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: CYCLODIPEPTIDES FROM PSEUDOMONAS AERUGINOSA MODULATE THE MAIZE (ZEA MAYS L.) ROOT SYSTEM AND PROMOTE S6 RIBOSOMAL PROTEIN KINASE ACTIVATION
Review round: 1
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: Regarding the cyclodipeptides (CDPs) used in this study, authors refer to Ortiz-Castro et al. (2011), who isolated and identified three CDPs ( cyclo(L-Pro-L-Tyr), cyclo(L-Pro-L-Val), and cyclo(L-Pro-L-Phe).  First, autjors should show include mass spectra of purified CDPs fractions and also indicate if they used a particular CDPs of a mixture of the three isolated CDPs.

Authors should also clarify the selection of  used concentrations should be given, e.g.,12 uM of auxin.
Validity of the findings: In this study, authors showed that cyclodipeptides (CDPs) promote the growth of maize plant  by modulating the root system architecture. The CDPs  treatment elicited a dose-dependent increase in ZmS6K phosphorylation, suggesting the involvement of the TOR-S6K signaling pathway in the growth promoting effects of CDPs.
Although the study indicates a correlation between CDPs treatment and S6K phosphorylation, more analyses are needed to validate the involvement of TOR-S6K signaling in root growth promotion. In this regards, authors should test if the CDPs treatment increases TOR kinase and the ZmS6K activation resulted in S6 ribosomal phosphorylation

The activity of plant S6Ks has been shown to increase in response to auxin, also CDPs require a canonical auxin signaling pathway for activity (Ortiz-Castro et al. 2011). Thus CDPs induces S6K activity may be auxin dependent, please discuss it.
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: CYCLODIPEPTIDES FROM PSEUDOMONAS AERUGINOSA MODULATE THE MAIZE (ZEA MAYS L.) ROOT SYSTEM AND PROMOTE S6 RIBOSOMAL PROTEIN KINASE ACTIVATION
Review round: 2
Reviewer: 1

Output:
Basic reporting: 4B. In response to my MAJOR CONCERN 4, the authors have provided as supplementary information (Fig5wb2raw.png) the second blot western blot for p-ZmS6K. However, they did not provide the corresponding Western blot for unphosphorylated S6K that was requested during the first reviewing.  It is therefore not possible to claim that “The experiment was repeated twice with similar results” (legend Figure 5). Indeed, Fig5wb2raw.png suggests different results than Fig 5, particularly regarding line 2 and 7. This might be due to differences of amount of protein loaded or S6K amount, hence the importance of the unphosphorylated S6K control.

If the authors have difficulties with the anti-human-phospho-p70S6K-Thr389 antibody, they might have better results with a plant specific antibody like ab207399.

5B. In response to my concern 5, the authors should simply write in the manuscript the amount of DMSO used.
Experimental design: no comments
Validity of the findings: no comments
Additional comments: The authors have properly responded to my comments exept for comments 4 and 5.


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

Input:
Title: CYCLODIPEPTIDES FROM PSEUDOMONAS AERUGINOSA MODULATE THE MAIZE (ZEA MAYS L.) ROOT SYSTEM AND PROMOTE S6 RIBOSOMAL PROTEIN KINASE ACTIVATION
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The authors made amendments that considerably improved the manuscript, and I consider that it is OK for publication.


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

Input:
Title: EARLY AGENIAN RHINOCEROTIDS FROM WISCHBERG (CANTON BERN, SWITZERLAND) AND CLARIFICATION OF THE SYSTEMATICS OF THE GENUS DIACERATHERIUM
Review round: 1
Reviewer: 1

Output:
Basic reporting: Latin names should be in italics; see for example line 212 (m. interossei), line 213 (m. extensor carpalis), line 433 (m. fibularis longus).
Experimental design: no comment
Validity of the findings: no comment
Additional comments: One of the best papers on rhinos I reviewed during the last year. Well-done.


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: EARLY AGENIAN RHINOCEROTIDS FROM WISCHBERG (CANTON BERN, SWITZERLAND) AND CLARIFICATION OF THE SYSTEMATICS OF THE GENUS DIACERATHERIUM
Review round: 1
Reviewer: 2

Output:
Basic reporting: As a whole, the manuscript is well organized and clear, but there are some sentences or paragraphs for which I suggest some modifications in order to clarify the idea or the authors’ position (e.g., concerning systematics).
The Introduction is brief and synthetic, and I suggest not revealing the precise taxonomic results (see PDF).
Figures are relevant, of high quality and well described. I would only suggest alluding to them sometimes within description for some of the highlighted features.
Experimental design: The presented work is a review of fossil remains previously published many years ago, which leads to a new taxonomic interpretation of some of them. Some are figured for the first time. In addition, some comments on the controversial systematics of the genus Diaceratherium are also included. It is in this issue where I suggest a more detailed author’s opinion to avoid incongruences in the text (see PDF).
The review is based on morphological and metrical comparisons with the known European species of the epoch (Early Miocene).
I think metrical data could be gathered in one or more tables instead of being included within the descriptions, and maybe some of the compared specimens could be added to reinforce the affinities expressed in the text.
Concerning Table 3, it only shows the length of the metapodials of the discussed species. I think it would be more informative showing the whole available dimensions of each metapodial, both those studied (dimensions within the text) and those compared (see comment in PDF about Table 3).
Validity of the findings: The presented comparison (concerning morphology and size) supports the taxonomic results, but better explanations in the Discussion section are needed and tables could be improved to be more informative (see PDF). Particularly, the discussion on the validity of species of Diaceratherium needs to be expanded and reviewed to avoid incongruences.
Additional comments: I think all my suggestions do not imply major revisions of the manuscript, but most would be necessary before publishing it.
Only if the editor consider it necessary, I could evaluate the revised version.
The attached PDF shows all my comments and suggestions.


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: EARLY AGENIAN RHINOCEROTIDS FROM WISCHBERG (CANTON BERN, SWITZERLAND) AND CLARIFICATION OF THE SYSTEMATICS OF THE GENUS DIACERATHERIUM
Review round: 1
Reviewer: 3

Output:
Basic reporting: The English expression is excellent, at least for the non-native English speaker I am. References are up-to-date and the state of the art correctly exposed. The structure is straightforward and rigorous. I have no comment about the figures and raw data are shared. Results are relevant, new, and well supported, based on the material described.
Experimental design: This manuscript meets the technical requirements of PeerJ and it matches its Aims and Scope. The research question is relevant and the results answer a 70 year-old question on the taxonomic identity of Wischberg rhinocerotids. Furthermore, this MS provides an interesting discussion on the genera Diaceratherium and Pleuroceros, with tantalizing perspectives at the European scale. I have found neither scientific nor technical flows and ethical issues are not into question. Methods are OK and the rationale is crystal clear.
Validity of the findings: I do agree with all the taxonomic assignments and commend the authors for solving this 70-year old puzzle. Figures and measurements are provided. Repeatability is ensured by clear and thorough description, following a proven descriptive protocol. Statistics are not applicable here (but see my suggestion on body weight estimates).
Additional comments: This is an interesting and useful work, providing new robust data on a classic locality from Switzerland. I have minor comments and suggestions aiming at improving the manuscript. You will find them below, sorted by order of appearance in the manuscript. I have also uploaded an annotated version of the merged pdf (mainly used for typos).

-line 36: please provide the original words abbreviated as USM;

-lines 100-108 (and Table 8 + Palaeoecology…): I would advise to be more careful with body weight estimates based only on lower molars, due to tremendous disparity in body shape and stoutness among rhinocerotids, likely to range from tapir-like to dachshund-like while having similar tooth size (Fortelius, M. 1990. In: J. Damuth and B.J. MacFadden (eds.), Cambridge University Press, Cambridge). It would be really useful to further provide estimates based on postcranial elements, such as long bones, metapodials (Fortelius, M. and Kappelman, J. 1993. Zoological Journal of Linnean Society, London 108: 85–101), and/or astragali (Tsubamoto, T. 2014. Acta Palaeontologica Polonica 59 (2): 259–265);

-lines 204-209: what is the general shape of the anterior side (high, low, narrow, wide)?

-lines 218-249 and 455-530: I would recommend to use your specimens from Wischberg as a reference and to overturn the corresponding sentences, such as “The referred specimens have a developed external groove and an angular trigonid on lower molars, contrary to representatives of Diaceratherium” (line 220).

-lines 360-361, 601 (and throughout the MS): please homogenize spelling (UK [centimetres / palaeobiogeographical] or US [characterized]);

-line 419 and throughout the MS (e.g., lines 517, 524):
“astragal” shall read "astragalus" or "astragali" (plural);

-line 447: does “lacking” mean broken or absent?

-line 517: shall read “collum tali”;

-Figure and table captions: to be complemented by providing a family-level assignment [“the rhinocerotid” or “(Perissodactyla, Rhinocerotidae)”] so that they are fully self-explanatory.


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: COMPARATIVE TRANSCRIPTOMICS REVEALS THE DIFFERENCE IN EARLY ENDOSPERM DEVELOPMENT BETWEEN MAIZE WITH DIFFERENT AMYLOSE CONTENTS
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment.
Experimental design: Enhance description of the research question (end of Introduction).
Validity of the findings: Enhance concluding remarks at end of the manuscript.
Additional comments: The current manuscript “Comparative transcriptomics reveals the difference in early endosperm development between maize with different amylose contents” is a solid work that compares two maize lines with different amylose content at different stages of endosperm development through transcriptomics analysis. It provides valuable data for research in the area by suggesting key genes and regulatory networks that are fundamental of this stage. It already suggests some interesting ideas on the role of some genes in starch synthesis, but the major work remains to be done – exploration and confirmation of the provided data. It is overall well written, with some exceptions.

