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: MIR-421 IN PLASMA AS A POTENTIAL DIAGNOSTIC BIOMARKER FOR PRECANCEROUS GASTRIC LESIONS AND EARLY GASTRIC CANCER
Review round: 1
Reviewer: 2

Output:
Basic reporting: Despite the goal and the message of the work is clear, english must be absolutely revised.
Furthermore, the authors should improve introduction to give sufficient field background to the reader. Several papers investigated the expression of circulating miRNAs as early markers of gastric cancer and precancerous lesions and of GC progression (Chen TH, et al. Circulating microRNA-196a is an early gastric cancer biomarker. Oncotarget. 2017 Dec 7;9(12):10317-10323. doi:10.18632/oncotarget.23126;  Zhang J et al. Circulating MiR-16-5p and MiR-19b-3p as Two Novel Potential Biomarkers to Indicate Progression of Gastric Cancer. Theranostics. 2015 Apr 5;5(7):733-45. doi: 10.7150/thno.10305. eCollection 2015; Wang XW et al. MicroRNA network analysis identifies key microRNAs and genes associated with precancerous lesions of gastric cancer. Genet Mol Res. 2014 Oct 27;13(4):8695-703. doi: 10.4238/2014.October.27.10). Importantly, Zhang and co-workers, initially screened out differentially expressed miRNAs by genome-wide miRNA profiling microarrays and validated some of them in a larger cohort of patients. Authors should quote this article and discuss the results obtained on the basis of those already existing in the literature.
Experimental design: The research question is relevant and meaningful, however the authors should keep in mind that scientific works already exist in which the expression of some microRNAs has been analyzed as markers of early diagnosis and precancerous lesions. As these miRNAs are different from miR-421, the authors should validate and compare their level of expression, sensitivity and accuracy with that of miR-421 and discuss these results.
Methods are described with sufficient information.
Validity of the findings: Data are robust and statistically sound. Patient cohort is numerically good to reach the conclusion. Conclusion is well stated and linked to the riginal research question.
Additional comments: The work is well designed and structured, however it should be improved both in the introduction and in the results section: The authors should consider the other works in the same field of research and analyse the expression of the miRNAs already published as potential markers of early diagnosis of GS and precancerous lesions in order to compare thei expression with miR-421. These results should also discussed.


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: MIR-421 IN PLASMA AS A POTENTIAL DIAGNOSTIC BIOMARKER FOR PRECANCEROUS GASTRIC LESIONS AND EARLY GASTRIC CANCER
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The authors reviewed the work according to my comments.
In particular they revised the form, added some important references and expanded the introduction to make the experimental part clearer.
I think the work is now suitable 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: SYMMETRY PREFERENCE IN SHAPES, FACES, FLOWERS AND LANDSCAPES
Review round: 1
Reviewer: 1

Output:
Basic reporting: The study's methods and results were clearly articulated. There were a few minor grammatical errors. I would encourage the authors to give the manuscript a careful proof read when resubmitting.

In the paragraph where symmetry as a fitness cue is discussed (starting line 75) it would be worth mentioning conflicting data, from a more recent study (Pound et al. (2014). Proceedings of the Royal Society of London B: Biological Sciences, 281) which calls this account into question.

In the following paragraph (starting line 83) the authors could make clearer the link between Vessel's work on agreement and the current work. Are the authors implying that symmetry is a more salient feature in faces and this accounts for agreement in this context (vs. architecture or artworks)?

The authors may want to consider adding an article in the section on symmetry of flowers, which shows that bumblebees seem to innately prefer bilateral symmetry (Rodríguez et al (2004). Symmetry is in the eye of the ‘beeholder’: innate preference for bilateral symmetry in flower-naïve bumblebees. Naturwissenschaften, 91).

The final paragraph of the results section (starting line 436) would be better placed in the discussion as it is alluding to theoretical considerations of the results. Also, might it not be the case that Grzeckowski et al's (2017) research on illusions suggests that they are different mechanisms underlying these various illusions. This would not necessarily entail that each individual is unique, but more that each illusion is unique. I think Vessel's research on agreement is more pertinent to the question of whether aesthetic taste is individual.
Experimental design: The research question was somewhat clear, however I would have liked to have seen better justification for the categories of stimuli chosen (beyond that they have been the subject of previous research). Why specifically flowers and landscapes? Also, it would be worth making clear from the outset (the first paragraph) why looking across different stimulus categories aids our understanding of the aesthetic value of symmetry.

There were no concrete experimental hypotheses given. While I appreciate this research is somewhat exploratory, I do think on the basis of prior research and with consideration of the conceptual implications of these various stimulus types, the authors could provide predictions of the kind of pattern of results they anticipated.
Validity of the findings: The use of linear mixed effects modelling was effective and patterns in the data are clearly visualised.
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: SYMMETRY PREFERENCE IN SHAPES, FACES, FLOWERS AND LANDSCAPES
Review round: 1
Reviewer: 2

Output:
Basic reporting: In line 46: is stated "positive" response to symmetry on an implicit task. I think would be better to talk about association in that context.

