted above, some lack of information/lack of clarity make it difficult to assess the methodology as currently written.

The authors have provided an ethic approval statement. This study appears to conform to the Declaration of Helsinki, although I could not find a copy of the consent form in the confidential supplemental materials.  I did not see any identifiable information in the files.  There were no unnecessary or unethical aspects to this study.
Validity of the findings: The above comments notwithstanding, the data appear robust. Conclusions are well stated and linked to the research question and current findings.
Additional comments: This paper is on an important and interesting topic.  The research question makes intuitive sense, and is well supported by the introduction.

However, some important aspects of the methodology were not clearly described, making it difficult to evaluate the quality of the data.

1.	It would be really helpful to clarify the Delphi process in more detail, for those of us unfamiliar with it.  For example, I am unclear on the differences between rounds 2 and 3. Were items removed? Or were participants simply alerted to items that had low agreement? Were participants prompted to reconsider/change their response if there was low agreement? Were any items removed prior to round 4?  If not, I'm unclear on the rationale for showing the list a third time.  A thorough description of this process would be extremely helpful to the reader.  Some of this information may be in Figure 1, but I found it very difficult to interpret without accompanying explanation in the text.
2.	In Round 4 were participants providing agreement with the themes, rather than the individual items?
3.	Line 177: The results jump from Round 1 to Round 4, with no discussion of Rounds 2 or 3. This may make sense within the Delphi process, but there should be some explanation of the reason for this for the reader.
4.	Lines 139-143.  I'm unclear on what is being described here.  It sounds as though the results from round 1 are being compared to the results of round 1, and also to a systematic review?  Were items from the review then added to the list as well?
5.	What was the rationale for including experts from non-COPD specific backgrounds?  I initially assumed that knowledge around COPD was what defined them as experts.  A bit of background on the value of including these non-COPD experts would be very helpful, as the reason for their inclusion isn't explained/justified in the current introduction.
6.	Related to the above, how was the number of COPD and non-COPD experts determined?  It seems that the non-COPD expert category would be extremely large given how they were defined (e.g. likely 100’s, if not 1000’s of individuals worldwide). How was this winnowed down to a manageable number?
7.	Line 100: More detail is needed on the types of websites and documents that were reviewed to identify experts.  Was there specific criteria?  Were search terms used? Or was this more of a convenience sample?
8.	In the results section, the authors do not describe the items/themes that were rated with high agreement.  Although this info is provided in tables, it is still worth summarizing in the text.
9.	Lines 212-229: This is all good information, but it comes before any in-depth description of the results of this study itself, which is absent from the Results section.  This information would be much more useful after a more thorough description of the specific findings of the Delphi process - e.g. what specific items/themes were important/unimportant, beyond MVPA.
10.	Lines 47-48: these are good points, but the references are presented in such a way that it is unclear which refer to the general population, and which to people with COPD
11.	Line 64: this sentence is worded a bit awkwardly. Are you saying that people with COPD view fatigue as having a bigger impact on QOL, and feel that daily activities are not affected by the disease?  I suggest rewording, or perhaps breaking into 2 sentences, to make the meaning more clear.
12.	Lines 73-74: The phrase " two countries where differences in factors to influence behaviours exist " is unclear to me.  Do you mean the social and built environment differ between the two countries?
13.	Did the authors consider looking at the views of different GOLD stages?  It seems like stage I and Stage 4 could have very different views on what is important.
14.	Were the questionnaires/results translated into Dutch by the authors?  Did they have anyone back-translate the questionnaires to confirm they were consistent in both languages?
15.	Line 200: was there disproportionate focus on increasing PA, or disproportionate agreement?  Since the results aren't summarized in the text, it’s hard to know exactly what is being referred to.


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: IMPROVING PHYSICAL ACTIVITY, SEDENTARY BEHAVIOUR AND SLEEP IN COPD: PERSPECTIVES OF PEOPLE WITH COPD AND EXPERTS VIA A DELPHI APPROACH
Review round: 1
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: - In the methodology (line 97), the authors state that patients were included if they had at least mild COPD as confirmed by spirometry. Do the authors have these spirometry data available? Were both COPD groups matched? (e.g. in terms of FEV1) Do the patients cover a wide spectrum of COPD patients in terms of obstruction severity? Is there information available on the number of acute exacerbations patients experienced in the past 12 months? These might have implication on the generalization of findings and is not reported in the table.

- How did the authors assess the number of days patients were active in the previous week? Was this evaluated with a questionnaire or objective data monitoring. Please specify in the methodology section. In case the authors used a questionnaire to analyze the level of physical activity, this should be mentioned in the 'limitations'-section.

- Line 147: After round 2, low group agreement was defined as [IQR] ≥2. On Line 151, low group agreement in round 4 was defined as IQR>2. Is there any rationale for this difference in cut-off value?
Validity of the findings: No comment
Additional comments: In this manuscript, Lewthwaite and colleagues identified a wide range of factors that are judged as important to improve behaviour (physical activity, sedentary behaviour and sleep) by patients with COPD (both from Australia and the Netherlands), COPD-experts and non-COPD specific experts via a Delphi approach. The investigators conducted a very well established and properly reported qualitative part. However, there is missing information to characterize both COPD groups more in detail (in terms of severity of airflow obstruction, past history of acute exacerbations, objective physical activity data,…) Addition of a section ‘clinical importance’ might be of great (additional) value to the manuscript in which the authors might summarize which specific themes they would include in the implementation of future interventions and how these themes should be tailored for each patient group.

Minor comments:

Table 1:
- Did the authors investigate any significant differences within each patient and expert groups? This was not reported but can play an important role in explaining differences in themes within each group.

Fig 2:
The percentages of the COPD-E and the Non-COPD-E groups are not clear. In the COPD-E group, 76 experts were invited of which 17 were include (22%). All those who were included participated to round 1A (which is still indicated as 22%). In round 1B, only 14 experts participated. However, this is indicated as 82%. This is not in line with the use of percentages in both patients group: Eighty patients from Australia were invited from which 29 were included (36%). Of these patients, 26 participated to round 1A (90%). If the same strategy would be applied as in the experts groups, the % would be 32.5 for round 1A in the Australian patients.

- Line 211: ‘Increasing MVPA is the same as reducing SB.’ The authors might consider to add the reference of Biswas et al., Ann Intern Med 2015. In their manuscript, they provide further evidence for the independent effect of sedentary time on health outcomes (independent from physical activity).

- Line 261: The authors refer to several behavioral change techniques. Can the authors further elaborate on the possible overlap of their findings with behavioral change techniques proposed in literature?


