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: IS HIGH-INTENSITY INTERVAL CYCLING FEASIBLE AND MORE BENEFICIAL THAN CONTINUOUS CYCLING FOR KNEE OSTEOARTHRITIC PATIENTS? RESULTS OF A RANDOMISED CONTROL FEASIBILITY TRIAL
Review round: 1
Reviewer: 1

Output:
Basic reporting: -	The article has been written in English clearly and accurately. No major errors are observed.
-	The introduction shows sufficient background to understand the possible functional benefits of a high intensity interval training (HIIT) and a therapeutic alternative for older adults with knee osteoarthritis (OA).
-	I suggest this modification to the title: Is high-intensity interval cycling feasible and more beneficial than continuous cycling for knee osteoarthritic patients? - Results of a randomised control feasibility trial.
-	Table 1: Apply t-test for independent samples in the baseline characteristics of the groups.
Experimental design: -	The manuscript complies with the Aims and Scope of PeerJ.
-	The research question is well defined.
-	The research complies with the ethical code of the international research community and includes a record of the protocol: “The protocol for this study was approved by the Bond University Research Ethics Committee (RO1776) and registered at the ANZCTR (trial registration number ACTRN12616000273482)”. Two groups were included: an exercise group (EG) and a control group (CG).
-	The research has criteria that may increase the likelihood of type 1 error: the HIIT group presents less age and a higher proportion of male participants than the MICT group. This difference in age and sex can generate better results in favor of the experimental group (HIIT), as can be seen in the results of Table 3 (TUG). It is suggested to apply a t-test for independent samples to determine if there are significant differences between the groups in the baseline characteristics.
-	L237: The abbreviation BIA is observed for the first time, without indicating the complete word.
Validity of the findings: -	The results are novel and significant for the understanding and effectiveness of the training / rehabilitation protocols of patients with knee OA. However, the results and conclusions should be taken with caution, since the research has a high probability of committing a type 1 error. Consider the comment presented in "Experimental design".
-	L272-278: Two of nine participants experienced adverse effects on the HIIT group, which corresponds to 18% of the participants analyzed. This corresponds a considerable percentage and even more when the sample size is small. I recommend being cautious when supporting the HIIT group over the MICT in relation to the reduction of adverse effects, considering that the main purpose of the investigation was to examine and compare the feasibility and safety. I recommend including a sentence on this comment between L328-338.
Additional comments: The research is novel and original since it attempts to solve a recurrent research problem in patients with knee OA. This research tries to understand the possible functional benefits of a HIIT and a therapeutic alternative for older adults with knee osteoarthritis (OA) through variables associated with the reliability and functionality of the patient. However, the research has criteria that may increase the likelihood of type 1 error: the HIIT group presents less age and a higher proportion of male participants than the MICT group. That can generate a high probability of observing significant results when they really are not.


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: IS HIGH-INTENSITY INTERVAL CYCLING FEASIBLE AND MORE BENEFICIAL THAN CONTINUOUS CYCLING FOR KNEE OSTEOARTHRITIC PATIENTS? RESULTS OF A RANDOMISED CONTROL FEASIBILITY TRIAL
Review round: 1
Reviewer: 2

Output:
Basic reporting: Please to read my general and specific comments attached in the document.
Experimental design: Please to read my general and specific comments attached in the document.
Validity of the findings: Please to read my general and specific comments attached in the document.
Additional comments: Please to read my general and specific comments attached in the document.


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: IS HIGH-INTENSITY INTERVAL CYCLING FEASIBLE AND MORE BENEFICIAL THAN CONTINUOUS CYCLING FOR KNEE OSTEOARTHRITIC PATIENTS? RESULTS OF A RANDOMISED CONTROL FEASIBILITY TRIAL
Review round: 2
Reviewer: 1

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


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

Input:
Title: IS HIGH-INTENSITY INTERVAL CYCLING FEASIBLE AND MORE BENEFICIAL THAN CONTINUOUS CYCLING FOR KNEE OSTEOARTHRITIC PATIENTS? RESULTS OF A RANDOMISED CONTROL FEASIBILITY TRIAL
Review round: 2
Reviewer: 2

Output:
Basic reporting: I agree with the final version submitted by the authors.
Experimental design: I agree with the final version submitted by the authors.
Validity of the findings: I agree with the final version submitted by the authors.
Additional comments: I agree with the final version submitted by the authors.


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

Input:
Title: GLOBAL OPPORTUNITIES FOR MARICULTURE DEVELOPMENT TO PROMOTE HUMAN NUTRITION
Review round: 1
Reviewer: 1

Output:
Basic reporting: This paper is well-written and provides really interesting food for thought regarding the potential for expansion of mariculture activities in alignment with appropriate policies to address nutritional deficiencies. The authors are clear about the limitations of such a broad approach and its individual components as proxies (e.g. ratio of aquaculture production to fisheries production as a proxy for infrastructure). There are lots of insightful and interesting parts throughout the discussion and conclusion. The structure is clear and the data are presented effectively.

Minor comments:
- Convert Final opportunity score rank table from landscape to portrait
- Add accents to Bene throughout the manuscript (sometimes you do, sometimes you don't)
- Lines 307-313 - you switch back and forth between "aquaculture" and "mariculture" here; is this intentional?
- I'd delete the first sentence of this paragraph (Line 361-362) and just lead with the second, which is more important and stronger.
Experimental design: The authors are clear about their methodology, why some countries needed to be excluded due to data gaps, and the risks of aggregation (e.g. the loss of local / regional diversity within countries). The sensitivity check on variables is good, and the transparency resulting from the sharing of all data is helpful. See above for concerns about including all coastal countries in this analysis and conflating reliance/preference, etc. The proxy for infrastructure (capture fisheries / aquaculture production) also becomes problematic in countries with small production volumes, as FAO data is most accurate for large-volume fisheries and aquaculture production and becomes shakier at smaller volumes (i.e. a small island state may have very precise tonnage reports for tuna fisheries, but far less accurate reports for smaller or less well-monitored fisheries, especially artisanal and subsistence fisheries), yet the tuna catch would likely be exported and have little nutritional contribution to local communities, while the artisanal/subsistence fisheries would be largely missed, while being more important in this regard.

In short, the authors have made it clear that this is a big picture, rough assessment, and I believe it has value as such. But perhaps a bit more attention to the data limitations and their implications for the different components of the index would be helpful.
Validity of the findings: The inclusion of highly industrialized countries (where populations have access to diverse foods rich in micronutrients) and developing countries (where access to such foods is more mixed) within the same index caused a number of problems throughout the paper. The definition, for instance, of "reliance" on seafood for this entire suite of countries is problematic, as it certainly become "preference" rather than reliance for populations with access to diverse foodstuffs and the means to purchase it. Highly industrialized countries that consume lots of seafood, but which have access to any number of other foods that could provide this nutrition are unlikely to face micronutrient deficiencies related to fluctuations in fish availability or price.  Likewise for nutritional opportunity, according to the index, countries like Venezuela and Syria are among those with the least nutritional opportunity, ranking below countries like Denmark and Canada. More clarity may be possible by simply dividing the index into several categories of countries (e.g. based on income levels), or in your graphics by using coloring similar to Golden et al. (2016) that draws emphasis away from countries with little reliance on seafood for nutrition. Or perhaps just focus on coastal, low-income food deficit countries?

Another issue that was missing was a greater focus on the actual mariculture species being produced (you hint at this on lines 337-338). It is useful that the authors highlight a divide between high volume and high value production, and also the disconnect between mariculture production levels and nutritional outcomes. Yet I wonder about the argument for including high value mariculture species in this analysis. Residents in low-income food deficit countries are unlikely to be able to afford high value mariculture outputs. Adding further information to the introduction or discussion on which mariculture species are the best candidates (i.e. low cost, high nutritional value, generalist, less susceptible to disease, etc) for reducing food insecurity and nutritional deficiencies would be helpful to frame this issue.
Additional comments: This is really interesting and exciting work! Good luck with it! While having a global index with lots of countries may feel nice, I have a hunch that limiting your index to LIFDCs or least developed countries, etc may help to clarify some of your arguments and conclusions, and feed into clearer policy recommendations. Good luck!!


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: GLOBAL OPPORTUNITIES FOR MARICULTURE DEVELOPMENT TO PROMOTE HUMAN NUTRITION
Review round: 1
Reviewer: 2

Output:
Basic reporting: Very good
Experimental design: Very good
Validity of the findings: Very good
Additional comments: This is a really useful study using a novel approach to show where mariculture has the largest potential to influence human nutrition.
Ln. 62. Please note that the per capita consumption of 20.3 is the highest aver recorded.
And while I agree that nutritional importance of fish is part of the explanation, I think you should add somthing to the effect that the main driver has been innovatins that has reduced production cost and increased competitiveness not only towards marine fish, but also other foods (Smith et al,, 2010 Science; Kumar and Engle, 2016, Reviews in Fisheries and Aquaculture).
Ln 76. While this is true for many species, it it should be nuanced as e.g. Belton et al (2018) show that a significant portion of aquaculture has made seafood more available in countries like China, Bangladesh, Indonesia and Egypt. Still this do not affect you main question about mariculture, as the main species here are freshwater species. However, it do suggest that mari-culture can become important.
Ln 156. I realize that all metrics has to be somwhat pragmatic given the data availability, but I think you should also acknowledge weaknesses. For instance, cultural preference is also an expression of tradiotinal availability, and there are numerous examples of how eating habits change in surprising directions with economic opportunity with Japan´s declining seafood consumption and increasing meat consumtion as an example. I think your economic metrics are a bit more problematic than your other metrics as they are even more grounded in what has been, and not in what is possible.
Ln. 235. I assume that also exludes nations with significant fisheries in large lages like Uganda? Please note.
Ln 262. This may not be too surprising since aquaculture does not happen to a large extent in developed countries.
Ln. 266. Latin America is really interesting since that is also the region where the relative growth is predicted to be fastes in most general aquaculture production forecasts such as the WorldBand FAO Fish to 2030 project (Kobayashi et al 2015, Aquaculture Economics and Management).
Ln 376. I think that you somewhere need to state cleare that there are also other reasons for why aquaculture production may grow in a country such a natural endowment etc., that may lead aquaculture to increase also places that your model does not indicate. Asche and Smith (2018, Food Policy) is a recent example of a study that argue that increased aquaculture production is largely a function of incentives. Howevert his may also increase availability for nutiritional poor, as seafood to developing cointries are increasing quite rapidly. For instance, Nigeria is now the world´s 11th largest seafood imorter by quantity.