I have some comments and then a list of small errors to be corrected.

1) At the end of the Abstract, add some potential benefits that this knowledge (timing of endosperm development) has for agricultural production or any other application. Also clarify your working hypothesis at the end of the Introduction. That includes clarifying what is “other essential metabolisms” (line 120).

2) Line 41: what do you mean with “maize starch is one of the best-quality starches, with a purity that can reach 99.5%”. This may be linked to extraction and purification protocols, which may be well developed for maize but not for other crops. Please clarify.

3) Gene and mutant nomenclature are not consistent nor accurate. Example: line 65 (stands for the rest of the manuscript): globby-1 should be in capital letters, similarly to other gene names. In addition, the gene should be named GLOBBY1, and the mutant globby1-1 (example: Costa et al 2003). Also check for consistency in italics when referring to a gene (example lines 81-82). In addition, make sure to describe names of proteins and their roles throughout the text. Indicate what mutants are you referring to (example: line 104-105)

4) Lines 291-294: clarify in the Results section how the 16 clusters were obtained, and how many genes are included in each cluster.

5) Lines 547-554 consider rephrasing the entire paragraph, it is confusing in some parts and the English clearly needs to be enhanced. Consider as well stating again the agronomic importance/applications of future advancement of this research line.

6) Figure legends:
- figure 1B does not show a “vertical axis”, consider rephrasing
- figure 7: consider describing the different colors and to what genes are they referring to

And then some small details:
- line 26, add “with” after “correlated”
- line 44: I would say endosperm development starts “after” double fertilization, and not “through” as stated now. Please clarify when during plant development the endosperm starts being formed and when is it fully established.
- lines 46 and 50 (stands for the rest of the manuscript): double check text for consistency, example cellularisation VS cellularization
- line 89: state the name of the SSIIa and SBEI proteins
- line 104: add “in” before “nonmutant”
- line 105: clarify if higher levels of amylopectin (in the wx1 mutant) result in lower amylose content
- line 130: consider rephrasing, the way it is stated now it seems that the discarded material was frozen in liquid N2 and used for RNA extraction
- line 189: “Eleven DEGs…” correct the rest of the sentence
- line 234: add “developmental” after “four”
- line 273/274: double check the DEG numbers that are detailed in the text VS the numbers show in Suppl Figure 3 (example SD609 4905 VS 4914)
- line 299: add “were” after “clusters”
- line 485: add “seen/described” after “been”


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: COMPARATIVE TRANSCRIPTOMICS REVEALS THE DIFFERENCE IN EARLY ENDOSPERM DEVELOPMENT BETWEEN MAIZE WITH DIFFERENT AMYLOSE CONTENTS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The present manuscript submitted by the Xue and Guo et al., is an attempt to enhance our understanding of spatiotemporal regulation of starch biosynthesis in maize plant using a comparative transcriptomics approach. Authors selected two contrasting varieties of maize, of which one is having high amylose content (HS68) and the other one has a normal (SD609). The global transcriptome has been carried out at 4 developmental stages day after pollination (DAP 5, 10 15, 20) in both cultivars. As a result, thousands of genes were identified differentially regulated during 4 developmental stages of the two varieties. Of these, authors identified core genes involved in starch synthesis pathway in both maize varieties with limited success. For example, this is surprising that O2, an endosperm-specific TF affects the expression of starch biosynthetic pathway genes (SSIIa, SSIII, SBEI) reportedly shows high expression in transcriptome and qRT-PCR but not present in the core gene group. Overall, the present manuscript is well organized, written and presented. I think this manuscript deemed fit for publication after minor revisions.

First and foremost, I would like to see more information from this huge chunk of transcriptome data. It can be meaningful if authors can perform promoter analysis of clustered genes. What kind of common up-stream regulatory elements are involved in regulating the starch content in HS68 and SD609? This is an important question to know.

There is a discrepancy in no. of genes mentioned in the text (Line no. 248) and shown in supplementary table S3. In table S3, the no. of genes are mentioned as 28144 for HS68 genotype at 20 DAP but in the text, it is 22358 for HS68. Which one is correct?.

What is the FPKM value? Authors should abbreviate in the text at least at the first occurrence (Line no. 241).

Fig. 1C is a bit complicated, I guess it should redraw to make it simple and self-explanatory.

Line no. 269, please mention the name of two specific developmental stages.

Supplementary figure 3 is quite confusing. Authors say 1193 (7%) genes were differentially regulated between SD609 and HS68, what does it mean? In the same figure, 4919 (31%) genes were uniquely expressed in SD609 and 2002 uniquely expressed in HS68. All these genes are differentially expressed. How it (1193) has been derived from the total no. of genes 15922?

Line 271-274, this sentence needs to be rearranged for better clarity.

Line no. 277-278, what does it mean by the “Material-specific DEGs were also found to regulate the same biological process.” What does the word “material-specific” imply?

In table S7, author should remove the clutter by filtering the data on the basis of FDR value (<0.05). By this way, data can be viewed and interpreted in a better manner.

In line no. 282, authors used the word "maize species" I suggest "varieties" or "cultivars" will be the correct word.
Experimental design: For better clarity, the experimental design needs to be illustrated schematically.
Validity of the findings: Altogether, the findings are backed by the data. The data is analyzed appropriately and statistical parameters are robust. I do not see any problem in this section.
Additional comments: The present manuscript submitted by the Xue and Guo et al., is an attempt to enhance our understanding of spatiotemporal regulation of starch biosynthesis in maize plant using a comparative transcriptomics approach. Authors selected two contrasting varieties of maize, of which one is having high amylose content (HS68) and the other one has a normal (SD609). The global transcriptome has been carried out at 4 developmental stages day after pollination (DAP 5, 10 15, 20) in both cultivars. As a result, thousands of genes were identified differentially regulated during 4 developmental stages of the two varieties. Of these, authors identified core genes involved in starch synthesis pathway in both maize varieties with limited success. For example, this is surprising that O2, an endosperm-specific TF affects the expression of starch biosynthetic pathway genes (SSIIa, SSIII, SBEI) reportedly shows high expression in transcriptome and qRT-PCR but not present in the core gene group. Overall, the present manuscript is well organized, written and presented. I think this manuscript deemed fit for publication after minor revisions.

First and foremost, I would like to see more information from this huge chunk of transcriptome data. It can be meaningful if authors can perform promoter analysis of clustered genes. What kind of common up-stream regulatory elements are involved in regulating the starch content in HS68 and SD609? This is an important question to know.

There is a discrepancy in no. of genes mentioned in the text (Line no. 248) and shown in supplementary table S3. In table S3, the no. of genes are mentioned as 28144 for HS68 genotype at 20 DAP but in the text, it is 22358 for HS68. Which one is correct?.

What is the FPKM value? Authors should abbreviate in the text at least at the first occurrence (Line no. 241).

Fig. 1C is a bit complicated, I guess it should redraw to make it simple and self-explanatory.

Line no. 269, please mention the name of two specific developmental stages.

Supplementary figure 3 is quite confusing. Authors say 1193 (7%) genes were differentially regulated between SD609 and HS68, what does it mean? In the same figure, 4919 (31%) genes were uniquely expressed in SD609 and 2002 uniquely expressed in HS68. All these genes are differentially expressed. How it (1193) has been derived from the total no. of genes 15922?

Line 271-274, this sentence needs to be rearranged for better clarity.

Line no. 277-278, what does it mean by the “Material-specific DEGs were also found to regulate the same biological process.” What does the word “material-specific” imply?

In table S7, author should remove the clutter by filtering the data on the basis of FDR value (<0.05). By this way, data can be viewed and interpreted in a better manner.

In line no. 282, authors used the word "maize species" I suggest "varieties" or "cultivars" will be the correct word.


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: COMPARATIVE TRANSCRIPTOMICS REVEALS THE DIFFERENCE IN EARLY ENDOSPERM DEVELOPMENT BETWEEN MAIZE WITH DIFFERENT AMYLOSE CONTENTS
Review round: 2
Reviewer: 1

Output:
Basic reporting: I am glad to see that the authors have addressed the questions and concerns raised by reviewers and editors sincerely. They have thoroughly revised the manuscript wherever necessary. Authors have replaced the figure 1C by a better explanatory figure. They have added the upstream regulatory element analysis (Fig. 9) that further strengthen the manuscript. The language is revised, factual errors are rectified. At this juncture, the manuscript is deemed fit for the publication.
Experimental design: This section is well written with every detail so that it can be reproduced.
Validity of the findings: Now manuscript is in good shape and findings are clear with regard to high starch biosynthesis related transcriptomic responses.
Additional comments: As above.