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: IMPROVING PHYSICAL ACTIVITY, SEDENTARY BEHAVIOUR AND SLEEP IN COPD: PERSPECTIVES OF PEOPLE WITH COPD AND EXPERTS VIA A DELPHI APPROACH
Review round: 1
Reviewer: 3

Output:
Basic reporting: No comment
Experimental design: no Comment
Validity of the findings: no comment
Additional comments: This Delphi study is researching an important clinical area identifying the perspective of individuals with COPD and experts on what is considered important to improve behaviour and identifying areas of difference between these groups. Being an international collaborative study strengthens the findings and the overall, the manuscript is very well written, clear in the steps of the Delphi process and draws sounds conclusions based on the results found.  Within the discussion, each of the key points have been well discussed in relation to existing literature, with the inclusion of factors unique to each country involved in this study, highlighting a global difference in strategies which may be applicable in future studies. The authors have highlighted the main strengths and limitations.

A few minor points for clarity are detailed below.
Abstract
Unclear what less commonly before point 3 means? Can the authors clarify this point?
Methods: a clear outline of the methods has been provided. Each step in the Delphi process is clearly articulated, with appropriate process of collation of themes by multiple pairs of authors.
Minor point
Page 11, line 122 – participant’s should read participants’
Line 132 – prior should read priori


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: IMPROVING PHYSICAL ACTIVITY, SEDENTARY BEHAVIOUR AND SLEEP IN COPD: PERSPECTIVES OF PEOPLE WITH COPD AND EXPERTS VIA A DELPHI APPROACH
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No Comment
Validity of the findings: No Comment
Additional comments: All of my previous comments were thoroughly addressed.


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

Input:
Title: IMPROVING PHYSICAL ACTIVITY, SEDENTARY BEHAVIOUR AND SLEEP IN COPD: PERSPECTIVES OF PEOPLE WITH COPD AND EXPERTS VIA A DELPHI APPROACH
Review round: 2
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: no comment
Validity of the findings: The authors have addressed all concerns
Additional comments: The authors have addressed all concerns well.


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: PALMITIC ACID INDUCES NEUROTOXICITY AND GLIATOXICITY IN SH-SY5Y HUMAN NEUROBLASTOMA AND T98G HUMAN GLIOBLASTOMA CELLS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The structure is acceptable, but the titles of some paragraphs and figures demand re-considering and re-naming. The manuscript contains too many unreasonable abbreviations. Cytometrical data (FCS files) are not presented in Raw data.
Experimental design: Cytometrical analysis needs elucidation: additional information on minimal quantity of analysed events in samples and the order of analysis of obtained cytometrical data.
Validity of the findings: Microscopic photos and cytometrical figures demand improvement.
Additional comments: The manuscript is interesting and necessary for the biomedical science in general, but needs major revision for improvement. The results obtained are believable, important and well discussed, but need some corrections in their presenting. Please, find my comments in attached PDF file.


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

Input:
Title: PALMITIC ACID INDUCES NEUROTOXICITY AND GLIATOXICITY IN SH-SY5Y HUMAN NEUROBLASTOMA AND T98G HUMAN GLIOBLASTOMA CELLS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript contains many typographical, punctuation and other errors. Also English is not clear, unambiguous and professional throughout the manuscript. Please be more conscientious. Some examples of those errors:

1.	When authors use abbreviation for the first time, they must give the full term (in line 27 for CNS, in line 85 for BMI).
2.	Scale bars are absent in Figure 2. Moreover, it seems that pictures of T98G untreated and 300 μM PA treated cells were made with using different magnification.
3.	Full stop is absent in line 474 in the end of sentence.
4.	In Figure 1 is not clear which one of the diagrams describe 24 h and 48 h of treatment.
5.	In some points authors give unnecessary information (in line 231it is enough to write just final concentration).
6.	In Figure 4 E what does percentage of cells means? It is not clear from diagram.
Experimental design: Research question of this manuscript is well defined and relevant, but methods described with sufficient detail not in all cases. Also there are some concerns on experimental design.

Why authors chose treatment for 48 h for apoptosis detection? To my knowledge is too long. Besides, did they do all necessary controls for these experiments?

Also it is not clear why authors chose 300 μM treatment with 300 μM PA, OA or LA for 24 for ORO staining.

Authors did not give concentration of paraformaldehyde (Line 195) and model of camera. Please check is duration of fixation that you give is real (it seems too long).

Authors did not include Western blot for γ-synuclein protein expression after establishment of stable cells in the manuscript, but I expect to see it.
Validity of the findings: Data is mostly robust and statistically sound, but appropriate controls for apoptosis detection are absent.

Authors did not discussed in details why PA, OA and LA can increase viability of cells. What can be molecular mechanisms of this?
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: PROANTHOCYANIDINS IN SEED COAT TEGMEN AND ENDOSPERMIC CAP INHIBIT SEED GERMINATION IN SAPIUM SEBIFERUM
Review round: 1
Reviewer: 1

Output:
Basic reporting: English is easy to understand but some grammatical errors are still spread all through the document and should be corrected.

The overall structure is correct, but the content within each section does not always match the sections (for instance, results stated in the introduction, methods detailed in the results section)
Experimental design: The design is not presented clearly and we understand parts of it, by mixing text of the material and method sections, text of the results section and elements in the figure and legends. A clear synthetic presentation is required.

No indications is provided regarding statistical analyses, although some statistical tests results are indicated on the figures.
Validity of the findings: It is difficult to judge as some elements are missing on the design description.
Additional comments: The manuscript needs thorough reworking to improve all sections of the documents, developing the state of the art, better presenting the objectives, completing the material and methods, centering the result section on the results by moving additional methodological details to the previous section,  and focusing the discussion on the interpretation by synthesizing the statement of the conclusions rather than repeating detailed results.

Therefore, I recommend to reject the manuscript for publication, with possibility to submit again following completion of the modifications. This seems feasible, as some of the necessary elements can be found dispersed in the text at the wrong place or in the figure legends. It seems that no additional experimental work nor analyses should be done, but a huge effort to correctly and fully explain the design, the methods, the results.


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: PROANTHOCYANIDINS IN SEED COAT TEGMEN AND ENDOSPERMIC CAP INHIBIT SEED GERMINATION IN SAPIUM SEBIFERUM
Review round: 1
Reviewer: 2

Output:
Basic reporting: The standard of English writing in the paper is clear enough to be understandable, but needs editing and correcting in numerous places to improve clarity, especially for non-English readers.  I have not given specific examples because there are so many.

The Introduction and background show the context and background, and give the aim(s) of the investigation.  It would help the reader if some more detail of the internal structure of the seed was given, particularly how the endosperm is related to the embryo.  The two layers of the seed coat (outer tegmen, inner testa) are described, but the position and structure of the endosperm relative to the embryo is not.  From the photos supplied, it appears that the endosperm surrounds the embryo.  If that is the case, the endosperm could potentially provide a mechanical constraint on the embryo, preventing germination, as is the case in other species.  In this species, the results of the investigations suggest that this is not the case.  But more detail about the endosperm is required here.

The structure basically conforms to PeerJ standards.  There is a lot of repetition of material between sections, so that parts of results are given in the Introduction, parts of Methods are repeated in Results, and details of Results are repeated in Discussion.  Examples of this are given in the Comments to Authors below.