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

Input:
Title: THE RICE XA21 ECTODOMAIN FUSED TO THE ARABIDOPSIS EFR CYTOPLASMIC DOMAIN CONFERS RESISTANCE TO XANTHOMONAS ORYZAE PV. ORYZAE
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript entitled “The rice XA21 ectodomain fused to the Arabidopsis EFR cytoplasmic domain confers resistance to Xanthomonas oryzae pv. oryzae" challenges the idea that the endodomain of plant plasma membrane localized pattern recognition receptors determine downstream immune response and the ectodomain is only responsible for pathogen molecule recognition. Using rice XA21 receptor (conferring resistance to bacterial pathogen Xoo virulent strain PXO99A) as an example, the authors propose the ectodomain of XA21 confers disease resistance and triggers immunity as far as a functional kinase present in endodomain. The authors introduced the background sufficiently, pointed out key major results from their own lab and other labs supporting the previous idea, also introduced hints against previous idea (chimera receptor with endodomain of XA21 and ectodomain of EFR which can recognize EF-TU from Xoo failed to confer resistance to Xoo strain PXO99A), which provided rationale for performing this work. The data of this ms are of quality in general, and well presented by the three figures.
Experimental design: The authors generated transgenic rice plants expressing chimera molecule composed of ectodomain of XA21 and endodomain of EFR (named XA21:EFR), T1 and T2 generation plants of multiple lines showed resistance to Xoo strain PXO99A based on lesion measurements after leaf inoculation. The application of sulfate peptide RaxX21-sY from Xoo, which can be recognized by Xa21, can trigger ROS production and some PR gene expression in XA21:EFR rice cells. The experimental design is pretty straightforward.
Validity of the findings: The conclusion that XA21 ectodomain confer resistance to Xoo is supported by the results here, in general. Statistically sound.
Additional comments: Just some material description can be more specific. 1. In the methods part, the authors should state which promoter was used in generating XA21:EFR transgenic rice, and whether it is the same promoter used to generate previous EFR:XA21 transgenic rice. Because promoter difference may also contribute to the difference in confer resistance to Xoo. 2. In introduction, the authors should introduce that Arabidopsis EFR ectodomain can recognize EF-TU from bacteria including Xoo. This will help readers to understand that previous report on chimera receptor with endodomain of XA21 and ectodomain of EFR didn’t increase resistance to Xoo, conflict with the idea endodomain of PPR determines immunity response. 3. In results part, the nature of LOC_Os02g36190, 275 LOC_Os06g37224, and LOC_Os11g42200 is vague. Whether they are PR genes or stress induced genes is not clear. A brief description of the nature of these genes can help readers to evaluate whether they can represent immunity response.


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

Input:
Title: THE RICE XA21 ECTODOMAIN FUSED TO THE ARABIDOPSIS EFR CYTOPLASMIC DOMAIN CONFERS RESISTANCE TO XANTHOMONAS ORYZAE PV. ORYZAE
Review round: 1
Reviewer: 2

Output:
Basic reporting: Good introduction of the chimera literature.  Could maybe cut down the non-RD introduction, given it's not as relevant to a Xa21 vs EFR difference (they're both nonRD)
Experimental design: See point 5 in general comments
Validity of the findings: No major comments but see below
Additional comments: The manuscript Thomas et al. “The rice XA21 ectodomain fused to the Arabidopsis EFR cytoplasmic domain confers resistance to Xanthomonas oryzae pv. oryzae” describes experiments on transgenic rice lines that demonstrate an XA21 ectodomain is sufficient to confer RaxXsT response and Xoo resistance.  While several additional experiments and controls would have be nice, the work is generally sound and contributes to the field of membrane receptor kinases and structural requirements for conferring novel resistance.  There are some missing pieces of information, and several puzzling results are unexplained or uninterpreted – the points below should be addressed in a revision

1. In the discussion the authors interpret the resistance function of XA21:EFR relative to EFR:XA21 chimeras as a function of ectodomain interactors specific to Xa21.  This is possible (and exciting), but an alternate explanation is that the strength of receptor activation in an Xoo-rice infection context is simply lower for activating an EFR ectodomain vs an XA21 ectodomain, as discussed by Schwessinger et al. 2015a.  The authors should add this possibility to the discussion.  Is there any previous data that addresses this in an Xoo infection context? E.g. similarity of early responses by Xoo (not synthetic elicitor) in EFR vs Xa21 transgenics would weigh against my alternative hypothesis. (Also possible that Xoo masks its elf epitope somehow?)

2. In Figure 4, EFR and EFR:Xa21 transgenics are shown to respond to RaxX.  This strange result is not mentioned in the text or discussion.  Is this actually elf18 treatment??

3. The resistance phenotype in Fig. 1, reduced lesion length, looks robust and the use of segregating lines is well explained.  It would be very nice to also see reduced bacterial population and the use of a delta raxX strain as a negative control in these experiments.  I think these would be required revision experiments in most journals, I defer to the editor here.

4. The scale of RLU in Fig 3 should be consistent across the graphs, and Fig. S4 should be included in the main text for easier comparison.  Doing these steps would show that the ROS burst in the Xa21:EFR chimeric lines is much smaller than in native or EFR:Xa21 lines.  It’s interesting that they still are resistant despite lower ability to be activated by synthetic elicitors (as seen with marker gene activation as well in Fig 4).  The authors should note this general low magnitude of response in the results text.

5. Materials and methods is missing the protocol for bacterial infection.  Also clarify that the elf18 sequence used is E.coli

Other comments:
Line 189 mentions using both RaxX21 and RaxX21-sY; I think it was only sY in this paper?

Lines 309-313: It’s unclear how differential kinase domain interactors are consistent with driving an ectodomain-specific effect.  The data seem more consistent with these differential interactors not actually mattering for the actual resistance output, even if they affect ROS and other early events.


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: BIODIVERSITY OF THE GREAT BARRIER REEF—HOW ADEQUATELY IS IT PROTECTED?
Review round: 1
Reviewer: 1

Output:
Basic reporting: This manuscript is presented as a literature review around the theme of biodiversity of marine communities along the Great Barrier Reef (GBR). The authors have attempted to collate existing literature which reports on 1) the status of biodiversity, 2) processes threatening species diversity and 3) the extent of existing biodiversity monitoring and reporting on the GBR.
I found the basic premise of the study to be a worthwhile focus, i.e. highlighting the paucity of data at the species level for many communities and the need to rectify this given the expanding disturbance regimes that are affecting not only the GBR but coral reefs around the globe.
The inference of the paper is that it addresses all marine communities but the vast majority of the manuscript is about corals, which clearly reflects the expertise of the lead author. Hence I believe this study would be better if it explicitly focussed on coral communities to highlight in detail the issues the authors are referring to. By all means keep the tables which highlight issues across all taxa, which readers could use as a starting point into the literature on other communities but the detail necessary to drive home the points that the authors are trying to make is to be found in the coral literature.  I could even suggest some interesting wordplay to generate more interest in the topic e.g. along the lines of ‘Don’t forget the trees while trying to save the forests’.
The general writing style of the paper is unfortunately verbose and reads for the most part like a pep talk. For example the word biodiversity is mentioned 69 times in the main document – 82 times if the Abstract is included. I know that Biodiversity is the main theme of the paper but once the theme is established and framed in the Introduction then please refrain from literally shouting the term biodiversity at the reader every paragraph thereafter. There is also too much verbatim quoting from documents which continues to feel like the reader is being harangued about the importance/relevance of the authors study.
Experimental design: There are very clear methods around undertaking literature searches and I found the description of the survey methodology to be very unclear and very scattergun in approach. The question to be asked is “could I replicate the exact search process that the authors undertook?” I don’t believe I could, based on the description given. There needs to be a clear tabulation of themes, search terms used and search engines used. Then a summary of how the literature was then reviewed and the final documentation decided on.
Validity of the findings: As mentioned in my comments under basic reporting I found the manuscript to be far too emotive and repetitive in its messages to extract clear and concise outcomes from the work.
Additional comments: I found many statements that were unclear.
Line 87 – What is meant by biodiversity values being one of a complex array of multiple use values?
Line 98 - ……articles relating direct or indirect threats relating to the….(repeating of words – also seen in line 152, and again in lines 284 and 288 where the phrase ‘falls short’ is repeated)
Line 117 – Results section starts here
Line 131 – I don’t see the GBR as recovering from a legacy of impacts on crocodiles and whales? Populations of both of these taxa are healthy.
Lines 134-138 – more info on what is meant by habitats and species. % declines are stated but without baseline info included. E.g. coral cover has declined by 0.5% year – is this for the past 20 years or the past 50 years? Likewise for calcification rates.
Lines 162-164 – incomplete sentence and hence unclear – use the phrase ‘not only’ and then do not provide the subject this refers to.
Line 165 – Couldn’t find reference for Wilson (2007) in list.
Lines 172-175 I do not understand this sentence at all?
Line 323 – the comment “…where there is a large amount of uncertainty” needs clarification. The GBR is a well-managed system by national and international standards and the continued use of emotive terms without clarification continues the tone of this manuscript as a pep-talk.
Lines 327 and 330 – repeating phrase…”up-to-date understanding of biodiversity on GBR”
The conclusion – this section does not add anything to the document other than continue the emotive phrasing and entreaties of the rest of the manuscript.
Table S1 – in the section on Unsustainable fishing impacts, removal of herbivorous fish is mentioned as an unsustainable impact on the GBR. This is not as issue because herbivorous fishes are not targeted by Australian fishers, either commercial or recreational.