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

Input:
Title: THE EFFECT OF MODULATING THE QUANTITY OF ENZYMES IN A MODEL ETHANOL PATHWAY ON METABOLIC FLUX IN SYNECHOCYSTIS SP. PCC 6803
Review round: 1
Reviewer: 1

Output:
Basic reporting: This study reports a set of experiments aimed at the assessment the effect of altering the expression level of individual enzymes of a heterologous pathway, in the metabolism of a cyanobacterium, on the level of production of the product of that pathway. The manuscript is technically well-written, and the majority of the experiments has been carried out with state-of-the-art techniques. The design of the study, however, suffers from two basic weaknesses:
1] In a study in which the effect of variation of the expression level of heterologous enzymes is investigated, the factors limiting product formation should be clearly defined. Because of the choices made, this aspect is not properly covered: In many conditions presumably the availability of light will have been the limiting factor, which then makes it obvious that a poor correlation between product level and enzyme expression level is observed.
2] As a metabolic engineering study, the most relevant property to assay – next to final product level – is the amount of active enzyme present in the cells. This amount may differ from the total amount of enzyme-protein present, due to: misfolding of the overproduced enzyme,  post-translational modifications, etc. Information on this aspect is fully lacking in this study.
Experimental design: There are several technical imperfections identifiable in the manuscript. Examples are:
a)	The description of the physiological experiments (Cells were grown up to OD730nm of 0.5 and IPTG was added to final concentration of 1 mM. After 7 days, 1 mL of every culture was taken, the cells were spun down (4000 g, 10 min) and the ethanol content of the supernatant quantified by HPLC……..) is insufficiently detailed and does e.g. not allow one to calculate final product titers to be calculated. Disappearance of ethanol and/or acetaldehyde from the system is not taken into account.
b)	Genetic instability of the transgenic strains is not addressed, and the check of strain construction with PCR (lines 107-108) does not provide sufficient detail).
c)	Several figures and aspects of figures are redundant (e.g. Fig. 2 and background colors in Fig. 3A).
d)	The landmark study in this field (Gao Z, Zhao H, Li Z, Tan X, Lu X (2012) Photosynthetic production of ethanol from carbon dioxide in genetically engineered cyanobacteria. Energy & Environ Sci 5:9857. doi:10.1039/c2ee22675h) is not in the reference list. Presumably the titers in the current study are much lower than in this study.
Validity of the findings: Several single observations (on e.g. operon length, order of genes, effect of reading frame on RBS strength, etc.) are generalized without proper justification. Also, justification of the selection of AtpB as the base for normalization of enzyme expression levels is lacking.
Additional comments: My evaluation of this manuscript is that it needs a complete re-write and shortening, in which the limited number of lessons learned are clearly described.


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

Input:
Title: THE EFFECT OF MODULATING THE QUANTITY OF ENZYMES IN A MODEL ETHANOL PATHWAY ON METABOLIC FLUX IN SYNECHOCYSTIS SP. PCC 6803
Review round: 1
Reviewer: 2

Output:
Basic reporting: The article “Lessons learned from optimization of an introduced metabolic pathway in Synechocystis sp. PCC 6803” describes an interesting principle-based design approach to assess the mutual impact of different RBS variations on gene expression from a dicistronic operon in a model cyanobacterium. The authors used a selection of 5 well-established RBS variants to drive the likewise well-established ethanol biosynthesis pathway, consisting of pyruvate decarboxylase and alcohol dehydrogenase. This work is relevant to the fields of synthetic biology and metabolic engineering (in cyanobacteria), and it introduces an important aspect of synthetic operon design for optimized and balanced gene expression in cyanobacteria.
The language of the article is largely clear and unambiguous. Some exceptions are listed below.
Regarding the literature references, a few further suggestions are given below that might also improve the discussion.
The structure of the article is professional, all figures are relevant and of good quality. Minor concerns about figure captions are listed below.
Unfortunately, I have two major concerns regarding (i) the lack of physiological/ growth data and (ii) the lack of actual (unprocessed) raw data in the manuscript/ supplementary files.
(i) In all EtOH experiments titres were normalized to the cell density (OD730), without providing the (a) voluminetric titers and (b) the optical densities (except for Fig. 7). There is also an inconsistency between the main manuscript (figures) and the supplemental data, where apparently the same values are annotated as volumetric titres (e.g. Fig.3, 5, 6, 7- Ethanol (g/l)). This would make sense, if the OD values were ~1, but they should be provided in the manuscript.
(ii) The provided data in Supplementary file 2 are not actually raw, but already processed and normalized values that correspond to the provided diagrams. For sound reproduction of the data, all actually measured values would be required.

Abstract
1.	line 26: I suggest to use the more common ‘alcohol dehydrogenase’ instead of ‘acetaldehyde reductase’, and since the enzymes are named 3 times in the abstract, ADH and PDC might be used.
2.	line 29 : “operon design and modifications to native metabolism on pathway flux was monitored” … sounds too ambitiously worded for what has actually been done (and monitored). Lines 40-43 should be sufficient for this aspect
3.	line 38: “translational coupling can also occur in cyanobacteria.” rather: “…can be implemented by design in cyanobacteria…”

Introduction
The introduction gives a brief overview of the state of knowledge regarding the challenges and available solution in synthetic pathway design. The first passage (until line 54) might be improved by adding the aspect of intra- and intergenic ‘compositional context’ of genetic constructs, referring to the recent publications of Cambray et al., 2018 (https://doi.org/10.1038/nbt.4238) and Yeung et al., 2017 (https://doi.org/10.1016/j.cels.2017.06.001). These papers are also of relevance to this manuscript as they support the discussion of different relative protein levels using different CDS   (line 245).  Accordingly, in the next subsection (lines 55-63), the paper by Thiel et al 2018 (doi: 10.1186/s12934-018-0882-2.) should be cited as a work that has already addressed the influence of CDS in cyanobacteria.
4.	line 50: citation format. “For example, (Zelcbuch et al. 2013) clearly…” should be “For example, Zelcbuch et al. (2013) clearly…”
Methods
While the analytical part of the methods section is very elaborate, the cyanobacterial culture conditions should be described with more details and technical specifications, since these can be very critical to reproducibility of data derived from phototrophic conditions:
5.	General inconsistencies regarding µl/ml vs. µL/mL: microliter is mostly written ‘µl’, milliliter mostly ‘mL’
6.	General suggestion: Full plasmid sequences (at least exemplarily) might be provided as supplement data file (e.g. genbank format) or on an open data repository
7.	lines 86,90 and several time in lines 133-142: °C –> gap between number and unit sometimes missing
8.	line 84: “other cyanobacterial strains ‘derived thereof’…”
9.	line 84-86: more specifications of the cultivation equipment might be provided (shaker orbit, type of light source [fluorescence, diodes?])
10.	line 85: BG-11 ‘media’ - > ‘medium’
11.	line 86: how was 1% CO2 supplied (CO2 atmosphesre or bubble column)? Which light source (e.g. fluorescent vs. photodiodes, white light vs. specific wavelengths)? Which shaker was used?
12.	line 106: final concentration ‘of’
13.	line 115-123: was an internal and/or external standards used for calibration of HPLC analysis? How were the data calculated?
14.	line 127: extraction buffer
15.	line 127-128: 500 µL

Results and Discussion
16.	lines 189-191: I suggest to briefly introduce the basic pathway “Glycolysis  Pyruvate  Acetaldehyde  Ethanol” here for synthetic biologists without background in metabolic engineering. It could also be included in Fig. 2 for a clear overview
17.	also a schematic/representative plasmid map to briefly depict the vector composition and the modules could be added to Fig. 2
18.	lines 198-199: “These sequence variants were encoded within linker sequences (“RBS linkers”)”
	what does ‘encoded’ mean in this context?  should it rather be replaced by ‘attached to’ or ‘flanked by’?
19.	line 203: “shown to exhibit some IPTG-inducibility in cyanobacteria”. Rather provide a more specific or quantitative characteristic than “some inducibility”. Also change ‘cyanobacteria’ to ‘Synechocystis sp. PCC 6803’.
20.	lines 214-216: to give a quantitative idea of the ‘dynamic range’ of this RBS series, e.g. the maximal expression ratio/fold-change (RBS A vs. RBS E) should be provided
o	also in the following passages (lines 233-247; lines 250-259) quantitative statements regarding the respective expression ranges would improve the informative value of the data description

21.	Line 227-228: Synechocystis was “transformed” with the constructs, not vice versa
22.	Lines 244-245: this discussion can be supported with more recent literature (Thiel et al., 2018)
23.	Lines 263-264: “Comparing protein levels, Adh abundance was in fact more strongly correlated with Pdc abundance (R2 = 0.51).” Compared to which comparison are these protein abundances more strongly correlated?
Also: ADH-PDC protein correlation plot should be shown as well, since a clear statement is made here.
I suggest to discuss these data more carefully, since two similar R2 values are interpreted in a positive (R2 = 0.51 -> translational coupling) and a negative (R2 = 0.44 -> ADH overexpression might be redundant) way, respectively
24.	Lines 278-279: what does ICL stand for?
25.	Lines 284-291: also here, differential characteristics of DNA topology and/or RNA secondary structure might play a role in different expression levels
26.	Line 307: slr1395 -> italics

Figures and Captions
27.	In general: there are several cases, where there is a space missing between two words (e.g. Synechocystissp.) in several captions
Figure 1:
28.	 While the method section says that “The fluorescence was normalized
 against wild-type strain control and cell density.”, there is no indication for that neither in the diagram, nor in the caption. “Fluorescence (measured)” rather indicates raw data. The y-axis label and caption should be more specific.
29.	RBS in front ‘of’ eyfp