Figures are clear and relevant.  Figs 5  - 7 would benefit by the sub-figures being given letters (A – D), as in the preceding Figs, to assist referring to them in the text.  That way, the long technical names for the genes (eg SsDOG1) can be left in the figures, and omitted from the text in Results.  It would make the text in Results easier to read.
Experimental design: The authors have performed a carefully designed set of experiments to identify the cause of dormancy in Sapium sebiferum, and are to be commended for doing so.
They have framed a good research question which fills a gap in knowledge.
Methods are described in sufficient detail.  The experiments have been well- conducted with replication and proceed to test explicit mechanisms of seed coat dormancy know from the literature.
Validity of the findings: Their experiments are analysed correctly and interpreted correctly.  Their results support the hypothesis of germination inhibitors being present in the seed coat, and endospermic cap.  There may be an element of mechanical constraint by the testa (see comments in Discussion).  The Discussion as it stands is lengthy and would benefit from  re-writing to remove repetition of details of results and present their case more clearly (see comments below).
Additional comments: Introduction:
Lines 57 – 58: the two layers of the seed coat are described (outer testa, inner tegment).  Describe how the endosperm is related to the embryo (surrounds the embryo, lies alongside the embryo, or whatever is appropriate here)
Lines 63 – 73: this text is outline results, which should appear in Results.  Delete from here.
Lines 61 – 62.  Text about PA’s and nitrates: convert into a statement like ‘It is unclear whether PA’s respond to nitrate’

Methods
Line 104: ‘seeds were carefully uncoated with a scissor’.  Explain that this procedure (better described as ‘decoated’ or ‘seed coat removal by dissection’) left the endosperm + embryo as a unit, hereafter referred to as ‘decoated seeds’, or ‘endosperm + embryo’.  This makes it clear what the dissection of the seed coat left exposed.  Where experiments were carried out with decoated seeds, make this clear in Methods.

Results:
Lines 139 – 143: this repeats the methods of the experiment, which were given in Methods.  Do not repeat here.  Start with ‘We found that …’
Line 150: while it is true that water uptake increased with scarification, it is very important to state there that the control seeds also took up water without any scarification (18% in Fig. 1B).  This shows the seed coat is permeable to water.
Line 155: as the germination percentages are shown in Fig. 1C, there is no need to repeat them all in the text.  It is worth stating here that germination of intact seeds was 2%, and ranged 40 – 65% in scarified seeds.
Line 164: again, gives the detail of the methods, and so is unnecessary repetition.  Start with ‘We found that decoated seeds showed 85% germination …’
Lines 166 – 169: again, repetition of Methods.  Start with ‘We found that seed coat extract (SCE) …’
Repetition of Methods is continued throughout Results.   There is no need for it; the next important Result in this section is that SCE inhibited germination of decoated seeds.
Line 193 onwards gene expression section: this contains a lot of repetition of Methods, which needs deleting/moving to Methods.  In reporting the results, the names of the genes are very lengthy and confusing.  It would be better to report results such as the one in lines 199 – 202 as ‘The expression of a gene that positively regulates germination (SsDOG1) was significantly higher in SCE and PA treatments than in the control (Fig. 5)’.   Describe the genes in terms of what they regulate, and put the long names in brackets.  Alternatively, give the subfigures in Fig. 5 etc the letters A, B, C etc, and leave the long name on the Figures only (so refer to ‘Fig. 5A’ in the example above.
Line 194: ‘we found the crosstalk effect of seed coat with dormancy and GA,  …’.  Re-phrase as ‘We examined the relationship between seed coat dormancy and the gene expression of GA, ….’.  ‘crosstalk effect’ is used also in line 239, and this should be replaced  by ‘relationship between’ or similar expression.

Discussion

The Discussion is fairly long as it stands, and would be easier to follow if reduced.  It could be shortened by using the framework suggested below.  This sets up a logical framework based on known mechanisms of seed coat dormancy as set out in Baskin and Baskin (2014), that the results from S. sebiferum can be easily related to.

The Results for S. sebiferum show intact seeds are dormant,  imbibe water, and increase in germination if the embryo is removed from covering structures such as the seed coat and/or endosperm.  Seeds showing  this combination of features fit into the non-deep physiological dormancy category of Baskin and Baskin (2014).  The causes of dormancy in these seeds can lie in the covering structures, the embryo, or in a combination of both (Baskin and Baskin 2014).

If the covering structures control dormancy, there are a number of known mechanisms by which they do so: the covering structures are:
-	impermeable to water
-	mechanically constrain the embryo from germinating
-	contain chemical inhibitors of germination
-	prevent the exit of chemical inhibitors of germination present in the embryo
-	restrict oxygen diffusion to the embryo

The results the authors have can eliminate some of these mechanisms:

Water permeability: the intact seeds take up water and so the seed coat is permeable to water; if the seed coat was impermeable, water uptake of intact seeds in Fig. 1B would be effectively zero.

Mechanical constraint: if the seed coat is removed, germination does increase substantially, as would be predicted from this model.  Scarification of the seed coat with sulphuric acid improved germination.  The acid both weakened the seed coat, and degraded the PCA’s which the seed coat contains, and these inhibitors restrict germination of decoated seeds (see below).  So the seed coat, particularly the testa, may be exerting mechanical constraint on germination. The authors cannot eliminate this possibility with results they have, but, this could be tested by trying a non-chemical form of scarification (if possible), to see whether physically disrupting the testa resulted in improved germination.  If it did, the seed coat would appear to be exerting mechanical constraint.  If physical scarification does not improve germination, then the germination inhibitors in the seed coat model are restricting germination.

Covering structures contain germination inhibitors: the authors have built a strong case that there are germination inhibitors in the seed coat, that these inhibitors restrict germination of decoated seeds, that the endospermic cap also contains inhibitors, and that while removal of the seed coat results in 85% germination of the decoated seeds, removal of the endospermic cap results in 100% germination (ie, all the remaining seeds).  Moreover, the restricting effects of the seed coat extract and PCA can be reversed by application of GA3, H2O2 etc to decoated seeds.  And the gene expression data support the chemical inhibitor model.  It is also possible that the endospermic cap is mechanically restraining the extra 15% of seeds from germinating, but the evidence presented of changes in the colour (PCA content) of the endospermic cap support the chemical inhibition model.

Oxygen diffusion model: this is hard to test.  But the evidence above for the chemical inhibitor model makes limitation of oxygen diffusion by the covering structures unlikely

Prevention of exit of inhibitors from the embryo: again, unlikely given the evidence for the presence of inhibitors in the seed coat and endospermic cap.

If the above structure is used for Discussion, it will make it more coherent and easier for the reader to see how the evidence from the investigation matches against known dormancy mechanisms.