Quantifying, monitoring and managing biodiversity is extremely important but your review needs to be less a lecture about how important biodiversity is and more a clear examination - using corals – of how species level information can provide a much clearer picture of community dynamics in the face of disturbances. Proxies for biodiversity is a topic that needs much more work and this is a particularly relevant area to investigate – as you do – given the constraints with resources and funding that are the reality of field-based science. There is a fish paper that you have not discovered that provides an example of how surveys of the main fish families can be used to great effect to estimate total species richness of fish in a region. This would provide another example of how to get more value from surveys w.r.t biodiversity information. See Allen & Werner (2002) Env. Biol. Fishes 65:209-214.


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: BIODIVERSITY OF THE GREAT BARRIER REEF—HOW ADEQUATELY IS IT PROTECTED?
Review round: 1
Reviewer: 2

Output:
Basic reporting: Captured under general comments for the authors
Experimental design: Captured under general comments for the authors
Validity of the findings: Captured under general comments for the authors
Additional comments: Review of Richards and Day.

General comments.
This is a timely review. Great Barrier Reef Marine Park Authority and partners are progressing the Reef Integrated Monitoring and Reporting Program to support the Reef 2050 Plan. The ideas and recommendations presented by the authors of this paper will be relevant to the discussion of an effective program design.

The text can be tightened in a few places and I provide some specific recommendations below.

Line 37: World Heritage Status more due to outstanding universal value, of which biodiversity is an element. The Coral Triangle has higher biodiversity than the GBR.
L 43-46: This is much needed
L 50 – 55:  Agree, but I’m not sure more monitoring data will safeguard species. Recommendations for effective management could be based on existing data and models
L 55-61:  Great initiative, but again, monitoring may not be the limiting factor. In a time of more coral bleaching and other growing climate impacts (acidification, storms), the loss of habitat will mean less scope for biodiversity.  Monitoring the system more intensely will require resources (taken form management budgets) and may not provide solutions. Safeguarding key habitats could.

L 61-62:  Agree on the action, but I’m not sure more monitoring of “species of interest” is really effective action. What I’d say we need is a Value of Information analysis that helps us understand what new and targeted information we need to better manage the system to safeguard species and ecosystem values.
L 82-84.  This needs a reference.  I’d contest the idea that biodiversity buffers environmental variation. It would be more correct to say that biodiversity potentially comes with functional redundancy, which can increase resilience (Nystroem etc).
L 88:  Should read “Fernandez”
L 119-124.  Tighten the language here and sharpen the impact categories.  The word “unsustainable” is loaded unless you explain it.  Is “detrimentally” a word?
L 124:  Use “extensive” instead of “pervasive”
L 125-133.  Again, the text is loaded with adjectives. The far north has so far been spared from the land-use run-off issues of the wet tropics, but I don’t think the far north was ever considered pristine or a regional refuge.  Rather than referring to “unsustainable” activities (which needs to be demonstrated to be called unsustainable), instead use “cumulative pressures”.  You can use Anthony 2016 (Coral reefs under climate change and ocean acidification - challenges and opportunities for management and policy. Annual Review of Environment and Resources 41:59–81) as a reference.

L 161-174.  This section is spot-on. However, I was disappointed to see that the authors don’t follow up on these conclusions and recommend ways for targeted/focused monitoring to link with management strategies.  Instead, they leave the thread hanging and move on to talk about surrogates.
L 182:  Surrogates and proxies are just that – they cannot “accurately represent trends in biodiversity”.
L 192-205. As you point out yourself in L161-174, monitoring is a trade-off challenge, so reporting changes at the level of species for coral reefs that may have a million species (Fisher, Knowlton etc) is infeasible and could paralyse large-scale monitoring programs.  However, deeper taxonomic resolution at a few key sites could provide insight into biodiversity dynamics.
L 206-2013.  Again, a Value of Information assessment is needed. Up-scaled monitoring to measure everything may not be the most effective way to manage a system in stress and under limited resourcing.  Protecting key habitats and understanding risks to sensitive species that are likely to represent the responses of other species groups could be a cost-effective way to safeguard biodiversity without measuring everything.
L 206-247.  Agree coral cover is insufficient as a stand-alone indicator. All good points here that the science and management community need to hear (again).

L 247-294. Excellent section.  A new report that provides recommendations for the Reef 2050 review options is now available and could be used as a reference here. http://www.environment.gov.au/system/files/resources/3ae3225f-eb5c-418b-8889-e523a4fbb4e5/files/reef2050-plan-review-options-final-report.pdf
L 323-324. Explain what you mean by uncertainty.
L 330-331.  Biodiversity assessments could still be part of a pragmatic monitoring and management approach if stratified so that in-depth assessments are made at a smaller number of sites that overlap with key long-term monitoring stations.
L 354-367.  Good idea.  Such a program could also be complemented with citizen-science (i.e. crowd) analysis of detailed online photographs from monitoring stations. This means the world could essentially help monitor the GBR.


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: NATURE-DERIVED LIGNAN COMPOUND VB-1 EXERTS HAIR GROWTH-PROMOTING EFFECTS BY AUGMENTING WNT/Β-CATENIN SIGNALING IN HUMAN DERMAL PAPILLA CELLS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Writing. In general, the writing is good but can be improved in five parts.
1. Grammar errors. For example, the first sentence of the background in the abstract, the first sentence of the introduction, and the others in the text.
2. Some typos, for example, Abstract Method part, human dermal papilloma cells (hDPCs) is not correct.
3. Inconsistent abbreviations.
HFSCs in line 48, but to HFS in line 50.
DP cells, DPCs, human DPCs, and hDPCs.
V. negundo and Vitex negundo.
4. When you write RNA, please use lower case and italic. For example, when you check the mRNA levels of some genes like Lef1, you should write it in lower case and in italic. When you check its protein level, you should write it in capital.
5. Do not repeat your sentence in the abstract and introduction. For example, “Vitexins are a mixture of lignan compounds found in Vitex negundo, a widely used herb in China.”

Literature. More literature can be cited. For example, when you talk about hair reconstitution assay, the classical references should be cited. Also, references on Wnt signaling and its effect on proliferation of hair follicle should be cited.

Background/context. Can you briefly state why you would like to study the effect of vitexin compound 1 (VB-1) on hair follicle in the introduction part?

Arrangement of the manuscript. The structure of the manuscript is excellent, ranging from the phenotypic effect of VB-1 on in vitro cultured hDPCs, to the possible molecular mechanism, then to the in vitro organ culture effect. The figures are well arranged. However, the font in the figures can be enlarged to visualize.
Figure 3B should be Figure 3E; Accordingly, Figure 3E should be Figure 3B.

Results. The results are relevant to the hypotheses, although future study should be performed. For example, proliferation can be examined by immunostaining of some markers for Figure. Importantly, the VB-1-treated DP cells can be transplanted into the nude mice with epithelial cells, to observe if their hair induction ability is enhanced.
Experimental design: In the manuscript titled “Nature-derived lignan compound VB-1 exerts hair growth-promoting effects by augmenting Wnt/β-catenin signaling in human dermal papilla cells”, Luo et al., reported that vitexin compound 1 (VB-1) has a hair growth-promoting effect. This is an original primary research within the aims and scope of this journal.

The authors ask how VB-1 promotes hair growth. They first treated the cultured hDPCs with different doses of VB-1, and found that 0.1uM VB-1 promotes hDPCs proliferation. Then they identified that the expression of some DP signature genes and Wnt signaling pathway genes is changed in hDPCs after VB-1 treatment. They also treated the human scalp hair follicles with VB-1 in an in vitro organ culture system, and found that VB-1 significantly promotes hair follicle growth. This is a rigorous investigation using appropriate methods to examine the effect of VB-1 on hDPCs proliferation and on hair follicle growth.
Validity of the findings: The effect of VB-1 is more studied on other systems such as cancer cells, but not on hair follicle. So, this study has its novelty and impact on the potential future clinical application. The results and conclusions are appropriately made based on their scientific findings, and through enough replicate. However, there are still some statements needed to be improved.

1. Line 51-53, please make sure if the statement is correct. Are the outer root sheath and matrix cells generated from bulge stem cells with anagen initiation?

2. Result part. Figure 3, please clarify why the Axin2 mRNA levels are different from the protein levels after VB-1 treatment.
Also in Figure 3, the sampling times are inconsistent.
A descriptive error in the legend for figure 4B. This result does not show the percentage of hair follicle elongation.