Figure 2:
30.	The caption should contain more information to be self-contained

Figure 3:
31.	In the methods section the construct series were named “RBSx_eYFP and RBSx_EtOH”. For consistency, I suggest to stick to this specification in this (and the other) caption(s)
32.	“Mean average ethanol titres of each cultures…”  “…culture…”

Figure 5:
33.	This caption should refer to Fig. 3+4

Figure 6:
34.	The caption lacks the information of OD normalization, which is indicated on the Y-axis. According to the data in suppl. File 2, these values are g/L values. These inconsistencies need to be checked throughout all figures/data

Supplementary Material

Supplementary Table 1
35.	Synechocystis and E. coli  italics
36.	the BASIC prefix and suffix should be labeled (which is in the forward and reverse sequences?)
37.	the PA1lacO1 reverse sequence is completely in bold

Supplementary Table 2
38.	for ‘RBS E’ the annotation ‘RBS*’ should be added to the ‘additional information’ column (according to Heidorn et al)
39.	the table caption should explain the capitalized nucleotides
Experimental design: The experimental design is overall sound and well-chosen to address the central question. The use of state-of-the-art techniques, in particular regarding cloning and protein quantification is further inspiring for follow-up projects in the field of cyanobacterial synthetic biology.
However, a major drawback of the design is a lack of physiological analysis, particularly regarding growth characteristics of the examined strains. In lines 221-222 the authors write: “Further, we wanted to correlate the output to protein levels and understand the impact on growth.” Growth characteristics are not provided, except for the final experiment, where all strains harbored the same RBS combination for PDC and ADH (Fig. 7C). And here, only endpoint data are provided.
Particularly regarding the potential acetaldehyde toxicity, there might be a bias in growth and biomass accumulation between strains with low and high ADH levels. All EtOH data are (according to the main document) normalized to optical density. If higher PDC abundance would result in higher acetaldehyde titers and potentially growth inhibition, similar (or even lower) acetaldehyde to EtOH convertion rates might result in higher apparent per-cell EtOH titers. This is certainly important for the conclusion that “abolishment of
reductase over-expression resulted in the greatest ethanol productivity”. Therefore, growth curves – or at least endpoint OD730 values – need to be disclosed in order to exclude a bias from severe growth effects.
Validity of the findings: The data are statistically well analyzed. However, there are inconsistencies regarding the normalization of EtOH titres between main document and supplement. More transparency regarding physiological data/ growth would be important to substantiate the interpretation of the results.
See also previous sections.
Additional comments: I suggest to run a replicate experiment with selected strains in order assess and discuss the physiological outcome (i.e. growth curve vs. EtOH accumulation) of the different PDC/ADH titres over time.


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 EFFECT OF MODULATING THE QUANTITY OF ENZYMES IN A MODEL ETHANOL PATHWAY ON METABOLIC FLUX IN SYNECHOCYSTIS SP. PCC 6803
Review round: 1
Reviewer: 3

Output:
Basic reporting: The title “Lessons learned from ..” fails to represent the manuscript and looks like that of review articles. Several keywords such as “ribosome binding site sequences”, “translational coupling”, and “operons” should be included.
Experimental design: Line 267, “The relative abundance of Pdc was generally higher than Adh regardless of which RBS was used”. The claim was not supported by the experimental data since ” Protein quantification was performed based on relative peak intensities of the analysed protein” . Please determined the expression levels of Pdc and Adh proteins by using the suitable standard peptides.
Validity of the findings: Fig. 3 The data shown in Fig 3B is not required since the results are summarized in Fig. 3A
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: THE EFFECT OF MODULATING THE QUANTITY OF ENZYMES IN A MODEL ETHANOL PATHWAY ON METABOLIC FLUX IN SYNECHOCYSTIS SP. PCC 6803
Review round: 2
Reviewer: 1

Output:
Basic reporting: The manuscript has improved a lot and I am happy that the authors addressed all concerns, either by supplying the requested information/ changes, or by convincing argumentation.

However, I have a remaining concern regarding the statement in line 222-223: "The strains exhibited minimal differences in growth curves and final optical density values"
According to the added supplementary data (Fig. 2) there are actual, gradual differences of up to ~30% in the final OD730.  This is not exactly 'minimal' and should be described more accurately, since the ‘impact on growth’ is clearly defined as part of the study (line 209) . In particular, strains harboring RBS B for PCD and RBS C for ADH show higher final ODs compared to the rest.

The growth curve diagram in Suppl. Fig 2 refers to data on a local drive, which is not remotely accessible (and hence no graph is visible!); I therefore suggest to provide these data in a supplementary spreadsheet or in an online repository, in accordance with the journal's 'open data' standard. Also, raw data of the ODs for the YFP normalization (Fig.1) are not yet provided.

I suggest to publish the study ASAP after some brief revision of the mentioned points.
Experimental design: OK
Validity of the findings: Underlying data not fully provided, See 'Basic reporting'.
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: ENDOGENOUS TESTOSTERONE CORRELATES WITH PAROCHIAL ALTRUISM IN RELATION TO COSTLY PUNISHMENT IN DIFFERENT SOCIAL SETTINGS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript should be proof-read by a native English speaker. There was awkwardness and grammatical issues throughout. Furthermore, the paper did not follow a clear style, particularly in regards to numbers and use of parentheses.

I also find the terminology throughout the paper confusing. Altruism seems to be used interchangeably with favoritism. This does not seem accurate. It also seems like altruism is being used when spite should be used. In terms of the behavioral ecology literature, altruism is doing something beneficial for someone else (+) at some cost (-) to yourself. Spite is doing something detrimental for someone else (-) at a cost (-) to yourself. This convention seems not to be followed in this manuscript. This makes both the intro and discussion sections confusing. See for example: Bshary R.& Bergmüller R.. 2008. Distinguishing four fundamental approaches to the evolution of helping. J. Evol. Biol. 21, 405–420 (Box 1.1).
Experimental design: Generally, the experimental design was appropriate. However, the writing of it, at times was confusing.
Validity of the findings: The validity of the finding were challenging to interpret, as the results of the ANOVAs were not presented in a typical way. The authors, at times, focused on main effects when there was a significant interaction and did not report or explain the simple main effects of that interaction. I am also a bit unconvinced about findings that did not reach statistical significant and had a small effect size. P-values are not everything, but if it is not significant and there is a small effect size, this is rather unconvincing.
Additional comments: Line 69: What do you mean by unique scale? That is unclear. If you are trying to say this is uniquely human, I disagree. If you mean to a large extent, I do agree.

Line 72 – you go straight into the behavioral econ literature, but I am sure this has been shown in other contexts. Perhaps this would be worth mentioning.

81 – I would remove “than their own”

Line 83-84 – This is what I was talking about in my above comment. Perhaps giving an example would make this more impactful.

86 – I think you need to define exactly what you mean by altruism. Do you mean behaving in a way to benefit someone else at a cost to yourself? Or do you mean ingroup favoritism as in line 71? I don’t think you can use altruism and favoritism interchangeably. I suspect what you really mean here is favoritism. I think bringing in altruism might confuse the issue.

108-109: Clarify -  “testosterone has recently been proposed to play an important role in mediating the effect of testosterone”

125-130 – This is a very dense sentence. I would consider making this multiple sentences.

137 – missing word: “even in the absence…”

147 – Given world politics at the moment, I think you need a better justification about why political parties would not have a strong tribal identity. It seems like your explanation in lines 151-155 could also potentially apply to political parties.

160 – please clarify why you did this. Was it to make arbitrary groups? How was performance divided? How explain this in the methods. I might avoid talking about the maze until then so it is more clear.

217 – use of parentheses

238 – This is a bit unclear. Are you using the data from your previous study?

250 - use of parentheses

251-252 – this would be more clear without the use of a double negative. Is problematic rather than is not unproblematic.

255 – I am not sure of your use of the word punishment. This typically mean a consequence of behavior. Here, would they be punishing themselves by being generous? I am not following this.

263 – So you are looking at respondent reactions rather than proposer offers. This is why I was confused in my above comment. You need to say that particiants are the respondent. I assumed they were the proposer.

277 – People often think of the potential motivations for the UG as either inequity averse or selfish. However, selfish behavior could also be a rationally maximizing strategy. As you can’t disentangle these potential strategies with the UG, I don’t like the term pro-selfs. At the very least, you should include an acknowledgement that this might not be selfish, but rather rational. In other words, people acting rationally, may not be responding to inequity at all. This is in contrast to your statement that they have a high tolerance for unfairness. I would prefer you call them self-oriented subjects as you sometimes do in the discussion and figures.

284 – Were novel pictures used in both sessions? In other words, in session 2 did people see some of the same faces they saw in session 1? Would this effect the results?

296 – I am a little confused about why you talk about session 2 at all if you are not going to analyze it.

Line 329 – This actually suggests you are correct and they are being selfish. If rational, they wouldn’t reject offers differently between in-group and out-group. However, when you are setting up the study, the logic from my comment at line 277 maybe should be addressed.

Line 328 or so – You say you ran an ANOVA, but you are not reporting the ANOVA results. This should be reported. Actually, I now think that you are sort of mixing the methods and results sections. I would not report what the pro-selfs did until the results section. It is a bit strange to report some, but not all, statistics here.

348 – Why use nonparametric statistics for post hoc comparisons with an ANOVA?