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: PROANTHOCYANIDINS IN SEED COAT TEGMEN AND ENDOSPERMIC CAP INHIBIT SEED GERMINATION IN SAPIUM SEBIFERUM
Review round: 1
Reviewer: 3

Output:
Basic reporting: Seed dormancy in plants is one of the bright examples of contradictions between natural selection and adaptation of plants to unfavorable environmental conditions on the one hand and artificial selection and breeding purposes tasks on the other hand. One of the ways to manipulate with seed dormancy is based on knowledge of genes underlying this trait. The results of biochemical, physiological and genetic investigations of seed dormancy in Chinese tallow, described in the considered article, are of importance.
Experimental design: The article represents original primary research within aims and scope of the journal. Methods described with sufficient detail & information to replicate. I suggest to add description of methods of statistical analysis (in the current version these methods are mentioned in figures legends only).
Validity of the findings: Data is robust, statistically sound, & controlled.

Conclusions are well stated,
Additional comments: Seed dormancy in plants is one of the bright examples of contradictions between natural selection and adaptation of plants to unfavorable environmental conditions on the one hand and artificial selection and breeding purposes tasks on the other hand. One of the ways to manipulate with seed dormancy is based on knowledge of genes underlying this trait. The results of biochemical, physiological and genetic investigations of seed dormancy in Chinese tallow, described in the considered article, are of importance.
The article can be published after some revision.

1. Article title: I recommend to change to “Proanthocyanidins in seed coat’s tegmen and endospermic cap inhibit seed germination in Sapium sebiferum”.
Chinese tallow is first of all the ornamental plant and a source of oil used for some industrial purposes. Yes, it is considered as a promising energy plant, but it is not sufficient to call it “the bioenergy plant” in the article title. It is the overstatement.

2.  Introduction.
Lines 40-42. Not some, but many plants contain proanthocyanidins in seed coats, and the relationship between PAs in seed coats and seed dormancy was first discovered in wheat. In the model plant Arabidopsis, mentioned in lines 40-42, this relationship was described much later. I think it is important to say in the beginning of the Introduction section that the role of PAs for seed dormancy was first discovered in wheat. As early as in 1914, Nilsson-Ehle showed that red seed coat color in wheat is associated with extended dormancy period in comparison to that in white-grained wheat. In 1958, Miyamoto&Everson showed that red pigment of seed coat is a substance derived from catechins (PAs).

3. Materials and methods:
Line 135. Here methods of statistical analysis should be described.

4. Conclusions
Some practical conclusion are required. How the knowledge obtained in the current study can be used in breeding.

5. Supplementary materials
I did not find description of supplementary materials, supplementary figures legends, headings of tables presented in excel format, etc.

6. English
The English language should be improved (over the whole text). Some examples where the language could be improved include Line 18 (“highly” should be removed or replaced), Lines 96-98 (Seed coat proanthocyanidins contents were analyzed <here preposition is needed> conventional HCl–vanillin assay (Herald et al. 2014) and pure proanthocyanidins (UV≥95%) was <”was” should be replaced by “were”> used as a reference.), Line 93 (“was” should be replaced by “were”), Lines 372-374 – the current phrasing (and using “…which may …” 2 times in 1 sentence) makes comprehension difficult.

7. Additional changes:
S. Sebiferum should be changed to S. sebiferum (over the whole 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: PROANTHOCYANIDINS IN SEED COAT TEGMEN AND ENDOSPERMIC CAP INHIBIT SEED GERMINATION IN SAPIUM SEBIFERUM
Review round: 2
Reviewer: 1

Output:
Basic reporting: Text is greatly improved; specific comments (most minor) are given below.  Repetition of text present in the earlier version has largely been eliminated.  The structure of the Introduction and Discussion is improved greatly.
Experimental design: Sound: as per my previous review.
Validity of the findings: The experiments are analysed correctly and interpreted correctly.  Their results support the hypothesis of germination inhibitors being present in the seed coat, and endospermic cap.  There may be an element of mechanical constraint by the testa.
Additional comments: Specific comments to authors

Lines 37 – 40: this currently reads as though the hard (water impermeable) seed coat plays a role in all of the other seed coat dormancy mechanisms.  It would be clearer to re-word these lines as eg. ‘The seed coat can play a role in regulating dormancy.  Know mechanisms by which the seed coat regulates dormancy include the prevention of water uptake by an impermeable seed coat, or inhibiting gas exchange (McGill citation), or by mechanically constraining the embryo, or by containing germination inhibitors (B & B citation’.

Line 48: italicise Rubus

Line 52: PA’s located in the seed coat can  …’ (replace ‘distribution’ with ‘located’)

Line 72: insert ‘outer’ before ‘testa’ and insert ‘inner’ before ‘tegmen’.  This reminds readers of the relative positions of the two seed coat layers.

Line 73: Better re-worded as ‘The tegmen encloses the endosperm, which in turn encloses the embryo’.

Line 73. ‘Tallow tree seeds had good water permeability, but the seed coat structure at the side of the radicle …’ replace with ‘Tallow tree seeds readily imbibed water but the seed coat at the site of the radicle appeared to ….’

Line 79: insert ‘germination’ in front of ‘inhibitors’

Line 79: Re-word ‘Which layer of the seed coat …’ as ‘It is currently unknown which layer of the seed coat …’

Line 83: re-word ‘Can the application of an exogenous ABA …’ as ‘We tested whether exogenous application of an ABA …’

Line 87: replace ‘unveil’ with ‘demonstrate’

Line 103: replace ‘was purchased’ with ‘were purchased’

Lines 121 – 2: delete sentence ‘All things were weighed by  …’ (unnecessary detail, can be omitted)

Line 145: replace ‘scar’ with ‘cut’

Line 148: replace ‘replicons’ with ‘replicates’

Lines 191 – 5: the text ‘S. seberiferum has a hard seed coat  …  and seedling growth’ is all repetition of information given previously in the Introduction and Methods, and can be deleted.  The heading for this section indicates what experiment is being talked about.  Start text with ‘We found that sulphuric acid  …’

Line 201 - 2:  it is very important here to state that the untreated seeds themselves showed substantial water uptake, and the current text does not draw this important finding out.  Re-word as eg ‘Water uptake of untreated control seeds was 16 – 17% (authors to give the %) over X hours; seeds treated with sulphuric acid for longer periods of time showed increasing water uptake to a maximum of 20% (authors check %) after 60 minutes of acid treatment…’ Then give details of significant differences as currently done.

Line 206: insert ‘from’ after ‘ranged’ (ranged from 40 – 60% …)

Line 222: insert ‘a’ in front of ‘dark brownish colour  …’

Line 234: change ‘We examine …’ to ‘We examined …’

Line 292: replace ‘played the main role in ‘ with ‘were the major controllers of dormancy in  …’

Line 296: the listing of possible mechanisms of seed coat dormancy need to have ‘can’ inserted in front of each one: ‘..they can be impermeable to water, they can mechanically constrain … can contain chemical inhibitors  ….can prevent the exit of  …’

Line 305: replace ‘this model’ with ‘the model’

Line 307: insert ‘which degrades the germination inhibitors ..’ after ‘scarification with sulphuric acid’, to read ‘but scarification with sulphuric acid which degrades the germination inhibitors greatly improves germination’.