3. In method part, authors wrote “150 anagen hair follicles isolated from 3 different volunteers were cultured with each concentration of VB-1”. However, in Figure 4 legend, authors wrote that “three independent experiments are shown (n=30)”. Which is the correct one?

4. Discussion part, line 205-206, authors wrote that “However, VB-1 at some low doses had proliferation-promoting effects in cultured hDPCs, suggesting that VB-1 represses the proliferation of cancer cells.” How and why can you make this suggestion which looks impertinent?
Additional comments: None


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

Input:
Title: NATURE-DERIVED LIGNAN COMPOUND VB-1 EXERTS HAIR GROWTH-PROMOTING EFFECTS BY AUGMENTING WNT/Β-CATENIN SIGNALING IN HUMAN DERMAL PAPILLA CELLS
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: In this study, Luo and colleagues revealed that VB-1, extracted from Vitex negundo, a widely used herb in china, can significantly promotes the proliferation of human dermal papilla cells (hDPCs) in a concentration-dependent manner. They found that DKK1 is obviously down-regulated by VB-1 in hDPCs, and the increased active β-catenin and decreased Axin2 protein levels suggest VB-1 facilitates Wnt/β-catenin signaling in hDPCs in vitro. Moreover, the expression of DP signature genes are also upregulated after VB-1 treatment. They further showed that VB-1 promotes human HF growth by HF organ culture.

Overall the findings presented in this study are potentially interesting and novel. Before I can recommend publication, several issues require clarifications and/or further experimentations.

1. It will be informative if the authors can detect whether VB-1 affects the other hair growth-related genes (eg. TGFβ1).

2. The authors claimed that VB-1 improves the hair-inducing properties of human dermal papilla cells. It would be useful to assess whether VB-1 also promotes the expression of other important DP signature genes (such as ALPL and VCAN ).

3. The labels of figures (such as Fig3a, c, d, and f ) is indistinct. And the font in the figures should be uniform.

4. In Fig.1A and Fig.4A, please use “μΜ” instead of “uM”.

5. There are some typos and grammatical errors throughout the manuscript. It is essential to correct all the errors in the revised version 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: NATURE-DERIVED LIGNAN COMPOUND VB-1 EXERTS HAIR GROWTH-PROMOTING EFFECTS BY AUGMENTING WNT/Β-CATENIN SIGNALING IN HUMAN DERMAL PAPILLA CELLS
Review round: 1
Reviewer: 3

Output:
Basic reporting: 1.The introduction should be written in more detail. Previous studies show that VB-1 induces apoptosis of cancer cells. This function of VB-1 should be mentioned in the introduction. In the last paragraph of introduction (lines 82-89), please clearly state your hypothesis and propose how to explore it.
2.The format of the reference does not meet the requirement of the journal. Please correct.
3.The time points shown in Figure 1A and 1B are inconsistent. Authors count the number of hDPCs at day 3, day 6 and day 9 in Figure 1A but at day 2, day 4 and day 8 in Figure 1B. Also,  “After culture for 8 days” is more accurate than that “During the 9 days culture” in lines 152-154.
4.In Figure 2, the gene name should be written in italic.
5.In Figure 4A, the length of HFs treated with 10 μM VB-1 is shorter than that treated with 1 μM VB-1. But opposite results are showed in Figure 4B. Please clarify.
6.Please describe your statement more accurately in the manuscript. For example, “activity” can be changed to “capacity” in line 47.
7.Please write your abbreviations consistently and correctly. For example, in line 50, HFC should be HFSCs. In line 56, DPCs should be DP cells, or dermal papilla cells (DPCs). In line 67, WNT should be Wnt. In Figure 1A and Figure 4A, uM should be μM. Please check the whole manuscript and correct them.
8.Please discuss more why 0.1 μM VB-1 promotes hair growth and hDPCs proliferation but has an opposite effect in high concentration.
Experimental design: 1.In the MM section, please briefly introduce how you did the real-time PCR. Please show the information for Tubulin antibody.
2.Authors aim to detect the effect of VB-1 on the proliferation of hDPCs by counting the number of hDPCs. I think the evidence is weak. Please add other methods to support this. For example, Brdu can be used to mark the proliferating cells.
Validity of the findings: Speculation is welcome, but should be more accurate. The authors aim to demonstrate that VB-1 promotes the proliferation of human dermal papilla cells (hDPCs) in a concentration-dependent manner (lines 149-150). Actually, VB-1 might promote the proliferation of hDPCs but not in a concentration-dependent manner because a high concentration of VB-1 inhibits the growth of hDPCs.
Additional comments: In this manuscript, authors showed that Vitexins promote hair growth by augmenting Wnt/β-catenin signaling in human dermal papilla cells. Although the manuscript needs to be improved, it is convincing that VB-1 can promote the growth of human hair follicles, which may provide a therapeutic strategy for treatment of alopecia.


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: ASYMMETRIES IN MOTHER-INFANT BEHAVIOUR IN BARBARY MACAQUES (MACACA SYLVANUS)
Review round: 1
Reviewer: 1

Output:
Basic reporting: I believe the manuscript is self-contained and provide relevant results to hypotheses. However, I have a number of specific concerns.

Lines 170-171: One or two citations after “e.g.” would be enough here. Otherwise, it looks like you cite all the studies which use LI scores, while there are a great many of them.
Line 231: “…consistent behavioural lateralization in mother - infant interactions might have first appeared in hominids…” This sounds strange in light of recent studies showing pronounced lateralization of mother - infant interactions in at least 14 (!) non-primate mammal species. Please see the references below.
Karenina K, Giljov A, Ingram J, Rowntree VJ, Malashichev Y. 2017 Lateralization of mother–infant interactions in a diverse range of mammal species. Nature Ecol. Evol. 1, 0030.
Giljov A, Karenina K, Malashichev Y. 2018 Facing each other: mammal mothers and infants prefer the position favouring right hemisphere processing. Biol. Lett.14: 20170707.
Karenina K, Giljov A, De Silva S, Malashichev Y. Social lateralization in wild Asian elephants: visual preferences of young and mothers. Behavioral Ecology and Sociobiology, 72, 21, 2018.
Karenina K, Giljov A, Malashichev Y. Lateralization of mother-infant interactions in wild horses. Behavioural Processes, 148, 49-55, 2018

You cited only one of these studies without any mention of what has been shown. This is not necessary to cite and discuss all the studies on non-primates, but at least Karenina et al., 2017 should be cited and mentioned in the text since it first shows that lateralized mother-infant interactions emerged long before primates. In addition, you may consider a brief discussion of Giljov et al., 2018 since it shows lateralized behaviour of walruses closely resembling cradling in primates.
For reader’s more complete understanding of the topic, it would be useful to mention (at least briefly) the suggested perceptual origin of lateralization in mother-offspring interactions. You investigate the impact of mother-infant lateralized positioning on motor biases, but the influence of sensory modalities should not be neglected. The role of sensory lateralization has been argued in the studies on non-primate mammals (Karenina et al., 2017). Moreover, recent studies of human infant cradling/holding bias (poorly cited, by the way) support the perceptual origin of this kind of lateralization (e.g., Todd, B. K. & Banerjee, R. Lateralization of infant holding by mothers: A longitudinal evaluation of variations over the first 12 weeks. Laterality 21, 12–33 (2016)).
Authors may also consider discussing other kind of lateralized social behaviour of primates and humans - embraces, a kind of 'adult cradling'.
Boeving, E. R., Belnap, S. C., & Nelson, E. L. (2017). Embraces are lateralized in spider monkeys (Ateles fusciceps rufiventris). American Journal of primatology, 79(6).
Packheiser, J., Rook, N., Dursun, Z., Mesenhöller, J., Wenglorz, A., Güntürkün, O., & Ocklenburg, S. (2018). Embracing your emotions: affective state impacts lateralisation of human embraces. Psychological research, 1-11.
Experimental design: Lines 148-150:  This may be an important confounding factor since suckling bouts may happen in a sequence (series). That is, behaviourally there was one suckling event (i.e. an infant approached the mother’s tit for suckling), but it was stopped and started again several times for some reasons. In that case, you obtained a series of data which were not independent from each other. The series of data should not be mixed with separate (independent) data points. From your description, it seems to me that you recorded as separate data points both the contacts with the nipple during one suckling interrupted by a few seconds pause and different suckling events between which an infant was involved in other activities or even was away from its mother. From statistical point of view this is not particularly correct. Analysis of separate approaches to mother’s tit for suckling would be much more preferable here, to my opinion. I suggest this cannot be changed already, but try at least to describe the data collection technique in more detail.
Lines 152-154: Were the sequences of reaching events properly recorded and analysed (e.g. several reaching events when sitting at one feeding point and in the same posture were considered as one sequence, then a subject changed location and a new sequence began)? Please describe, how did you separate independent reaching events? I believe that you cannot just record every single case of reaching, but there should be some clear criteria to separate the events. Otherwise, the degree of independence between the data points will vary significantly.
Validity of the findings: Lines 183, 188, 192 and 202: Given the authors used non-parametric tests, none of which is using mean LI's, the reporting of mean LI seems to be redundant. In my opinion, median values would be more appropriate here, because of non-parametric distribution of data. The same applies to Figure 1.
Lines 185, 194 and 204: Given a small sample size (7 subjects in both cases) I recommend authors avoid reporting binomial tests here and simply stick to the existing statements of individual laterality distribution.
Lines 216-221: Threshold levels of significance for correlation coefficients must be adjusted for multiple comparisons by Bonferroni's correction. Will be there a significant correlation between the LI scores for nipple preference and food reaching after this correction? May be need to reconsider also the discussion (lines 286-292).
Additional comments: Authors are recommended to provide more discussion of their findings in the context of recent studies of lateralized mother-infant interactions in both primates and non-primate mammals. The widely assumed association between lateralized infant’s position and perceptual lateralization should be mentioned. To my opinion, the data recording protocol lack standardization in terms of distinguishing of separate behavioural events. I suggest describing the data recording procedure in more detail. If Bonferroni's corrections were made this should be clearly stated, but if it was not made the validity of these results is questionable.