396 – Was approachability also done in study 1? If no, why not?

400 – In what way was it extraordinary? Do you mean an outlier?

406 and other places – please follow the style of PeerJ. Number 1-9 should be spelled out unless used with units. https://peerj.com/about/author-instructions/

431 – Were the total points available still 10? If so, why did you not let them make generous offers?

Section 2.2.6 – Same comment as the previous statistics section. It feels as if you are mixing results in here. I would prefer you to talk about all of study 1, then all of study 2.

Section 3.1. I feel the way you are presenting the ANOVA is confusing. You should address interaction effects before main effects, as some main effects may not be interpretable depending on the interaction. You need to explain the significant interactions.

479 – You identify a trend here, but this is not significant and the effects size is weak. Therefore, this is not convincing to me as a trend that should be reported.

480 – You seem to be talking about the three-way interaction, but only of team and offer, not at each level of testosterone.

482 – Do you mean there were no significant simple main effects? A post-hoc is typically only for one independent variable whereas simple main effects look at the how the level of each IV is effected at each level of the other IVs.

485 – The test statistic, Z, should be reported as well.

486 – Why do you include the p value in line 485 but not 486? Be consistent.

Section 3.1 – I think a graph of the significant interaction would help the reader interpret your results.

503 – Again, this is not significant and only has a small effect size. Therefore, I am not sure you should interpret this. A graph would help.

522 – Supposedly? If this is not your interpretation then this should be cited. Otherwise I am not clear why you said that.

543 – Please interpret the interaction. A graph would be beneficial.

Section 3.2 – It would help the reader to include means and SEM when you talk about where the differences are in the ANOVA.

569 – I am not following the logic of why the correlation coefficient would decrease and that would show a similar strength…

599 – use of parentheses

607 – I know that p-values are not the only way to evaluate effects, but when you have a trend, to really be convincing, the effect size should be large. Again, this is a small effect size.

599 – This is the first time you have mentioned social discounting.

701-702 – forgoing points to punish others would be spite, not altruism

763 – I again don’t understand why you are calling this altruism when it seems to be spite. Spite seems to me to be a more parsimonious explanation.

766 – Do you mean to begrudge the other player?


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: ENDOGENOUS TESTOSTERONE CORRELATES WITH PAROCHIAL ALTRUISM IN RELATION TO COSTLY PUNISHMENT IN DIFFERENT SOCIAL SETTINGS
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: underpowered. The authors have a 2 (offer) x 2 (group) x 2 (decision strategy) x 2 (testosterone - median split) design for study 1, and a  2 (offer) x 6 (rank) x 2 (decision strategy) x 2 (testosterone - median split) for Study 2.  Study 1 had n = 61 men, while study 2 had n = 34 men.  See my comments in the attached.
Validity of the findings: Nice that the findings from Study 2 are similar (though weaker and more variable) than those of Study 1
Additional comments: please see attachment for more specific 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: ENDOGENOUS TESTOSTERONE CORRELATES WITH PAROCHIAL ALTRUISM IN RELATION TO COSTLY PUNISHMENT IN DIFFERENT SOCIAL SETTINGS
Review round: 1
Reviewer: 3

Output:
Basic reporting: The authors present a well-written paper on the role of endogenous testosterone levels, intergroup membership, and individual differences attitudes toward inequity in predicting rejection behaviors in the ultimatum game. The authors seemingly replicate their findings across two intergroup settings- one using a minimal group paradigm and another using a naturalistic, political context.

Below, I state some broad comments for the authors to improve their paper:

The authors highlight inconsistencies in the testosterone literature concerning ultimatum game findings. However, they do very little to integrate their study both in the introduction and discussion within the context of those inconsistencies. The authors find that both inter group context (in-group vs. out-group) and the individual difference of decision-making strategies toward inequity serve as possible moderators to explain the inconsistent testosterone-rejection behavior findings. The authors could consider using that framing to integrate their research with the extant literature.

There is little theoretical rationale provided for examining the role of fairness-related decision-making strategies in testing their hypothesis.
Experimental design: There is little information on why the cued memory recall paradigm was included in the study, until I reach the results section of Study 2. What was the theoretical rationale? Was this measure primarily used as a manipulation check? Also, why did the authors not consider looking at the testosterone x group interaction for this particular DV?

What was the rationale for the authors measuring testosterone in the morning and not in the afternoon (testosterone usually shows stable hormone concentrations between noon and 5 pm)? Also, did participants self-administer these hormone samples? In which case, how were the samples stored and transported?
Validity of the findings: The authors perform parametric inferential statistical analyses but follow-up those overarching tests with non-parametric analyses. Is there a rationale for doing so? If there is, the authors may consider including that in their paper. The authors collected nested data, and multilevel modeling may afford them more degrees of freedom in examining such data. If anyone on the author-group possesses expertise to conduct those analyses, I would highly encourage them to do so.

Minor comment: In Study 1- the authors report fairly small degrees of freedom for their error term in spite of having 61 participants and a repeated measures design. I may be missing information that was reported previously that explain these small degrees of freedom and/or the authors conducted analyses within a sample sub-group which may be another alternative explanation for the smaller degrees of freedom. It will be nice to know why the testosterone x team x offer interaction had such small degrees of freedom.

To test the relationship between testosterone and parochial altruism, the authors conducted post-hoc analyses among proself individuals by assessing non-parametric correlations between testosterone levels and unfair offer rejection (ingroup vs outgroup difference scores). These analyses are exploratory (in my view) but extremely interesting. However, I would appreciate if the authors first reported the overall interaction between testosterone x decision-making strategy type predicting rejection rates before delving into simple slope analyses where they could examine correlations between testosterone and rejections across the individuals with different strategies. Further, for the sake of transparency, I also encourage the author to report the 4-way interaction between testosterone x offer x group x strategy in their main analyses.

It is unclear to me if the authors did indeed find an interaction between testosterone x group x offer in Study 2? They do not clearly report whether this interaction was present before they proceed to examine the correlations between testosterone and rejection rates. It would be ideal if the authors followed the same/similar reporting order and style as Study 1 for ease of comprehension for the reader.

My biggest concern with this paper to do with the sample sizes used by the authors to test fairly complex and a large number of statistical analyses (although they do correct for their p values during post-hoc analyses). To demonstrate their effects, the authors also adopt a split-by-condition approach, which in turn reduces their statistical power substantially. However, the replication of the findings broadly across both study 1 and study 2 gives me some confidence in their findings. I encourage the authors to acknowledge sample size as a limitation in their discussion section. I do think that examining the boundary conditions of testosterone’s relationship with parochial altruism is a valuable contribution to the psychoneuroendocrinology literature and hope that the authors will consider conceptually replicating these findings in larger samples in the future.

Further, in order to test the replication of the effects reported across both studies, the authors may consider conducting a mini-meta analysis within their paper. Or figure out a way to combine both datasets and examine the combined effects of the testosterone x group x offer interactions and correlations between testosterone and rejection rates across proself vs. inequity averse groups.

In general, I enjoyed reading the discussion section of the paper, especially the portions that integrated their current work (submitted to this journal) with their prior research (which I am familiar with). However, because the study only comprised of male participants, I would encourage the authors to speculate about how their findings may extend in a mixed sex or all female sample.
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: ENDOGENOUS TESTOSTERONE CORRELATES WITH PAROCHIAL ALTRUISM IN RELATION TO COSTLY PUNISHMENT IN DIFFERENT SOCIAL SETTINGS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have greatly improved the manuscript from the first draft in terms of presenting the theory, using consistent and clear terms and the presentation of results. The ANOVA tables were particularly helpful. With these changes, this now meets the standards of PeerJ. There were still a few typos and I mention these in the general comments section below.
Experimental design: Experimental design was appropriate.
Validity of the findings: With the inclusion of the ANOVA tables and the way the results have been revised, the conclusions appear to be valid. I have three minor questions.

Line 473 – Does this mean that low T men are more sensitive when offers are close to fair? Why would they discriminate between the ‘fair’ offer types less than men with high T?

Line 606 – why would this be? Does scoring high enough on the German political interest scale correlate with treatment of humans in economic contexts? Perhaps people interested in politics are more conscientious than others

Results – when you followed up the ANOVA with a post-hoc t-test, did you correct the p-value to account for multiple analyses? You mention you did this in line 516, but it is unclear whether than applies to all analyses, or only the one you reference in this paragraph.
Additional comments: 147 – offers

176 – extraneous parenthesis

181 – You haven’t mentioned the use of photos yet, so this may be unclear. I would add something to the effect of: ..same number of new faces that were used to represent players in the UG.

212 – extra “at” – at home the morning of

379 – incorrect word – the structure of the…

415 – here you use player A and player B for the first time. Unless this is used later, I would just say proposer and respondent.

739 – correlation, not correlations

772, 801 – author names are both in text and in parentheses


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: ENDOGENOUS TESTOSTERONE CORRELATES WITH PAROCHIAL ALTRUISM IN RELATION TO COSTLY PUNISHMENT IN DIFFERENT SOCIAL SETTINGS
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: the authors have done a good job addressing my concerns. I applaud the authors for making their data freely available. The paper makes a nice contribution to the literature.