Lines 307- 8: the conclusion here about the relative strengths of mechanical constraint vs germination inhibitors could be re-worded as ‘These results suggest that any mechanical constraint by the seed coat on germination is minor, compared to the major role played by germination inhibitors in the seed coat’.


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: PROANTHOCYANIDINS IN SEED COAT TEGMEN AND ENDOSPERMIC CAP INHIBIT SEED GERMINATION IN SAPIUM SEBIFERUM
Review round: 2
Reviewer: 2

Output:
Basic reporting: Text is thoroughly revised
Experimental design: The statistical methods are described now
Validity of the findings: Data is robust, statistically sound, & controlled.
Additional comments: The authors have made thoroughly the necessary changes. Article in its current form is acceptable for publiction.


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: COMPARISON OF PATHOLOGICAL CHARACTERISTICS BETWEEN SELF-DETECTED AND SCREEN-DETECTED INVASIVE BREAST CANCERS IN CHINESE WOMEN: A RETROSPECTIVE STUDY
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1.	This study use the China breast screen program as a natural experiment to explore the prognostic characteristics pre and post, and between those self-detected and screen-detected. In the introduction section (second paragraph), please clarify whether there are similar studies done or not and if not, how this study fill in the literature gap.
2.	The English language is still a little awkward and confusing in a few places. One example is that in the result section (Line 147), the word “comparable” means similar/equivalent whereas the P-value is actually <0.05. The sentence in the method section (line 93-94) also looks awkward.
3.	The abstract “Methods” section should also contain description of your statistical analysis approach. The P-value format should also be corrected (i.e. 0.000 should be <0.001).
Experimental design: 1.	This retrospective study used patient medical records from three different periods, which derived from different sources (paper documentation and electronic documentation). Please clarify whether there is quality control process of abstracting the data. In addition, were the missing data due to the use of electronic documentation? The records from the latter two periods (1995-2000, and 2015) showed higher % of missing data which happened to be the two periods that used electronic documentation. Or was there a systematic reason that caused the missing data?
2.	There are a few issues with the author’s statistical analysis approaches. First, raw cases of screen-detected patients and self-detected patients should not be compared directly. The increases of screen-detected cases might be due to more women being screened in 2015, and fewer women with symptoms visited the hospital in 2015. The other similar studies cited in the manuscript (Ref [13], [22], etc.) did not compare the raw cases. I suggest taking out comparison from the second paragraph in the result section (line 130-139), as well as in the discussion section (line 156-167).
3.	Multiple Imputations: this approach is based on multivariate regression. I am concerned about the validity of the regression because there are very few variables provided in the study. Please clarify what variables you put into the model.
4.	Unadjusted comparison of prognostic factors between self-detected and screen-detected is not sufficient. The other Asian studies cited in the manuscript all showed significantly different patient characteristics between the self-detected group and screen-detected group, and some of these characteristics may also affect prognostic factors. Although very few patient characteristics were available in this study, the author could still try to adjust for age or output age-standardized distribution of prognostic factors. As shown in Table 2, the age range is big and considerably bigger in self-detected group (compared to screen-detected group) especially in earlier period.
Validity of the findings: 1.	As mentioned above, the raw cases of self-detected and screen-detected should not be compared directly and therefore the discussion (line 156-157) is not valid.
2.	This study used data from Tianjing Medical University Cancer Institute and Hospital, which is only a small subset of the national data. Given the huge regional variation of cancer incident rate in China, the screen-detected from this study may not be representative of China. Therefore, the conclusion (line 210) that “this study indicates that the proportion of screen-detected patients in China remains lower than that in other Asian developed countries and regions” is not valid. The results from this study cannot be generalized to the whole country.
Additional comments: 1.	Please revise or take out the comparison of raw number between self-detected and screen-detected cases.
2.	I suggest adding in and controlling for some patient characteristics if you have them available.
3.	Please review the paper and make sure the format of the numbers (i.e. P-value) is correct.
4.	Please carefully refine the conclusion generated from your results.


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: COMPARISON OF PATHOLOGICAL CHARACTERISTICS BETWEEN SELF-DETECTED AND SCREEN-DETECTED INVASIVE BREAST CANCERS IN CHINESE WOMEN: A RETROSPECTIVE STUDY
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1. In this manuscript mentioned there are studies worldwide have indicated that screen-detected patients have more favorable survival outcomes compared with patients that present with self-discovered breast cancer symptoms. Western and some Asian developed countries include Korean, Japan, Singapore. Please emphasize that this study use long term and huge database. Because used total sample were 3,014 female patients with invasive breast cancer. Describe and emphasize these database represent a trend of Chinese breast cancer.
2. It is probably more appropriate use the term of “characteristics of the cancers detected by screening and those by self-exam.” The term “prognostic factors” is more appropriate for comparing survivals while the factors too are prognostic factors.
3. Line 33, “changed” seems to be less desired than “differed.” There are many more grammatical errors and writing style issues.
4. Figure 2, the lines are difficult to differentiate between the groups. Please change them to color-lines or other format.
Experimental design: 1. The study design is not very clear to me. If it were to examine the effects of the screening-policy implementation, the authors ought to consider the factors associated with the implementation as this is an intervention-based cohort study. If they wanted to compare the cancers detected by self-exam and those by screening, then what study design did they have?
2. The 1995-2000 had too few (absolute number) screening-detected cases. The lack of statistical significance (P=.08) may be associated with the small sample size. In fact, because n=4, Fischer exact test should have been used.
3. What are the factors associated with screening for breast cancer? Those factors may have driven or linked to the differences shown in the study.
Validity of the findings: In this manuscript, Zhang Qi et al reported comparison of prognostic factors between self-detected and screen-detected invasive breast cancers in Tianjin, China.
They compared the prognostic factors at diagnosis between self-detected and screen-detected cases of invasive breast cancer in Tianjin, use three databases were selected breast cancer diagnosed in 1995–2000, 2010, and 2015 groups. Data include patient’s clinical history and prognostic factors, plus the cancer detection methods.
They found Screen-detected breast cancer was associated with favorable prognostic factors.
The results in this study, confirmed the benefit of early detection through a screening program for breast cancer in Tianjin, China.
Additional comments: This study is well written, there are comments to change before 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: COMPARISON OF PATHOLOGICAL CHARACTERISTICS BETWEEN SELF-DETECTED AND SCREEN-DETECTED INVASIVE BREAST CANCERS IN CHINESE WOMEN: A RETROSPECTIVE STUDY
Review round: 2
Reviewer: 1

Output:
Basic reporting: All prior comments were addressed. Good job!
Experimental design: Most comments were addressed appropriately, except for the final multivariate regression analysis. It looks like the current regression use T stage, N stage, and histologic grade as continuous because only one Odd Ratio was provided for each variable. However, these three variable should be used as categorical variable and there should be one Odd Ratio for each category except for the reference group. Since there are small cells for certain categories, you can consider combining some of the categories into bigger group. If the distribution of age is not normal or extreme, can also consider categorizing it into several groups as well.
Validity of the findings: All comments addressed appropriately.
Additional comments: The revision is overall good. But need to make some adjustment to the multivariate logistic regression (using T stage, N stage, and historic grade as categorical variable) and modify the corresponding "Result" and "Discussion" part accordingly.