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: ASYMMETRIES IN MOTHER-INFANT BEHAVIOUR IN BARBARY MACAQUES (MACACA SYLVANUS)
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: (1) In the Introduction, considering the increasing evidences on primate mother-infant behavioral asymmetries, it is better to present one Table containing the basic information on all previous related findings.

(2) In the Methods, on line 162-163, it is better to show how to identify the hand preference when the z score equals 1.96.

(3) In the Discussion, on line 248-249, it is better to show clearly what is the "previous one on rhesus macaques" and what about the age range in that study.

(4) In the Discussion, it is better to make more explanation on the consistency between findings in this study and that in previous studies (e.g. line 225-228, line 237-240).

(5) In the Discussion, it is better to cite the findings on hand preference for other behavioral tasks in the focal species (e.g. Regaiolli et al. Hand preference on unimanual and bimanual tasks in Barbary macaques (Macaca sylvanus). American Journal of Primatology. DOI: 10.1002/ajp.22745).


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: PLASMA PROTEOMIC ANALYSIS OF SYSTEMIC LUPUS ERYTHEMATOSUS PATIENTS USING LIQUID CHROMATOGRAPHY/TANDEM MASS SPECTROMETRY WITH LABEL-FREE QUANTIFICATION
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1. The English language should be improved and hence an extensive revision of English by native speaker is recommended  (e.g. lines 355, 357-359, 368-371, 378-379, 395-397).

2. The discussion needs to be reorganized and more concise. The acute phase proteins should be discussed together (combine line 355-367 and 391-399 with line 327-334). Next, another proteins connected to inflammation should be mentioned and at the end of the
discussion others proteins should be discussed. It is good to group discussed proteins in relation to their similar function/pathway/process.

3. The mass spectrometry data should be available online and deposited to the ProteomeXchange Consortium via the PRIDE partner repository.

4. The information about standard deviation (SD) should be added to Table 3 as it is stated in method section (data are presented as a mean + SD).

5. Labels A, B, C should be added to Figure 4 and 6.

6. KEGG pathway analysis (Figure 5) indicated that 9 differentially expressed proteins were connected to complement activation. However, there is no information in the discussion which of identified proteins (and why) are engaged in complement activation. This could be potentially important in the pathogenesis of SLE. The same issue is for proteins related to coagulation.
Experimental design: No comment
Validity of the findings: 1. My main concerns regarding this paper are about statistical analysis of the data. In method section the authors did not mention that they performed p-value adjustment with Benjamini-Hochberg False Discovery Rate (FDR) (p<0.05) correction in order to test for multiple hypothesis. If it is not tested, the obtained results could not be reliable.

2. Why did the authors use three statistical test (independent t-test, paired-t-test, ANOVA) to determine the differentially expressed proteins between two groups? And p-values presented in Table 3 are related to which test?

3. How could the authors explain the huge discrepancies in fold changes between presented two methods of analysis for clusterin and hemoglobin beta subunit?
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: PLASMA PROTEOMIC ANALYSIS OF SYSTEMIC LUPUS ERYTHEMATOSUS PATIENTS USING LIQUID CHROMATOGRAPHY/TANDEM MASS SPECTROMETRY WITH LABEL-FREE QUANTIFICATION
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1. Language is generally clear and professional.  A couple of places need attention for language: (1) line 240 first paragraph of the Results section, there seems to be a word missing "the proteins at least two of the three..."  (2) line 325, first paragraph of the discussion also seems to have a word missing in phrase "significantly enriched by involved in the innate..."
2. Literature references are sufficient.  However reference #17 is an abstract, not a published article, and I would suggest that it is not suitable to cite unless there is a related full publication.
3. Structure, figures and tables are OK with the exception of Figure 1: for this type of study it is not necessary to show such a diagram and it can be eliminated.
4. Data are shared.
5. Relevant results presented; hypotheses presented.
Experimental design: 1. This is original primary research.
2. The research question is well-defined and it does fill a gap though it is in large part just confirmatory of other studies.
3. Technical approaches are appropriate; use of human subjects is ethical.
4. Methods are describe in sufficient detail.
5. Need to get more information about antibodies to dsDNA; the text states that all the patients were positive but the table of this information (Table 1) indicates a range of values.  Furthermore, we need to know what is considered the normal range for the laboratory.
6. In table 1 it is not necessary to indicate "disease status" as the SLEDAI score, which is elevated, confirms the active disease condition.
Validity of the findings: 1. Findings appear valid; as the authors note these findings will need replication by another approach or in another patient group to confirm.
2. Data are appropriately controlled; the patients are compared to a control group with similar features.
3. Conclusions are clear.
4. The utility of these findings as prognostic/diagnostic markers is at this stage speculative but possibly this is a direction for future research and the conclusion in this regard, as stated in the abstract, is appropriate.
Additional comments: In general these findings demonstrate utility of a technique that might uncover novel or informative markers and pathways in lupus.  At the present stage, the results are not especially novel; the pathways uncovered are ones that are generally well-recognized to contribute to lupus pathogenesis, such as those associated with complement and the acute phase response.  It is not clear yet that such an approach has diagnostic or prognostic utility over existing and available tests.


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: PLASMA PROTEOMIC ANALYSIS OF SYSTEMIC LUPUS ERYTHEMATOSUS PATIENTS USING LIQUID CHROMATOGRAPHY/TANDEM MASS SPECTROMETRY WITH LABEL-FREE QUANTIFICATION
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors revised the paper thoroughly according to my suggestions and I do not have additional comments.
Experimental design: No comment
Validity of the findings: The authors revised the paper thoroughly according to my suggestions and I do not have additional 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: FINE GRAINED COMPOSITIONAL ANALYSIS OF PORT EVERGLADES INLET MICROBIOME USING HIGH THROUGHPUT DNA SEQUENCING
Review round: 1
Reviewer: 1

Output:
Basic reporting: Insufficient background and context is provided for this study. Hardly any data are actually presented.
Experimental design: It is unclear how this study addresses a new (or even valid) research question as presented. Insufficient methodological detail is provided including basic information regarding which hypervariable region was sequenced.
Validity of the findings: Very little data is presented and the interpretations and conclusions of what is presented are not well validated.
Additional comments: The authors characterize the microbial community in an inlet by taking weekly samples for a year. The study is poorly presented, the data are not clearly shown, and the context and conclusions stated are inappropriate given the existing body of knowledge regarding aquatic microbial communities. It is unclear what hypervariable region was sequenced, the data are not publicly available, and it is not clear how the authors leveraged a weekly sampling strategy since hardly any data are actually shown.

Broadly, the abstract is a bit long, although within the journal guidelines, but could be made more clear by condensing general descriptions and adding in more statistical values. Discussion of phyla is not very informative. In contrast, the introduction is relatively brief and should be expanded to provide more literature, especially regarding next-generation sequencing studies of bacterial communities, to better contextualize this study. The results section is among the strangest I have ever reviewed as nearly no actual data are presented and analyses. This must be corrected. Furthermore, it is unclear why the authors are broadly evaluating communities at Phylum and Class levels, which are poorly informative and lend no value beyond what already exists in the literature. Similarly, the discussion has no focus and requires more specific direction and organization.

Specific comments
Abstract:
line 34: Please remove the sentence, "Over 1.4 million 16S rRNA V4 reads generated a total of 16,384 Operational Taxonomic Units (OTUs) from the PEI habitat". This is too minute a detail for the abstract.
line 37: Italicize Bacillus.

Introduction
lines 87-91: This is a rather long and confusing sentence. Suggest splitting into two.
line 94: Please revise as "next-generation sequencing characterization" or something more accurate. As currently written, I believe this overstates the scope of the characterization.
line 100: Please expand the introduction to provide some rationale for these hypotheses.
line 106: Please revise this final sentence. While this study does differ from those cited, there are many others studies that have performed NGS characterization of bacterial communities in various aquatic ecosystems.

Methods
The sampling strategy needs to be clarified. It is not clear how weekly sampling for a year at two sites results in 82 samples. This should intuitively be 52 or 104 samples. Additional information is also necessary describing the PCR amplification e.g. primers used, numbers of cycles, any barcoding, etc.
line 116: Correct "taken".
line 120: Correct to "by vacuum filtration".
line 136: Remove "raw". The first two sentences here are also redundant.
line 140: At what similarity were OTUs called?
line 159: Why is regression analysis treated separately from other statistical analyses? And what is the justification for a = 0.10 here?
line 164: These should be referred to as POTENTIAL pathogens.