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

Input:
Title: NICOTINAMIDE RIBOSIDE EXERTS PROTECTIVE EFFECT AGAINST AGING-INDUCED NAFLD-LIKE HEPATIC DYSFUNCTION IN MICE
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comments.
Experimental design: No comments.
Validity of the findings: No comments.
Additional comments: In their manuscript, Han and colleagues report the effects of nicotinamide riboside, a nicotinamide adenine dinucleotide precursor, on development of age-induced non-alcoholic fatty liver disease and inflammation liver in mice. Identifying novel, effective therapeutic compounds for liver diseases is an important goal, and the paradigm used in the present study is interesting to investigate potential new therapeutics. The experiments seem to be well performed and the results are sound, but some points need to be addressed by the authors.

1.	In the keywords section, please do not use abbreviations;
2.	Overall, there are quite a few grammatical errors along with sentences that are difficult to interpret. This manuscript would benefit greatly from additional proofreading and editing; For example: the word “that” sometimes do not necessary (ex. first phrase od introduction section).
3.	The introduction section has much information, it is good and interesting, but the introduction needs a connection to improve the read;
4.	The introduction could use additional background information about the role of NR in other diseases;
5.	The last paragraph of introduction section, need demonstrate the objective, not results of manuscript; Please, in the phrase “In this study, we demonstrate that 18 months…” the word “demonstrate” need correction;
6.	Why the authors used only female? A complementary  study with male will be interesting;
7.	The authors have three groups, but I think that a group with young mice + NR improve the study;
8.	The NR compound causes toxicity in young mice? Are there some reports in the literature?
9.	Please provide a reference to “Mice were transcardially perfused with 4 ℃ saline”. Why not PBS?
10.	Please provide information about the induction of non-alcoholic fatty liver disease, only the age caused this disease?
11.	Please provide all F values, P values of data analyzed; The authors could make a table;
12.	Why the authors did not use the young group in the immunohistochemical:
13.	Please correct the phrase in the discussion section “The principal findings arose from the present study”.
14.	 The hepatic word is discussion section is “heptic”;
15.	Please change “…we show the proof that…, by “ In the present study, we show…”; and “Vitamin” by “vitamin”


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: NICOTINAMIDE RIBOSIDE EXERTS PROTECTIVE EFFECT AGAINST AGING-INDUCED NAFLD-LIKE HEPATIC DYSFUNCTION IN MICE
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript is well-structured with a substantial content of data and based on significant background.
The English language is used clear, though some words/expression could be changed (i.e. crux (l. 69); proofs (l. 76); hepar (l. 41)).
Experimental design: The data are original. In some aspects, there are similarities (in context) with the study “Nicotinamide riboside attenuates alcohol induced liver injuries via activation of SirT1/PGC-1α/mitochondrial biosynthesis pathway” (DOI: 10.1016/j.redox.2018.04.006), however, the results are different.
A major issue, that needs to be mentioned in methods/discussion, is the use of female mice for the study. The authors should mention why only female mice were studied, and potential hormonal interference in the experimental data.
Validity of the findings: The discussion is well stated with the findings of the study. In addition, the authors also mention future goals for the study.
Additional comments: The manuscript seems to be original, it is original and exciting. However, a major issue needs to be elucidated, regarding why only female mice were used. In the current form, the manuscript should be accepted with some clarifications.


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: TESTING THE FUNCTION OF DROMAEOSAURID (DINOSAURIA, THEROPODA) ‘SICKLE CLAWS’ THROUGH MUSCULOSKELETAL MODELLING AND OPTIMIZATION
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: REVIEW
My identity: Denver Fowler

This manuscript presents a musculoskeletal model of the hindlimbs of dromaeosaurid dinosaurs, seeking to address the question as to the function of the hypertrophied claw on digit II. The model showed that the highest forces occurred during crouched postures, rather than the extended position of the hindlimb required for some suggested behaviors. The study concludes that the D-II ungual is not well suited for exerting much force, and is more supportive of a grasping function.

The manuscript is well written and structured. I appreciate the author attempting to directly address some of the functional hypotheses using modeling of the hindlimb.

My comments concern only content, mainly related to definitions of prey bodysize (lack thereof in this MS), and characterisation of how body weight of the predator is transferred on to its victim when the predator is standing on top of prey. Other than these two small issues the manuscript is good. I would suggest acceptance with minor revisions.

COMMENTS (also see annotated word doc)
I don't think the abstract is clear enough about the difference between body weight for prey restraint (Fig 1. F; ie. Fowler et al, 2011 large and very large prey), and the suggestion that body weight is transferred to the claw tip for use in making longer wounds (fig 1 C, D):

" Interestingly, the magnitude of force capable of being produced at the claw tip was relatively small, arguing against it being used for transmitting a large proportion of body weight into a substrate. Collectively, the results best support a grasping function for digit II (e.g., restraint of small-bodied prey), although other behaviours involving flexed hindlimbs cannot be excluded."

At first read, this sounds like a rebuttal to our suggestion that a dromaeosaur might stand on top of its prey to hold it down using its body weight (as modern birds of prey do; Fowler et al., 2009; 2011). I suppose that our papers are perhaps not clear enough when we state that the inside toe (D-II) is better placed to exert body weight of the animal on to prey. As I have mentioned in the comments on the manuscript DOC, when a modern bird of prey is standing on top of its victim, its body weight pins down the victim - the body weight is not transferred only through the claws, but through the toes and metatarsus too (especially when the metatarsals contact the prey. This was well illustrated for birds of prey by Goslow, 1972). However, I understand that the current manuscript abstract is only talking about the D-II claw. I think this could be made more clear, because I don't think the findings of this analysis refute our suggestion that bodyweight helps pin down the prey, only that the bodyweight is not entirely/mainly applied through the  D-II ungual.

There needs to be a definition of what is meant by prey size categories - we do this in Fowler et al. (2009), and extend it in Fowler et al. (2011). We define prey size relatively: if prey can be contained within the foot of the predator, then it is a "small" prey item. If the prey cannot be contained within a foot, but can be held down by the body weight of the predator (if the predator stands on top of it) then it is "large" prey. If the prey is so large that it cannot be held down by the predator, then we call it "very large" prey.

Defining prey size is quite important as in the conclusions and abstract it is stated " Collectively, the results best support a grasping function for digit II (e.g., restraint of small-bodied prey" - but I don't think "small prey" is meant in the same way that we would define it (Fig 1, F, would be considered "large prey" in our definition).

Finally, I agree that the claws are probably not exerting major force, but this does not mean that they are not useful for subduing very large prey (under our definition, see above). Imagine that a person is clinging to the back of an angry cow which is shaking about violently (as you might see in some western agricultural shows) - the person uses their fingertips to hang on for grip, they don't exert much force on the fingertips, and certainly don't hang all their weight on their fingertips (as a rock climber might), but they are maintaining grip. In this situation the person is acting similar to what we describe as "prey riding" - a behavior observed in birds of prey that attack what we categorise as "very large prey" (see Fowler et al., 2009; 2011; and above). Thus even without exerting strong force, claws help maintain grip on a prey items of "very large" size (indeed, all sizes).


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: TESTING THE FUNCTION OF DROMAEOSAURID (DINOSAURIA, THEROPODA) ‘SICKLE CLAWS’ THROUGH MUSCULOSKELETAL MODELLING AND OPTIMIZATION
Review round: 1
Reviewer: 2

Output:
Basic reporting: The article is well structured, but I have some issues with the figures - I appreciate these models output a great deal of information, but I am not sure that Figures 4-6 in their current form are the best way of presenting it.

The raw data have been shared, but not all of it is in an immediately accessible format. The code itself can be opened and viewed in any good text editor, but the associated data files cannot. Would it be possible to output the modelling data as .csv files rather than Matlab data objects?
Experimental design: Regarding the values for pennation angles, how was the initial range of values chosen? As part of your sensitivity analyses you used a more restricted range of values which was "comparable to the range observed in the digital flexor and extensor muscles of extant archosaurs". Could you please explain and justify then the range chosen in the initial system configuration which includes values well outside those seen in extant archosaurs? Why did you not use the values from extant taxa to begin with and then extend the range in the sensitivity analyses? As far as I can tell, all the other assumptions and initial values (flexor and extensor lengths, muscle volumes etc), appear well explained and justified so why pennation angles aren't is puzzling.
Validity of the findings: Whilst I believe the overall findings are sound, the methodological questions I raised in the previous section mean I do have some differences in interpretation.  I think the author should base their interpretations around results of the models with realistic pennation angles akin to those seen in extant archosaurs, and discuss what is currently presented as their main findings as a sensitivity analysis to test the effect of pennation angles outside of what we know from extant taxa. This would change the results somewhat – when restricted to more “realistic” pennation angles claw forces are substantially higher at extended angles and erect postures. However, at the more flexed angles the author suggests the claws operate, force are still highest with a crouched posture.
Additional comments: I think this is a valuable contribution to the literature, using biomechanical models to explore possible functions of the famous "killing claw" of Deinonychus and other raptors. The author explores a wide range of parameter-space and sensitivity analyses to identify those features, e.g. limb posture, which consistently correlate to relevant biomechanical measures of claw function. I do have certain reservations about the paper in its current form, which I have outline above, but addressing these should be feasible as (I think) they only require re-interpretation or re-analysis of the model outputs presented in the paper and supplementary information, rather than a complete rerun of the whole modelling process.