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

Input:
Title: COMPARISON OF PATHOLOGICAL CHARACTERISTICS BETWEEN SELF-DETECTED AND SCREEN-DETECTED INVASIVE BREAST CANCERS IN CHINESE WOMEN: A RETROSPECTIVE STUDY
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The authors have been answered all the questions and made the appropriate modifications.
I agree to accept your manuscript.
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: INCREASED CHEMICAL ACETYLATION OF PEPTIDES AND PROTEINS IN RATS AFTER DAILY INGESTION OF DIACETYL ANALYZED BY NANO-LC-MS/MS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Some parts need to be edited:

Page 11: Paragraph starting at Line 239 and ending at Line 244 -
Needs to be reframed as it is not immediately apparent to the reader what the authors are exactly trying to convey and how they are interpreting the results mentioned in this paragraph.

Page 11: Paragraph starting at Line 245 and ending at Line 253 -
This entire paragraph needs to be re-written as there are multiple instances of typographic and grammatical errors making it difficult to understand.

Page 11: Line 254 - "post-transductional" should be post-translational

Page 17: Line 388 to Line 390 is same as Line385 to Line387 - should be corrected appropriately.
Experimental design: The authors treated mice with 540 mg/Kg/day of diacetyl for 4 weeks to check the effect of this chemical on lung proteins. This amount used is substantial and difficult to imagine that anything near to this quantity is inhaled by workers who work in diacetyl manufacturing (as mentioned in the text).

The authors need to mention how they arrived at this quantity of the chemical and the length of time (4 weeks) of administration. Have they used different doses and/or length of time of administration? If so, then what is the cut-off (for both quantity and time) to observe a  significant change in the acetylation levels of lung-proteins.
Validity of the findings: No comment
Additional comments: No additional comments


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

Input:
Title: INCREASED CHEMICAL ACETYLATION OF PEPTIDES AND PROTEINS IN RATS AFTER DAILY INGESTION OF DIACETYL ANALYZED BY NANO-LC-MS/MS
Review round: 1
Reviewer: 2

Output:
Basic reporting: For the most part the paper is well written. There is a need for editing by a English native speaker. The paper cites pertinent literature and the results are consistent with the hypothesis that, in animal, diacetyl breakdown leads to radical formation as evidenced by the acetylation of histidinyl and arginyl side chains.

Figure 2. The figure I received for review has poor resolution. More importantly, it is difficult for the reader to figure out what is inside the purple ovals because the authors chose black as the color of the font. Please modify the figure to make sure that everything in it is readable.
Figure 3. The authors could use different colors instead of different hues of the same color. That would make it so much simpler to read.
Figure 4. Delete this figure; the information in it could be readily incorporated into the text. Should the authors argue for keeping the figure, please modify the y axis of part A by substituting 'Molecular Mass (kDa)' for the current 'Protein amount KDa'
The authors share the experimental data as supplemental information
Experimental design: The hypothesis that radical acetylation of proteins occurs in cells fed acetyl radical sources such as diacetyl was validated. The experimental design is straightforward and the data support the conclusion that diacetyl catabolism increases the level of acetylated proteins.
Validity of the findings: No comment
Additional comments: The idea of potentially expanding protein acetylation to include Arg and His side chains is of interest if it were mediated by enzymes. This paper should encourage investigators in this area of research to contrive experiments to test this possibility.


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: INCREASED CHEMICAL ACETYLATION OF PEPTIDES AND PROTEINS IN RATS AFTER DAILY INGESTION OF DIACETYL ANALYZED BY NANO-LC-MS/MS
Review round: 1
Reviewer: 3

Output:
Basic reporting: The article is clear with professional english used throughout and succinct.Data described supports hypothesis and is presented in unambiguous manner.
Experimental design: The experimental design is pretty well throughout and relevant network analysis is performed to study classes of proteins. Methods used for nano-LC MS/MS analysis, data collection and deconvolution are described clearly.
The authors should provide a explanation for using a very wide mass tolerance range: upto 1 Dalton and if that would have any impact of significance described between control and acetylated peptides.
Validity of the findings: no comments
Additional comments: No additional comments


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

Input:
Title: GEOLOGIC AND ANTHROPOGENIC SOURCES OF CONTAMINATION IN SETTLED DUST OF A HISTORIC MINING PORT CITY IN NORTHERN CHILE: HEALTH RISK IMPLICATIONS
Review round: 1
Reviewer: 1

Output:
Basic reporting: After going trough the review of the manuscript I find it suitable or publication in this prestigious journal.

I think one reference can be added to the manuscript on page 16, line 216-217:

Massey, D., Kulshrestha, A., & Taneja, A., (2013). Particulate matter concentrations and their related metal toxicity in rural residential environment of semi-arid region of India. Atmospheric Environment, 67, 278-286.
Experimental design: 'NO COMMENTS'
Validity of the findings: 'NO COMMENTS'
Additional comments: No additional comments


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

Input:
Title: GEOLOGIC AND ANTHROPOGENIC SOURCES OF CONTAMINATION IN SETTLED DUST OF A HISTORIC MINING PORT CITY IN NORTHERN CHILE: HEALTH RISK IMPLICATIONS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript entitled " Geologic and anthropogenic sources of contamination in the settled dust of a historic mining port city in northern Chile: Health risk implications" evaluated the concentrations and health risk of 12 metals in street dust samples of the port of Antofagasta, Chile. The data from previous studies and the current one are well combined in this paper and useful results are obtained eventually. However, it is not very well organized and needs a few corrections. This paper includes original data on this environmentally polluted port city. The English language is good. Certainly, there are some questions and comments that need to be considered. Overall, I recommend a minor revision for reconsideration.
The following comments should be taken into consideration by the authors:
Comment 1# The authors present the Tables and figures of result section in “materials and methods” section, which is not common and this makes finding texts related to presented data difficult. Please relocate tables and figures. Moreover, it is necessary to number the titles and take some sections as sub-section of the others. For example, Antofagasta Region background, previous sampling campaigns, and Study objectives should be sub-titles of Introduction section.
Comment 2# Lines 348-354: This information is more related to introduction or materials and methods section. Please move them to those sections.