Results
line 170: 82 samples were stated in the results, but there are 151 here. Please clarify.
line 174: Were samples rarefied to account for different numbers of reads between samples?
line 182: Regarding taxa present in >1% of samples, of course these are the most abundant. What information is this sentence meant to convey?
line 183: Why are none of the alpha diversity indices presented? Where is this data? And how are these indices not significantly different? Chao1 values are orders of magnitude greater than Simpson or Shannon indices.
line 187: Please revise this sentence to be more clear. Was August diversity greater or less than other months?
Figure 1: Please revise so that some taxonomic classification is presented along the x-axis. Otherwise, this figure is relatively meaningless.
line 196: The use of this 1% cutoff is very strange. Why not talk about actual abundances of taxa?
line 197: This sentence belongs in the discussion.
line 205-209: This is methods.
line 220: Please correct "than"
line 220-221: methods
line 227: Corrections for multiple comparisons should be performed when interpreting these results.
line 232: Why were analyses performed at class level? More specific taxonomic characterizations should be used to allow better interpretations of results. The entirety of this paragraph is also otherwise shown in the table. Please revise.
Chemical data: Why are these data not presented?
line 234-235: This description is not necessary.
lines 251-266: If the authors mean to address differences in relative abundances, these differences should be assessed statistically.

Discussion
line 277: What was the basis for this expectation?
line 282-284: It is unclear how this idea is connected to the rest of the paragraph.
line 287: Citing of unpublished sources does not provide a valid reference.
line 291: This statement is not accurate. The current study cannot confirm comparisons with other sample types that were not evaluated.
line 292: But the beginning of the discussion supposes that samples were not marine. Please clarify or explain.
line 309: If the authors wish to discuss rainfall data, it should be presented in the results.
line 339: What is the "microbial loop"? Please clarify.
Line 409-410: This statement is not accurate. The authors cite previous studies from their own lab as well as Staley, 2017 which previously characterized these commmunities. Please remove this sentence.


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: FINE GRAINED COMPOSITIONAL ANALYSIS OF PORT EVERGLADES INLET MICROBIOME USING HIGH THROUGHPUT DNA SEQUENCING
Review round: 1
Reviewer: 2

Output:
Basic reporting: Overall, the article is well-written and structured, most citations are appropriate, the raw data is available, and the article is self-contained. However, the following concerns should be addressed:

1. The introduction, especially the second paragraph, seems to be focused more on biogeochemical cycles and only describes potential pathogens in the marine system in one line. However, at least a third of the discussion is only on potential pathogens found in the data set. The introduction would be improved by adding more context in this regard.

2. While the majority of citations are appropriate, I find it odd that on line 78, three citations are used to show that a reef is directly offshore from the sampling site. I would think only one would be necessary or perhaps a map without any citations? In addition, on line 107, the study by Carsey et al is attributed to the year 2012, while in the reference list, this study is said to be in preparation.

3. The article is well-written and easy to follow overall, but there are a few inconsistencies in style and a few grammatical errors, including:
a. line 113, L does not need to be defined as it is the S.I. abbreviation
b. line 115, in situ should not be hyphenated and should be italicized
c. line 122, 'gel was run' is technical jargon
d. line 152, Kruskal-Wallis should be capitalized as it is in other mentions in text
e. lines 202 and 335, Portiera should be capitalized
f. line 237, there is a space needed between Flavobacteriia and (R2=0.21)
g. from line 210 – 250, p-values are sometimes reported as P, others as p, and others still as italicized p. I realize that different style guides suggest any of the above for p, but they should be consistent throughout the text.
h. line 277, Is ICW defined elsewhere?
Experimental design: The experimental design is sound, and the methods are detailed sufficiently for others to replicate the study except for a few areas, which are detailed below.

1. The primers used to amplify 16S rRNA genes should be explicitly stated. While the Illumina Sequencing Library Preparation guide does detail which primers are used, it leaves the readers to first consult this guide and then to consult the Klindworth et al paper for a full description of the primers used in this study. I would suggest directly citing the Klindworth paper.

2. The current study uses cd-hit to assign OTUs but fails to state which cut-off is used, which is up to the user when using cd-hit. Was the default (97%) cut-off used?

3. What was the justification used for having an alpha level at 0.10 for the Multiple Least Squares Regressions? Few of the significant results would be altered by using the more standard alpha level of 0.05. Even with the current alpha level, the statement that the relationship between Flavobacteriia and salinity is false as the stated p-value of 0.101 is above the alpha level used.
Validity of the findings: The findings and conclusions are logically stated and are borne out by the data and statistical analysis. I only have one issue that require clarification.

1. The present study refers to Chloroplast as a class. As far as I am aware, Chloroplast is an organelle and not a class of bacteria, which the text seems to make it out to be. Does this class designation refer to all 16S sequences that were assigned to belong to various chloroplast genomes? Please clarify what is meant by class Chloroplast and do these sequences annotate as specific types of eukaryotic algae.
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: FINE GRAINED COMPOSITIONAL ANALYSIS OF PORT EVERGLADES INLET MICROBIOME USING HIGH THROUGHPUT DNA SEQUENCING
Review round: 1
Reviewer: 3

Output:
Basic reporting: The language was clear and unambigous.
References were clear and correct.
The figures are not great and not very clear what they are trying to show. They need much more work in order to illustrate the message clearer.
I couldn't find a link or other to the SRA or other repo where the data is.
I couldn't really tell from the data presented that there was a difference between wet and dry - at least as presented. Especially from the NMDS plot.
Experimental design: These were fine. The plots need some cleaning up. But, the methods were will defined.
Validity of the findings: Once again, the plots really are confusing and don't say much relating to your message. I think it is there it just needs some more clear figures to show it.
Additional comments: Fix the figures to match the text better and you should be good to go.


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: FINE GRAINED COMPOSITIONAL ANALYSIS OF PORT EVERGLADES INLET MICROBIOME USING HIGH THROUGHPUT DNA SEQUENCING
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have significantly improved this manuscript upon revision.

I suggest the number of figures be reduced by combining Figures 1 and 2 into two separate panels of the same figure. Figures 4 and 5 as well as 6 and 7 could be similarly combined.

Finally, the raw data need to be made available in the Sequence Read Archive at NCBI or a similar depository and an accession number should be provided.
Experimental design: The description of the potentially pathogenic bacteria is still extremely difficult to interpret. As noted in specific comments, how these analyses were performed needs to be clarified since it is unclear in the revision which taxonomic level was being interrogated.
Validity of the findings: The authors would like to claim a fine-grain sampling strategy on the basis of weekly sampling, but the data presented represent only a monthly survey of the microbiota. As such, the authors need to clarify this, including revision of the title, to reflect the nature data presented.
Additional comments: Specific comments:
line 30: Change "will be" to "were".
lines 32-33: Monthly sampling is not uncommon, so the statement that this represents a higher frequency or finer scale than other studies is misleading.
line 40: What is meant by "significantly high abundances"? How was this level of significance determined? please also clarify at line 52.
line 49-51: The statement about N and P should be omitted unless this was evaluated statistically.
line 53: Suggest deleting the sentence starting "To our knowledge...".

lines 109-110: It seems a word is missing here.
line 111: The abstract states that monthly samples were analyzed, but weekly is stated her. Please clarify.

line 135-136: Please provide additional information how quality was determined. e.g., Did the samples that were dropped have low concentrations of DNA?
line 155: Should this be updated to QIIME 2?
line 178: Please provide a reference for the ANCOM test. (Mandal S, Van Treuren W, White RA, Eggesbø M, Knight R, Peddada SD (2015) Analysis of composition of microbiomes: a novel method for studying microbial composition. Microb Ecol Health Dis 26:27663)
line 182: Spell out PCoA at first use.
lines 193-200: This section is confusing as it appears genera were picked based on evaluation of order abundances. There is also the mention of OTUs. Which taxonomic level was evaluated?

line 255: Please specify "genera" instead of "taxa".
line 266: What is meant by "lower than expected"?
line 270-1: This sentence is redundant with the methods. Please delete.
line 278: Please specify "potential" pathogens.

line 325-369: Please break this section up into several paragraphs. I also suggest deleting the discussion of Rhodovibrio and Acholeplasma since the information presented is merely a description of the genus and is unrelated to the current study.


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

Input:
Title: FINE GRAINED COMPOSITIONAL ANALYSIS OF PORT EVERGLADES INLET MICROBIOME USING HIGH THROUGHPUT DNA SEQUENCING
Review round: 2
Reviewer: 2

Output:
Basic reporting: The manuscript is much improved from the initial submission. I appreciate all the work that was done to answer the questions drawn by the reviewers. While it has improved, I do have a few issues that I feel should be addressed before publication:

1. In the abstract, it says that the samples were collected monthly, however, in text, the samples were stated to be collected weekly.

2. The text states that 82 samples were sequenced, but the submission to the NCBI short read archive only has 81 different runs. Was one run poor quality or just lost in the fray during submission? I understand that not all of the runs were used for analysis.

3. Table 1 is formatted in such a way that makes it difficult to read. The OTU names are particularly useless. I'm not sure what it is supposed to be selling me on in its current iteration.

4. In figures 6 and 7, I assume that the historical means used are from the average of every month between 1948 and 2000 as they are flat lines. Is this really the best thing to compare monthly data to? Was this a mistake and was the average from each month supposed to be presented instead? Or is the average temperature and rainfall from month-to-month the same?
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: FINE GRAINED COMPOSITIONAL ANALYSIS OF PORT EVERGLADES INLET MICROBIOME USING HIGH THROUGHPUT DNA SEQUENCING
Review round: 2
Reviewer: 3

Output:
Basic reporting: The text looks much better. But, the pathogenic bacteria is a tough one for me. I would dial back the language on that a bit.
I think a plot on the pathogenic genera at the strain level would be very helpful. And, some brief language on how MiSeq isn't perfect and a reference to curtis huttenhower paper debunking the pathogenic bacteria in the subway.
I think it would be good to except with that infomation and some more cleaning of the figures/tables.
If they are fixed and I can't see them let me know.
Experimental design: The methods sections are greatly improved.
Yes, I think this section is up to par.
Validity of the findings: This was fine.
Additional comments: If they are fixed and I can't see them let me know.
Table 1 - use OTU numbers like OTU1 etc with the taxonomic id. These numbers as presented are useless and makes it hard to review or better yet plot this!