In addition to my comments above, I feel the graphs in Figures 4-6 could be improved - showing every single one of the 108 curves leads to very busy plots. I think plotting averaged curves with confidence intervals or min/max ranges for individual conditions would be better (i.e. in Figure 4A have three curves with confidence intervals, one for each posture etc.). Or perhaps running principal components analysis on the force-angle data (where the input into the PCA would be the value of force at each MTP angle)? This would also provide a means of comparing both curve magnitude and shape depending on whether you scaled the values or not.

I also wonder whether it is necessary to display the results for the full range (-65 to 60) of MTP angles? I assume they are mostly for illustrative purposes, but in Figure 1 the only behaviours shown in which the claws might actually reach substantial flexion are digging or holding small prey. All other behaviours show the claw either only slightly flexed, or extended. If posture and claw angle could be somehow codified for these different behaviours (perhaps with an added table?) I think this would make the links between the modelling results and conclusions about behaviour easier for the reader to see.

I appreciate the work the author has done in this paper and I believe it should be published. Whilst my comments should hopefully be straightforward to address I would prefer to re-evaluate any revised version, and so in-line with PeerJ reviewer guidelines I must return a verdict of major revisions.


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: TESTING THE FUNCTION OF DROMAEOSAURID (DINOSAURIA, THEROPODA) ‘SICKLE CLAWS’ THROUGH MUSCULOSKELETAL MODELLING AND OPTIMIZATION
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comments
Experimental design: No comments
Validity of the findings: No comments
Additional comments: I have reviewed the revised manuscript and I think it is greatly improved. I appreciate the work the author has done on this new version in response to my comments and those of my fellow reviewers. The author has managed to sufficiently address all of my points from my review of their original submission. I am particularly thankful for the additional clarification and discussion around pennation angles and general variable choice in models such as this, as well as the new table (Table 2). I think the manuscript should be published in its current form.


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: HEALTHY EATING PROMOTING IN A BRAZILIAN SPORTS-ORIENTED SCHOOL: A PILOT STUDY
Review round: 1
Reviewer: 1

Output:
Basic reporting: Overall, this was a longitudinal study that focused on improvements of adolescent athletes' dietary habits based on the integration of a garden, kitchen, and elective courses in the school.
There were a few areas that I thought needed to be addressed:
Abstract:

Methods: suggest removing the “an interventional study” and keep only the longitudinal. The second sentence of the methods needs to be revised, suggest, “This was considered a multi-component study as participants were exposed to a garden….
Include how often you collected the FFQ information.
Include the age range of your participants

Results: if you use increased, please use ‘decreased’ instead of reduced. This sentence, ”In relation to the dietary patters, in 2013 and 2016 two similar patterns were identified: a healthy pattern, composed by natural or minimally processed foods and an unhealthy pattern, characterized by processed and ultra-processed food items” is not clear. Please reword.

Conclusion: use same terms, in the background and methods it was referred to as a multi-component intervention. Technically based on the various interventions you included, it was not food only. Maybe it was a component of the curriculum, but a nutrition / health program is a better term. Even though it was mentioned school is an ideal environment, there was an increase in soft drink consumption and only 2% of the 83 participants reduced their snack intake. Also, this was only for athletes, so the conclusion has to be more for that group.

Introduction:
In the first paragraph, the authors first indicated that ultra-processed foods are high in fats and carbs, but for the minimally processed have high amounts of micronutrients. Be consistent with either the focus being on macronutrients or micronutrients. The reviewer understands the issues with ultra-processed foods, but there needs to be more information about adolescents, especially concerning adolescents who are athletes as that is the argument for this study.

There is discussion about school gardens and a little bit about kitchen. Also, there is not much about if these gardens are traditionally placed in high schools or research has been predominately in elementary schools nor the stats behind the success of these, even if they are short term.

Line 60: if there are statistics to indicate how low the consumption of fruits and vegetables are, include that within this sentence.

I would suggest you have an individual, not associated with this manuscript to read for grammatical/technical errors as there were quite a few.
Experimental design: In the background of the abstract, For the aim, healthier/healthy, etc is an obscure word when talking about diets. Suggest nutritious, nutrient-dense, etc. Also the aim is wordy, suggest “The aim of this study was to determine athletic adolescent’s food consumption after implementation of a multi-component school intervention.”

Methods:
Overall, this section was sparse. Further development should be on the gardening of what was included/taught, and the kitchen of what students did. Also, expand on what these 2 elective courses were, who taught them and the expertise of the individual teaching all these interventions. There needs to be more information about if the students participated in these events each week for the 3 years or how the curriculum was set up. Essentially what you had in table 1 should have been concisely written in this part.

Even though the participants completed a weekly FFQ, did they do it consistently for 3 years, please clarify. Also, ultra-processed would also include sports bars, energy drinks, etc. However, there does not appear to be that information on this FFQ, please indicate why that was not included on the questionnaire.
Validity of the findings: Results:
Lines 196 and 197, identify how many participants fell into one of these 2 patterns.

Discussion:
It was mentioned there was a correlation between healthy eating and soft drinks, but in this study, soft drink consumption rose regardless of if students ate more cooked vegetables and raw salad. There was not much explanation as to why these interventions may have led to participants eating more nutritious products and why they continued to drink regular soft drinks.
The other limitation is the small number of participants. Even though this is a different type of school, the results cannot be generalized to other adolescents.
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: HEALTHY EATING PROMOTING IN A BRAZILIAN SPORTS-ORIENTED SCHOOL: A PILOT STUDY
Review round: 1
Reviewer: 2

Output:
Basic reporting: I suggest having a native English speaker proofread the manuscript to catch several spelling and grammatical errors. For example, Introduction, line 58, should be “Data from a Brazilian survey… more candies”; line 86; line 176
It is unclear what line 60-62 adds. I am not seeing the connection between the paragraph beginning on line 65 and the subsequent paragraph content. I think the authors could remedy this by making an explicit link between adolescents, school sports and food in schools. Line 84 is repetitive.
Line 87 – I question the relevance of this line of background given that the study did not examine the effect of school garden programs on athletic training and performance.
The structure of the article conforms to an acceptable format.

Figure 2: it may be preferable to include a comma between letters for the foods for which there was more than one statistical difference over timepoints indicated as “ab”, “ac” (so “a,c” “a,b”)
The submission represents an appropriate ‘unit of publication’.
Experimental design: Authors should be more specific about what they define as “healthier” (line 92). So the students attend a school that emphasizes more sports activities and dedicates more of the curriculum to physical education practice and study. The authors raised the issue of marketing unhealthy foods to athletes. Are they suggesting that these students are more amenable to marketing messages?
What really makes this study different then is the study population. Otherwise it is really similar to previous studies that it is testing the effect of a school-garden intervention on dietary consumption.
Suggest providing more of a definition/description of what a “sports-oriented” school is. Please also provide greater detail as to what the students received in the different intervention components. For example, what occurred in the experimental kitchen and what were the elective subjects that were implemented?
Line 89 – wouldn’t this study have been more effective and been better able to address your point about the utility of the length of the intervention and the integration within curriculum if it had been designed to include some students 1) who participated for different lengths of time and 2) who received all components of the intervention, some components of the intervention compared with students who didn’t receive any intervention (a control group)?

Suggest revising line 97; students began in the study in 6th grade and were followed to 9th grade. The way this statement is currently written it sounds as if students in grades 6-9 participated.
Suggest making it clearer that the 450 students who were enrolled in the study was the parent study and that the current paper focuses on longitudinal change in 6th grade participants. Suggest providing additional detail on reasons for why 13 (102-89) students didn’t participate (was it that they didn’t agree to participate?). Furthermore, what is the definition of “participation” in the study? Is this being used synonymously with completed baseline data collection since all students presumably received the intervention?
Line 129 suggest clarifying as follows “food frequency questionnaire that asked about past week consumption of 12 food items” or similar. This was obtained on three separate occasions. How were the food items included on the questionnaire selected? Did the authors measure amount/quantity in addition to frequency?
Line 134 suggest indicating that dietary patterns will be described below.
Line 144 please provide more information on how the absence of consumption (zero days) was investigated.
Line 146 Is it defensible to derive a dietary pattern from only 12 items? I’m not sure I would use “dietary pattern” to describe what was being derived from the factor analysis because a “dietary pattern” usually refers to the sum of what someone is consuming. Is it really necessary to use a factor analysis when the authors were comparing natural/minimally processed versus processed/ultra-processed foods and it is fairly straightforward which of the 12 food items would be categorized into each group.
Line 159 It is not clear if the factor analysis was used each time for the three time points to derive patterns or if the patterns derived using baseline data were then applied to subsequent time points? Please clarify.
Line 193 I am really not understanding what the authors did with the factor analyses. Please clarify the approach. So factors were created at each of the three time points and they didn’t differ from each other? What does this tell you? Wouldn’t it have made more sense for the authors to create factors at baseline and then look to see if the scores for participants changed over time? In other words, did their factor score for “healthy diet” increase with greater time in the intervention?
Validity of the findings: Line 189 – confusing. Unclear what the Please re-word.
The authors are overstating their findings. Really there were changes for increasing raw salad and cooked veg (but only in the third year) and decreasing fried snacks.   Fewer students reported that they did not consume fruits over time. On the other hand, students consumed more soft drinks and processed meats. Results were mixed.
The authors should better discuss what the significance is of the study population.
Paragraph beginning on line 231 is repetitive. This paragraph includes results which should be moved to the Results section. This paragraph also includes statements requiring citations (line 242). Are the students in this school that emphasizes sports being marketed to?
Additional comments: The authors have not compellingly demonstrated what this study adds. If it is that a school-garden intervention is applied to a unique study population, then this should be further described. The methods are somewhat difficult to follow. This is a small study that collected limited data. The longitudinal nature is definitely a strength but the authors really don’t provide much detail on the intervention itself. The biggest problem I have with the manuscript is that the authors overstate their findings. The results are mixed at best and really don’t convincingly show a benefit of the intervention over time on dietary consumption.