Comment 3# Line 372-428: The section “Recommendations and considerations” is too long and including the unnecessary explanation for international readers. The authors also mentioned the summary of this section at the end of conclusion part that is enough for international readers. So, I highly recommend removing this section.
Comment 4# Figure 4: in legend please correct the second range as 0.5<AP<1 km
Comment 5# Please change “Work Methodology” title to “Methodology”.
Experimental design: Comment 1: Line 225: Please mention which size fraction was used in bioaccessibility tests and add QC data. For example, what Standard reference material/materials were used? What are results for them? How many replicates were used for each sample?
Comment 2: Please justify using Fe as the reference element and add it to line 192.
Validity of the findings: no comment
Additional comments: No additional comments


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

Input:
Title: GEOLOGIC AND ANTHROPOGENIC SOURCES OF CONTAMINATION IN SETTLED DUST OF A HISTORIC MINING PORT CITY IN NORTHERN CHILE: HEALTH RISK IMPLICATIONS
Review round: 1
Reviewer: 3

Output:
Basic reporting: The data is reported properly in the manuscript
Experimental design: Experimental design explained well in the manuscript except analytical and instrumental methods.
Validity of the findings: Findings are valid and presented well.
Additional comments: Current manuscript reports heavy metals levels and sources in street dust from Antofagasta region of Chile, where intensive mining activities exist. Current study reports an important data set in terms of assessment of human exposure to heavy metals. The manuscript is written well and data presented properly.  I recommend this manuscript to be published after correction of the following issues

Line 110-119. Please state the size fraction of dust samples that wer subjected to analysis.
Line 140-142. Authors consider atmospheric deposition as the sole pathway for deposition of metals and metalloid in street dust. How about heavy metals from vehicle emissions? In rest of the manuscript, vehicles emissions were never mentioned as a source, especially for Pb. Since the manuscript is dealing with heavy metals in street dust, contribution of vehicle emissions should seriously considered.

General comment

There is no information on analytical and instrumental procedures, i.e. sample preparation, instrumental methods, quality assurance/quality control, detection limits etc. Such information should be given briefly 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: A ROBUST METHOD FOR RNA EXTRACTION AND PURIFICATION FROM A SINGLE ADULT MOUSE TENDON
Review round: 1
Reviewer: 1

Output:
Basic reporting: - Typo on line 237: keep use of "p" vs "P" consistent
- Typo on line 275: "repeats" should be "replicates", correct?
Experimental design: - The authors did a great job in establishing previous research in the field regarding extracting RNA from tendon tissue. While RNA amplification (line 86) can be quite costly, especially for RNA Seq applications, there are more cost-effective methods of pre-amplification for qPCR. One such method is Specific Target Amplification (STA) offered by Fluidigm: https://www.fluidigm.com/search?query=specific%20target%20amplification. (Example reference: Jang, J. S., Simon, V. A., Feddersen, R. M., Rakhshan, F., Schultz, D. A., Zschunke, M. A., et al. (2011). Quantitative miRNA expression analysis using fluidigm microfluidics dynamic arrays. BMC Genomics, 12(1), 144). It would improve the article to refer to this technique.
- The authors presented results from other disruption methods (lines 187-199) including "enzymatic digestion, cryogenic grinding (manual and mill), shearing with a handheld homogenizer". Would you please provide additional details in the methods or supplemental methods regarding these procedures? For instance, which enzyme was used, concentration, time, etc.?
- When the sample was placed on ice prior to homogenization (line 203), was it in a solution such as trizol? Or was it in air? Please add this detail to manuscript.
- The authors state that homogenization times longer than 360 seconds could cause RNA degradation (lines 215-216). How much longer was required to degrade the RNA? This would be useful information to include.
- The authors stated in the methods (line 161) that nanodrop readings were taken. What were the 260/280 ratios for these samples? Was the RNA sufficiently pure with minimal contamination that may hinder RNA Seq library prep?
Validity of the findings: - I would suggest changing title for Fig. 3 to "Tendon pooling affects RNA yield but not quality" which better matches what is stated in the results section.
- While I agree that RNA Seq experiments can be conducted for RNA samples with RINs ~ 6.5, the authors should mention that this level of quality may not be sufficient for certain library preps. For instance, a RIN of 6.5 is borderline in terms of being sufficient for mRNA sequencing where PolyA selection occurs. Most manufacturers recommend RINs > 7 for this type of sequencing (e.g., Illuminia TruSeq RIN > 8, SMART-Seq RIN > 8, Exiqon RIN > 7). However, 6.5 is more than sufficient for total transcriptome sequencing using random priming libraries.
Additional comments: The authors present an inexpensive, simple method for extracting RNA with high yield and high quality from single murine tendons. As the authors stated, pooling of samples is commonly performed to obtain sufficient levels of RNA per biologic replicate. Being able to efficiently isolate enough RNA for downstream analyses without compromising quality or requiring expensive pre-amplification methods, would benefit the field.


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: A ROBUST METHOD FOR RNA EXTRACTION AND PURIFICATION FROM A SINGLE ADULT MOUSE TENDON
Review round: 1
Reviewer: 2

Output:
Basic reporting: This is a well-written manuscript. The topic of improving RNA yield from connective tissue is important in the musculoskeletal field.
Experimental design: The experimental design is excellent and the methods are well described. Although not possible to test every permutation, and some experiments were limited in sample size, this paper provides useful observations as a technical note.
Validity of the findings: The findings are useful in demonstrating their RNA isolation approach provides improvements over many of the currently utilized techniques.
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: A ROBUST METHOD FOR RNA EXTRACTION AND PURIFICATION FROM A SINGLE ADULT MOUSE TENDON
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Thank you for the edited manuscript. You addressed all of my previous concerns. This method will be very useful for the tendon field.


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: A ROBUST METHOD FOR RNA EXTRACTION AND PURIFICATION FROM A SINGLE ADULT MOUSE TENDON
Review round: 2
Reviewer: 2

Output:
Basic reporting: This is a well written study with appropriate references and objectives.
Experimental design: The experimental design is appropriate and the techniques are well described.
Validity of the findings: The findings are valid, and the data is robust and statistically sound.
Additional comments: This paper will be a nice addition to the tendon literature.


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: STRAIN-LEVEL GENETIC DIVERSITY OF METHYLOPHAGA NITRATIREDUCENTICRESCENS CONFERS PLASTICITY TO DENITRIFICATION CAPACITY IN A METHYLOTROPHIC MARINE DENITRIFYING BIOFILM
Review round: 1
Reviewer: 1

Output:
Basic reporting: Basic reporting is ok. Yet, I have some minor comments on figures and tables that should be considered:

Figures:
Please report in the figure caption, if you used SD or SE.
Figure caption for Fig 4 might be too long. Perhaps you could include relevant info from it in materials and methods.
Table 1: annealing T for narG1-primers missing.
Experimental design: no comment
Validity of the findings: Otherwise ok. One thing needs further clarification:
It remains very unclear, why the JAM1 is not reducing nitrite?? Based on Supplementary data 4 and 7 it is expressing Copper-containing nitrite reductase (EC 1.7.2.1). On the other hand in Supplementary document 6, you report that it is not expressing it. Why such discrepancy?
So, do JAM1 actually have gene for nirK-nitrite reductase and  it is also expressing it, but the nirK enzyme is not produced or is non-functional?
If JAM1 has nirK gene, what is the similarity of it with that of GP59? Are you then also sure that you really have designed GP59-specific nirK-primers?
Additional comments: General comments:
This is a very interesting and well-done study on Methylophaga strain that can do complete denitrification.
Besides comments above for Basic reporting and Validity of the findings, I have some additional comments  that are to be considered.
Abstract:
You could write it more clearly also in the abstract that JAM1 cannot do complete denitrification.
Could you mention something about the transcriptome results in the abstract?