Table 2
The words are running down on the next line. plot or fix

Figure 1 and 2
Please replot in ggplot2 and facet it to combine into one figure. Also make the y-axis larger

Figure 3
remove grey background - theme_bw(), would look much cleaner

Figure 4 and 5
Instead of stacked use a side by side taxa on the y-axis for the phyla it would be easier to read. The colors need to be fixed as well.

Figure 6 and 7
Please replot in ggplot2 and facet it to combine into one figure. Remove the default excel plot for a fresher and cleaner look.

Figure 8
Instead of stacked use a side by side taxa on the y-axis or facet it. The pink is a terrible color.

Figure 9
Same as figure 8 remove purple. However combining figure 8 and 9 would be better and a barplot on the overall abundance of bacillus including a narrow look at strains of straph and bacillus pathogens.

I think if these could be fixed then it's okay for me.


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

Input:
Title: FINE GRAINED COMPOSITIONAL ANALYSIS OF PORT EVERGLADES INLET MICROBIOME USING HIGH THROUGHPUT DNA SEQUENCING
Review round: 3
Reviewer: 1

Output:
Basic reporting: All comments have been successfully addressed.
Experimental design: All comments have been successfully addressed.
Validity of the findings: All comments have been successfully addressed.
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: FINE GRAINED COMPOSITIONAL ANALYSIS OF PORT EVERGLADES INLET MICROBIOME USING HIGH THROUGHPUT DNA SEQUENCING
Review round: 3
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The current iteration of the manuscript represents a marked improvement from the initial submission. The wording is much more clear, the rationale behind the study is evident, the methods are sound, and the findings are presented and interpreted in an appropriate manner. I have no further comments on what could be improved.

I would like to thank the authors for taking into careful consideration the comments from myself and the other reviewers.


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: SCREENING OF POLYHYDROXYALKANOATE-PRODUCING BACTERIA AND PHAC-ENCODING GENES IN TWO HYPERSALINE MICROBIAL MATS FROM GUERRERO NEGRO, BAJA CALIFORNIA SUR, MEXICO
Review round: 1
Reviewer: 1

Output:
Basic reporting: The phaC sequences from isolated strains (MF939169, MF939170, MG201834, MG201835) and also from direct sample amplification are not present in Genbank. Presumably they have just not been released yet. They need to be provided for review.
Experimental design: The aim of the study was to isolate novel PHA producing organisms from two hypersaline microbial mats from Guerrero Negro, Baja California Sur, Mexico, and also directly detect phaC genes from the samples by PCR amplification, followed by sequence characterization. PHA producing bacteria were successfully isolated by plating on four different agar media, identified by 16S rRNA sequence, and in some cases phaC genes amplified and sequenced. Sequences were also amplified directly from the samples, sequenced, and characterized.

Line 199: of 62 colonies obtained — indicate on which of the four culture media they were obtained. This information seems to be present in the strain designations in Table 2, but should be explicitly stated. As it stands, the information provided is inadequate. Also, it would be helpful to know which of the four media each of the isolates are able to grown on.
Validity of the findings: More information regarding the hypothetical protein sequences that were amplified should be provided. Unfortunately the sequence data has not been provided in review, so it is not possible to evaluate.
Additional comments: Line 201 — clarify that Gram-negative were also stained with Sudan Black

The isolate that didn’t amplify — perhaps try to amplify with some of the other phaC primers?

It should be discussed that the authors did not appear to isolate any of the strains that correspond to the sequences they were able to directly amplify, although this remains uncertain, as it was not 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: SCREENING OF POLYHYDROXYALKANOATE-PRODUCING BACTERIA AND PHAC-ENCODING GENES IN TWO HYPERSALINE MICROBIAL MATS FROM GUERRERO NEGRO, BAJA CALIFORNIA SUR, MEXICO
Review round: 1
Reviewer: 2

Output:
Basic reporting: The article is mostly written in clear and technically correct language. However, in places non-standard terminology is used, e.g. "codifying" instead of "encoding". The text needs to be checked for correct use of plural and singular forms as well as repetition of terms.

Existing literature is sufficiently presented and appropriately referenced, given that the existing research on the subject is not particularly abundant.

The structure of the manuscript is professional and concise. The article structure is clear and mostly easy to follow and relevant results are presented. However, it is not clear how the qPCR data contributes to the research questions.

Figures and tables are generally relevant and their presentation clear. However, readability of Figure 3 could be improved by reducing the number of different raster templates and using grey shades with fewer templates. In Figure 4 caption it is not clear what is meant by "Sequences showing discrepancies with 16S", please clarify.

The authors state all sequence data being deposited to GenBank, however I was only able to access 16S sequence data but not phaC sequence data.
Experimental design: The article presents clearly original primary research and forms a sufficient entity for a publication. Research questions are rather generally formulated and could be made more specific although research presented is exploratory by nature. The research is pursued with adequate technical standard, however I have few remarks mentioned below that need to be addressed.

Major comments:

1) It is unclear how pure cultures (lines 113-114) were obtained from the initial  serial dilution plates- how many purification steps were performed before genomic DNA recovery? If the genomic analysis was done directly on initial colonies the authors need to prove their initial colonies were monoclonal.

2) The non-specificity of primers used also in qPCR undermines the validity of environmental phaC2 gene detection. As the results of the qPCR analysis do not add much to the discussion or overall conclusions of the paper I suggest them to be omitted.

Minor comments:

1) Lines 139-140: As nested PCR strategy was used associated risk of PCR bias needs to be discussed.

2) Line 156 & Line 233: indicate BLAST version used

3) Line 165: how CD-HIT threshold was selected ?
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: EFFECTS OF NETTLE SLURRY (URTICA DIOICA L.) USED AS FOLIAR FERTILIZER ON POTATO (SOLANUM TUBEROSUM L.) YIELD AND PLANT GROWTH
Review round: 1
Reviewer: 1

Output:
Basic reporting: the article is well written.
L38: replace significantly with significant
L 91 : the use of the world “real is ambiguous. Do you mean certified?
In introduction add work by Virgilio et al 2014 "the potential of stinging nettle(Urtica dioicaL.) as a crop with multiple uses" who extensively described the chemical composition of Nettle.
Figure 6:  the use of the same letters to designate parameters and treatments is confusing. Please change the designation. For instance you can use Roman Numerals I, II, III for parameters or better to use parameters names: Height, Nb of leaves, Leaves length.
Figure 7: the names on figure need to be fully written in the legend
The use of the letter a in this figure is confusing. We do not know if it means no difference between treatments or no difference between parameters
Figures 8 and 9: legends are incomplete: numbers and letters need to be clearly corresponded with treatments, dates and water regimes.
Table 2: legend is repeated twice (in title and under the table). Chose a way and keep it consistent with other tables. Explain where points A, B, C, and D were to be found on the experimental site.
Table 5 title should be: “Average yield achieved in each treatment”. The information on  statistical tests should be found in legend.
No need for all these columns in this table. Leave only the first 5 columns (A, treatment, N, Mean, HSD, se).
Table 6: same remarks as for table 5
Table 7 and 9: I do not see a necessity for this table. What is mentioned in the text is enough
Table 19: is repeating same information as figure 7. Keep only one of them. If you choose to present it in a table, than the title needs to be changed to: “Chlorophyll A, B and total Chlorophyll content by treatment”. Keep details on statistical tests for the legend of the table.
Experimental design: In material and methods: describe agriculture management of the plots. Detail your fertilization program: Was any soil fertilizer (such as compost) used? Or did you rely only on foliar fertilization? Was the use of nettle slurry complementary or the essential fertilizer in your fertilization program?
Validity of the findings: No comment
Additional comments: A wide range of biologically based products are now available on the market that claim to boost crop growth and help plants withstand many plant diseases. However, there are few independent, scientifically-based studies to validate the efficacy of some of these products. This is one of them.
Manuscript well written,
solid and robust experiment,
Complete statistical analyses.


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

Input:
Title: EFFECTS OF NETTLE SLURRY (URTICA DIOICA L.) USED AS FOLIAR FERTILIZER ON POTATO (SOLANUM TUBEROSUM L.) YIELD AND PLANT GROWTH
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript is clear and well written. the literature is well expanded, the figures are clear and many. However, the total volume is too large for a published paper in a journal. The manuscript is closer to a thesis than a paper, which should be taken into consideration in order to make it a concise and easier to follow.
The parameters measured are many and could be reduced. For instance there is no need to measure Chlorophyll A and B if the hypothesis is the yield and growth effect of nettle slurry. Yield and growth data are sufficient to answer that question. The discussion section can be more suitable for such a detail, or even another paper altogether.
Experimental design: The design is acceptable, and is pertinent to the question. The research question is clearly formulated, and answers the lacking area on use of this type of slurry. The investigation however is too expansive and I mentioned previously.