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: HEALTHY EATING PROMOTING IN A BRAZILIAN SPORTS-ORIENTED SCHOOL: A PILOT STUDY
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have significantly improved this manuscript, but there were a few small areas that needed to be restructured:

Introduction:
Lines 58-61: Please integrate into the first paragraph as this paragraph reads out of place. Suggest not to start off with Furthermore, but instead, "Adolescent athletes consume a high quantity of low-nutritious quality foods..." Suggest to use 'refined carbohydrates' as adolescents who are athletes need carbohydrates, but the focus is on whole grain carbs.
Experimental design: Thank you for expanding on this section, for lines 112-113, even though it was good to include the types of foods offered per each meal and snack, what is the overall composition? Based on 2000 kcals, with 50% from carbohydrates, 20% protein, 30% fat? The reviewer is not familiar with Brazil's school breakfast/lunch program, so it would be good for other readers to know how different/similar the nutrient composition is compared to their respective country.
Validity of the findings: No comment
Additional comments: No comment


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

Input:
Title: HEALTHY EATING PROMOTING IN A BRAZILIAN SPORTS-ORIENTED SCHOOL: A PILOT STUDY
Review round: 2
Reviewer: 2

Output:
Basic reporting: Improved. Authors have taken reviews into consideration and made satisfactory edits to revised manuscript. Minor additional comments.
Line 112 and elsewhere “industrialized juice”; I am not sure what this is
Line 191 – better to use the Results section to refer to tables; use the discussion section to summarize or discuss implications of the data contained in tables
Experimental design: Improved. Authors have taken reviews into consideration and made satisfactory edits to revised manuscript. No additional comments.
Validity of the findings: Improved. Authors have taken reviews into consideration and made satisfactory edits to revised manuscript. No 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: HEALTHY EATING PROMOTING IN A BRAZILIAN SPORTS-ORIENTED SCHOOL: A PILOT STUDY
Review round: 3
Reviewer: 1

Output:
Basic reporting: The authors have addressed all the minor revisions identified by the reviewer. Overall, this is a great manuscript and study.
Experimental design: No comments
Validity of the findings: No comments
Additional comments: No comments


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

Input:
Title: IDENTIFICATION OF HUB GENES AND MOLECULAR MECHANISMS IN INFANT ACUTE LYMPHOBLASTIC LEUKEMIA WITH MLL GENE REARRANGEMENT
Review round: 1
Reviewer: 1

Output:
Basic reporting: In this manuscript by Zhang et al, the authors have used several bioinformatics tools to elucidate the molecular mechanisms of infant ALL with mixed lineage leukemia gene rearrangement (MLL-R). The objective was to identify potential biomarkers for the diagnosis and therapy of this deadly disease which has a low 5-year event free survival rate.

-The language used in this manuscript was unambiguous and easy to understand. The introduction contained enough background and explained why the study was conducted. This manuscript has been well researched as the literature references provided are extensive. There are some grammatical errors in this manuscript which are noted in the General comments section.

-The results were well presented but brief. More detail in terms of why, what and how should be added. The figures were of good resolution, but the figure legends were lacking in detail. Please add more methodology as well as any statistical analyses performed.

-The discussion section which explained the roles of each of the potential targets (from line 195 onwards) in this disease was well written. Please provide more explanation for the DEGs identified via GO analyses (lines 190-193).

-Lines 251-254 in the Conclusions section should be re-worded.

-All appropriate raw data has been made available.
Experimental design: The authors have used very well known bioinformatics tools to ascertain the molecular mechanisms of MLL-R infant ALL, which defers from childhood and non-MLL-R ALL. Several analyses including KEGG and GO were performed, including the addition of drug-gene interactions to identify and in some cases confirm the "hub" genes in this cancer. This study has original aims which falls within the scope of PeerJ.

-The research question was well defined and provides potential avenues for the identification and targeting of this disease.

-The choice of datasets for further analysis was appropriate and well matched in terms of sex and age so as not be confounding factors.

-Please check the p value for DAVID (line 102).

-More detail should be added to the drug-gene interaction methods. The choice of filters used should be better defined.

-Please add information regarding the statistical analyses (if any) performed.
Validity of the findings: This study fills some of the knowledge gap regarding the DEGs involved MLL-R infant ALL and their differences from other non-MLL-R cancers. The data is robust and well controlled. The normalizations and analyses performed have been sufficiently explained.

-The conclusion for this study states in like 254 that the identified genes "contributed to leukemogenesis, migration, and invasion of" but this is an overstatement since this has not been validated in this study. Please change that sentence to "may contribute".
Additional comments: The tense should be kept constant throughout the text. The following lines contain errors which should be rectified:

Line 18: No period between ALL and microarray expression.
Line 26-28: Needs more clarity.
Line 30: Please mention the KEGG pathways in which the DEGs are downregulated.
Line 33: Define DGIdb.
Line 165: What is the consequence of histone H3 lysine 4 methyation?
Line 223: Please change "to identify" to "and identified".
Line 236: Please comment on the role of CD44 as a marker of cancer stem cells.
Line 240: Please re-word this.
Line 242: Please add "the roles of" before ALB up-regulation.

Overall, please comment on the consequences of your discovery. This is a sound study, but may be lacking some validation.


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

Input:
Title: IDENTIFICATION OF HUB GENES AND MOLECULAR MECHANISMS IN INFANT ACUTE LYMPHOBLASTIC LEUKEMIA WITH MLL GENE REARRANGEMENT
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript is well-written.  However, the second sentence of the abstract is incomplete and requires revision.
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: IDENTIFICATION OF HUB GENES AND MOLECULAR MECHANISMS IN INFANT ACUTE LYMPHOBLASTIC LEUKEMIA WITH MLL GENE REARRANGEMENT
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript is well written in professional scientific language, with very few minor grammatical errors. There is sufficient reference to previously published work in the field with a few suggestions below .The figures are well made and have good descriptions and keys to make their understanding better. The results section is concise, well-written and correctly interprets the findings of their study.  There are a few minor flaws as described below:

(1)The introduction is well written and covers enough background about the differences and distinction between pediatric ALL and MLL-R infant ALL. While differences in survival rates and prognoses are mentioned, treatment strategies and current approaches to improve prognosis for this disease (such as work conducted by the Ishii group (Leukemia 2007,Leukemis 2007) needs to be referenced).

(2) Line 18 : grammatical error: the line should read......To elucidate the molecular mechanisms of MLL-R infant ALL, microarray expression datasets were downloaded from the GEO database,......

(3) Figure 1: the x-axis below the figure can be removed or the font size needs to be reduced as it is not legible.
Experimental design: The study conducted in this manuscript fits the scope of the journal. The research question is well defined and addressing a major unmet need in the field of MLL-R infant ALL. The methods section is well written, providing enough background knowledge and details to the reader. The sample and control cohorts identified in this study are well-defined with no skewing factors such as gender or age disparities. The only comment here is

(1) Line 125: please specifically define what "criteria" is.
Validity of the findings: All raw data required to effectively replicate this study has been provided. The conclusions made are true to their findings. The KEGG and DAVID annotation and pathway analysis are very well written. There are few suggestions below on improving the discussion:

(1) Lines 178-182 :  Please mention speicifically that all downstream analysis from the DEG list including molecular functions and mechanism is in-silico as no in-vitro validation was performed in this study.

(2) NG2 has bee shown to be a marker for MLL-R infant ALL ( Behm et al, 1996, Blood, Hilden et al, 1997, Blood, Smith et al, 1996, Blood and Wuchter et al, 2000, Leukemia). The authors should comment on why this gene either did not come up as significantly de-regulated in their analysis or how their results correlate with the previous literature.

(3) The GO annotation using DAVID revealed pathways associated with general "hallmarks of cancer" and deregulated cell. While the authors do make some mention on how such findings could be specific to MLL-R infant ALL, more detail will be helpful in fully elucidating the significance of this study.

(4) Please add the appropriate references to line 245.
Additional comments: No comment.


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

Input:
Title: IDENTIFICATION OF HUB GENES AND MOLECULAR MECHANISMS IN INFANT ACUTE LYMPHOBLASTIC LEUKEMIA WITH MLL GENE REARRANGEMENT
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have made the necessary suggested changes.
Experimental design: No comment.
Validity of the findings: The conclusions have been modified according to suggestions.
Additional comments: All reviewer comments have been addressed. I look forward to validation of these genes in future manuscripts.


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

Input:
Title: IDENTIFICATION OF HUB GENES AND MOLECULAR MECHANISMS IN INFANT ACUTE LYMPHOBLASTIC LEUKEMIA WITH MLL GENE REARRANGEMENT
Review round: 2
Reviewer: 2

Output:
Basic reporting: All comments have been adequately addressed.
Experimental design: No comment
Validity of the findings: No Comment
Additional comments: No Comment