Introduction:
 line 58: Besides NO3-, also NO2-, NO and N2O are electron acceptors.
line 78: reference 13 could be positioned later in the sentence.

Materials and methods:
line 114: Why is FeSO4 added two times.
line 117: manufacturer for N2 gas?
line 119: filter-sterilized
line 126: 100 rpm??. could you spesify that they were on a shaker.
line 127-129. Did you work anaerobically during the transfers. How did you measure NO3-?
line 135: saline solution? Can you provide a more specific recipe or name for that??
line 141: How long were the colonies cultured?
line 143: How was NO3/NO2 measured?
line 145: do you mean full length 16S rRNA genes? Please indicate the primer regions or at least the primer names.
line 151: did you secure anaerobic conditions when injecting the cultures to bottles.
line 155. as prescribed? Where?
line 179: did you do size check of the amplicons? E.g. agarose gel electrophoresis.
line 193: Some numbers on the amount of genomic raw data and raw data fragment length could be interesting to see.
Results and discussion:
line 248-250. Why do you think there was this discrepancy with Laurin et al. (23)?
line 303-305. Why does JAM1 have higher dynamism of NO3- processing? Some genetic reasons that can be seen in the genomic comparisons?
line 330. …than IN the overall genome…
line 431. Why do you think there is the strain-specific difference in those encoding the synthesis of osmoprotectant ectoine?
line 440. “etc…” could be removed and the list could be closed without it
line 472: do you mean such accumulation of NO2- and not NO3-?


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: STRAIN-LEVEL GENETIC DIVERSITY OF METHYLOPHAGA NITRATIREDUCENTICRESCENS CONFERS PLASTICITY TO DENITRIFICATION CAPACITY IN A METHYLOTROPHIC MARINE DENITRIFYING BIOFILM
Review round: 1
Reviewer: 2

Output:
Basic reporting: This manuscript is generally well written, with only a few minor errors in syntax and grammar:
line 29 -- unsure if a culture can be "performed." Maybe inoculated, or set-up, or created
line 62 -- denitrification pathways
line 67 -- bacterial species and numerous protozoans
line 76 -- can carry out complete
line 91 -- lacks a gene
line 101 -- that is closely related
line 119 -- of filter sterilized methanol
line 130 referred to in the text
line 151 -- was added to the medium
line 175 -- was performed
line 227-228 -- rephrase sentence starting, "The intergenic sequences..."

The references and overall structure is clear and well presented. The article has a solid logical flow.
Experimental design: The experimental design is fine -- no comments for revision
Validity of the findings: The data are robust, however, there are a few areas where the significance and rational of the study can be clarified:

line 345 -- perhaps the related Methylophaga species derived from this referenced study can be highlighted in the Figure.

Section starting line 393 -- It is unclear how analysis of CRISPR regions determine whether one strain evolved from another. Can the authors put in a couple of sentences to explain the rationale and the associated references?

Why is the transcriptome data for the denitrification pathways in supplemental data? Also, the text and Table 4 indicate that gene expression in both strains was largely the same, even though the physiology of the strains was quite different. How is this difference reconciled?

The conclusions, particularly from line 474-481, do not reference the data that were collected such as the differences in growth of the isolates or differences (in any) that were found in the transcriptomes. The conclusion should have some reference to how these growth/gene expression parameters could influence enrichment under the conditions in the study. Also, this paragraph does not state why the findings of the particular study are important to further our understanding of methylotrophic denitrifiers. Rather, the conclusions are quite broad and ambiguous and not specific to this work. The authors can improve this last paragraph by stating the major findings of the work and the implications on the study system and the physiology of Methylophaga.
Additional comments: This is a good manuscript with a robust data set that substantially adds to our understanding of methylotrophic denitrifiers.


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: GENOME COMPARISON IMPLIES THE ROLE OF WSM2 IN MEMBRANE TRAFFICKING AND PROTEIN DEGRADATION
Review round: 1
Reviewer: 1

Output:
Basic reporting: Comments:

1. a couple of typos in the manuscript:
a) Page 14, line 266, "is more likely happened" change to "is more likely to happen".
b) Page 15, line 329, "likelihood" change to "likely".
c) Page 17, line 373, "strict" change to "strictly".

2. Question: Table 1
The data are very clean and significant. Do the authors have any clue why Bd21-3 (Brachypodium) exhibited some resistance to WSMV, but not zero under either of the 2 temperatures?
Experimental design: no comment
Validity of the findings: no comment
Additional comments: No additional comments


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

Input:
Title: GENOME COMPARISON IMPLIES THE ROLE OF WSM2 IN MEMBRANE TRAFFICKING AND PROTEIN DEGRADATION
Review round: 1
Reviewer: 2

Output:
Basic reporting: In this manuscript, the authors study the sequence and biological function of Wsm2, a gene of wheat genome goes against the wheat streak mosaic virus (WSMV). Total 93 promising orthologous groups are reported in wheat genome to further study Wsm2 ancestor locus. In addition, the protein degeneration and/or membrane trafficking, resulting from proteins encoded by Wsm2, is proposed to potentially contribute to the resistance of wheat with Wsm2 gene to wheat streak mosaic virus.

The author gains a huge amount of experiment data with years of field work as well as a comprehensive genetic analysis. In addition, the manuscript is clearly written and conclusions are reasonably drawn according to experiment data. Yet, there are several issues in this manuscript such as context arrangement and figure legend, which can cause the confusion and misinterpretation. Overall, the manuscript is publishable after these issues is fixed properly.

Issues
1.	In the first section of result, the authors choose Brachypodium to study the acquired resistance of wheat to WSMV by natural mutation. Brachypodium is reported as a good model for genome study of wheat, however, few evidences are provided to connect the Brachypodium with natural mutation of wheat, which weakens the rationale to choose Brachypodium. Unless more convincing evidences are given, this part should be either moved to supplement part or deleted since it is weakly relevant to the main topic of the manuscript.
2.	In the third section of result, it is pointless to include Brachypodium for identification of possible Wsm2 location in wheat since Brachypodium is lack of WSMV resistance as reported by author themselves in the manuscript.
3.	In line 71-73, the data of wheat reduction caused by WSMV infection is from a study in 1980s, which is somewhat outdated. The authors need provide up-to-date data about the effect of WSMV.
4.	The paragraph in line 311-319, should be moved into discussion part since it mainly focus in comparison between their results and others, which is the purpose of discussion.
5.	The content after “For example” in conclusion section should be deleted since it talks about the following work in future instead of draws conclusion according to experimental data.
6.	In the figure legend of table, the sample size of each condition should be clearly specified.
Experimental design: No comment
Validity of the findings: No comment
Additional comments: No additional comments