One note regarding your soil analysis: your results showed a low nitrogen level in the soil, the methodology should clearly state if nitrogen fertilizers were applied or not to correct the deficiency you reported. The effect of nettle slurry if present would probably be very subtle, the growth effect of nitrogen or lack thereof will be the dominant effect since nitrogen is the one of the more important determinants of yield. If you added nitrogen, please do mention it, and if not, then the question would be is foliar fertilization with a low nitrogen source even detectable in a low nitrogen soil.
Validity of the findings: Refer to the section above.
Data analysis is well presented and the methods are sound.
The conclusion is relevant to the discussed parameters.
Additional comments: The work is detailed and accurate. the design is well formulated and executed, however the scope is too expansive as mentioned earlier, and many parameters can be left out, to reduce the size and benefit the readers and saving time.
The remarks on nitrogen are important to take into consideration.


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

Input:
Title: EFFECTS OF NETTLE SLURRY (URTICA DIOICA L.) USED AS FOLIAR FERTILIZER ON POTATO (SOLANUM TUBEROSUM L.) YIELD AND PLANT GROWTH
Review round: 1
Reviewer: 3

Output:
Basic reporting: Some more references are needed in the introduction - see General Comments
Experimental design: no comment
Validity of the findings: no comment  but see General comment for a few details
Additional comments: I am aware that nettle slurry has been proposed as fertilizer and pest control in organic farming but I was not aware of any work proving its effectiveness.  Therefore, I find this work highly interesting and much needed.
The paper looks fine and should be published with minor revisions.

A few notes
The authors might spend a few words addressing a couple of important issues 1) cost of such treatment (how it may impact on the farm economy), 2) scale, that is… how much land it’d take to produce the amount of treatment to be provided to an ha of organic crops (ha/ha), that will expand the amount of land that organic farming will require to produce an unit of food

Line 38-40 “and their relation with agriculture (Michelsen, 2001).”
You may provide some more recent literature as well.  Michelsen (2001) is now a nearly 20 years old paper, which is the situation today? (in general I’d prefer to refer to a few references, not just one, that would better help to frame a statement/claim and may help to reduce presenting biased narratives coming from the view of a single author)

Line 40-42  “ Organic food consumption is associated with health beliefs and subjective well-being, which involves higher market values and demand (Apaolaza et al., 2018)”
There are review works that indicate that organic food is also associate with important benefits for human and environmental health
See for example
https://www.sciencedirect.com/science/article/pii/S0929139317302573
https://onlinelibrary.wiley.com/doi/abs/10.1002/jsfa.6578
https://onlinelibrary.wiley.com/doi/abs/10.1002/jsfa.6836

Line 42-43 “Moreover, in the next few years, agriculture will be pushed to become more sustainable as a global response to climate change.”
This statement seems a bit on the air… is organic farming an answer? In which terms? The authors should be aware that some scholars are critical concerning the sustainability of organic farming as, they claim, its low yield would force cropping a larger amount of land.
Of course, the paper is dealing with a specific issue, therefore the authors are not required to prove a review on the matter. Anyway, providing a few more information may help to improve the introduction.

Line 111 “ annual rainfall was 381.8 mm and it rained 58.5 mm” you may round those values

Line 351-361 “The chemical analyses performed during this experiment showed a very low total nitrogen content of the Urtica slurry (0.005%) and the Equisetum slurry (0.002)”
The authors may explain (referring in particular for the case of on farm self-made products) whether the place where plants growth/are harvested may significantly affect, or not, plant composition (concerning the effect tested in the paper). Could we expect important differences to occur comparing industrial and self-made products?


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: CRYSTALLIZATION AND STRUCTURE ANALYSIS OF THE CORE MOTIF OF THE PKS13 ACYLTRANSFERASE DOMAIN FROM MYCOBACTERIUM TUBERCULOSIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Yu et al report the purification of recombinantly expressed, full length Pks13 from Mycobacterium tuberculosis (MtPks13), together with the crystallization and structure determination of a 110-residue proteolytic fragment. The 110-residues are part of the acyltransferase domain in the polyketide synthases, that is responsible for substrate selection and transfer to a dedicated acyl carrier protein. The structure does not provide significant new information, as the crystal structure of the intact 52kDa MtPks13-AT domain is deposited in the PDB (PDB ID: 3TZW.pdb) and well described by Bergeret et al (2012) (doi:  10.1074/jbc.M111.325639). The authors should include detailed comparison to 3TZW.pdb, e.g. rmsd values (http://www.ebi.ac.uk/msd-srv/ssm/cgi-bin/ssmserver). The reported minor structural differences do not significantly support the authors conclusion that this structure represents an inactivated state of the enzyme, which would have been useful for drug discovery.

The manuscript would benefit from language proofreading from a native speaking colleague. In the crystallographic table are some points that require attention:
Sodium is stated as a ligand, but the submitted PDB file, does not contain a Sodium ion.
No rotamer outliers are stated, but the provided PDB-File has 2.4% rotamer outliers (according to wwwPDBvalidate)
The clashscore according to wwwPDBvalidate is 11, higher then current standards.


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: CRYSTALLIZATION AND STRUCTURE ANALYSIS OF THE CORE MOTIF OF THE PKS13 ACYLTRANSFERASE DOMAIN FROM MYCOBACTERIUM TUBERCULOSIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Yu et al. report the structure of the core motif of the acyltransferase domain of PKS13 from Mycobacterium tubercolosis. PKS13 is an important drug target to inhibt the cell wall biosynthesis of the bacterium.

Although the structural data seems solid the manuscript needs intensive improvement for publication. Some aspects are not clear (or clear enough). As an example, the strutcure reported here seems to be a degradation product of the full-lenght protein production. This is stated in lines 43-45 but it remains unclear if this is observed during protein purification or if degradation is occuring during the crystallization process? Another example: Why was Se-Met-labeled protein used although an MR model is available? The stucture comparison to the full length AT domain lacks important information like the rmsd. Additionally, an rmsd polt (rmsd vs residue number) might helpful.

The language of the manuscript serious corrections, e.g.

line 30:
.. locate in the C-terminal."

line 59:
...the C-terminal had a terminator codon."

line 111:
cell parameter units missing

line 120:
"In our structure..."

...

The research question of the article aims the desciption of different conformational states of the AT core domain. The structure reported here shows conformational changes in comparison to the full-length AT domain (pdb-code 3tzz) but it remains completely unclear, if these changes are really a different state of the protein, or just a result of protein degradation or just an artifact of the crystallization of a core protein. Further experiments and crystal packing analysis are needed. The manuscript totally ignores crystal packing influences.

The validity of the finding is my major concern of the manuscript in its current state. The authors claim that the conformational changes might indicate a different state of the enzyme. But from my point of view this is not experimentally confirmed.


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: CRYSTALLIZATION AND STRUCTURE ANALYSIS OF THE CORE MOTIF OF THE PKS13 ACYLTRANSFERASE DOMAIN FROM MYCOBACTERIUM TUBERCULOSIS
Review round: 1
Reviewer: 3

Output:
Basic reporting: This manuscript describes the 2.6 Å crystal structure of the N-terminal region of the Pks13 acyltransferase (AT) domain from M. tuberculosis. More extensive referencing needs to be provided, for example, concerning the description of catalytic mechanism of the condensing enzyme Pks13 and the work on phosphopantetheinyl (P-pant) transferase characterization and function in Mtb. The major focus of the paper is on the conformational changes occurring at the C-terminal extremity of this fragment with a possible link to the activation state of the enzyme. The crystallized fragment represents approximately one third of the AT domain, for which a structure has been published by Bergeret and colleagues (JBC 2012).
Experimental design: All the classical methods leading to structure determination have been described in details. The insert was cloned into a pET28 vector, transformed into E. coli BL21(DE3) cells, purified following a three-steps procedure, and then crystallized.

Major points
1-	There is no information on how this protein fragment has been selected.
2-	The use of the 4’-phosphopantetheinyl transferase Sfp from B. subtilis is not justified as modification occurs onto the ACP domain and not onto the AT domain, the subject of this work. Please explain
3-	The authors should justify the use of selenomethionine labeled protein considering that the X-ray structure of the full-length AT domain has been published (PDB code 3TZZ).
Validity of the findings: The Pks13 AT domain extends from amino acid 712 to amino acid 1035. The X-ray structure of a Pks13 fragment encompassing the full-length AT domain (amino-acid positions 597 to 1059) has previously been described (Bergeret et al. JBC 2012). Here, the crystallized fragment starts at position 717 and ends at position 826, in the middle of helix α11.

Major point
One structural feature of the full-length AT domain is the presence of a six-stranded β-sheet (β13-β12-β4-β5-β10-β11). Only residues forming the central β-strand β5 are present in the actual structure, at the C-terminal end. It is thus hard to imagine that the conformational changes observed in this region reflect a change in the activation state of the enzyme rather than a structural artefact arising from the absence of neighboring β-strands. In the absence of any experimental validation, this is pure speculation in my view.

Minor points
1-	Line 22 – Wrong reference
2-	Line 33 – Sfp from B. subtilis. Mtb also has two PPTases named AcpS and PptT.
3-	Line 48 – It seems premature to speculate on the orientation of the C-terminal helix considering that it is truncated.
4-	Line 96 and 111 – Missing ° and/or Å
5-	Line 111 and table 2 – Space group R32:H ?
6-	Reference section with numbering issues
7-	Figure 1 – Missing information on amino-acid positions, schematic representation in the legend
8-	Figure 2 – A structure-based sequence alignment with 3TZZ would be greatly appreciated. Also, indicate secondary structure elements on both sequence alignment and structure representation.
9-	Figure 3 – Legend and figures need to be modified. The term “channel-like cave” is not appropriate. A surface representation may be useful. A close-up view of figure 3b is necessary.
Additional comments: No additional comments