In line 59: when authors talk about "general aesthetic principles" authors must consider to include some more reference to cross cultural research like "Che, J., Sun, X., Gallardo, V., & Nadal, M. (2018). Cross-cultural empirical aesthetics. Progress in Brain Research, 237, 77-103. "

In line 74: Would be appropiate to clarify to which "tensions" is refered.

Figures are correct, but would be better if them showed each dot, maybe changing the transparency of the confidence inteval.
Experimental design: Experimental design is appropiate.
Validity of the findings: Perfectly replicable methods. Conclusions are attached to empirical task.
Additional comments: Would be interesting to take profit of mixed models and instead of comparing group means, use the individua coefficients throught coef(model)$subject. In that way, you are able to compare individual scores for each participant in different tasks.


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: SYMMETRY PREFERENCE IN SHAPES, FACES, FLOWERS AND LANDSCAPES
Review round: 1
Reviewer: 3

Output:
Basic reporting: No comment
Experimental design: (1) The paper requires improvement on conceptual rigour. It uses "symmetry" for "perfect bilateral reflectional symmetry", and "assymmetry" for "non perfect bilateral reflectional symmetry". The authors are well aware that symmetry is a perfectly defined geometrical property, that maybe of many different kinds: rotational, translational, inverse, helical... They also think of this property as "regularity" (in the discussion), without making it clear why. The complementary set, though, can be much diverse, with images classified as "non symmetrical" exhibiting high degrees of, say, rotational symmetry, as it is the case with the flowers set of stimuli. It would be advisable to objectively rate the images for the kinds of symmetry they exhibit in the first place. Images in the "non symmetrical" set may in fact be highly symmetrical. Think of the trisquelion, as an instance.
(2) While the authors take for granted the extensive literature on the influence of symmetry on beauty ratings, what they are interested in is in "symmetry salience" --this is what they ask the participants to rate. Salience in general is a context dependent property of images. Therefore, it is possible for an image to be objectively symmetrical but this symmetry not be salient. As a matter of fact, this is what happens with the flowers images, which are rotational symmetric, but this properties is not as salient as their bilateral symmetry. Some control of factors influencing salience was called for. On the other hand, a look at the stimuli set reveals that bilateral symmetry was present in all of them. Analogously, it can be said that turning coloured images into greyscale ones is not without consequence for salience ratings. Beauty and salience ratings of images in greyscale may not carry over to coloured ones. While for abstract patterns colour may not be particularly important, for flowers colour is probably more salient than any other property. Without control for these effects, the results can be full of confounds.
(3) While the authors acknowledge that perceptual judgments are idiosyncratic, they did not show to participants both images (symmetric and non-symmetric), but just one of them. This opens the possibility that the global results (that in general people prefer symmetric to non-symmetric images, with qualifications), do not hold for each person.
(4) I could not find the instructions given to the participants in the second task: did they mention "symmetry", "bilateral symmetry", "regularity", or something else? What they were asked to tell whether it was salient?
(5) The research question could much more ambitious. It reduces to check whether symmetry (or symmetry salience) influeces in the same way beauty ratings for different kinds of images (landscapes, faces, flowers, abstract patterns. But there is not much theoretical grounding for a hypothesis such as this one, as the authors acknowledge in the discussion.
Validity of the findings: (1) It would be better to report the direct ratings for the first two tasks included, as the sahere the same scale (0-10). Stardardization it is not required for relating both ratings, and it makes the results less intuitive.
(2) The third task -response time- is in a different scale and relating it to the first two dependent variables may require standardization, but it is not carried out in the analysis. It should be better explained what it contributes, given that it does not seem related to the saliency ratings per se.
(3) It would be advisable to proceed backwards: first to validate the set of images to be used as stimuli, then to ask the beauty ratings. It is doubtful that the second and third task provide the validation needed, given that the set of stimuli seem to have been chosed ad hoc.
Additional comments: The authors could be interested in further exploring the notion of regularity -better known in art studies as "rhythm", as the real pull of beauty ratings in naturalist settings, as was already proposed by the Gestalt school. Symmetry (some form of repetion) might just be a proxy for rhythm (repetition with a variation). On the other hand, exploration of non-linear effects of symmetry is also much needed (the influence of symmetry may be sensitive to the presence, or absence, of some other property).


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: SYMMETRY PREFERENCE IN SHAPES, FACES, FLOWERS AND LANDSCAPES
Review round: 2
Reviewer: 1

Output:
Basic reporting: n/a
Experimental design: n/a
Validity of the findings: n/a
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: SYMMETRY PREFERENCE IN SHAPES, FACES, FLOWERS AND LANDSCAPES
Review round: 2
Reviewer: 2

Output:
Basic reporting: All my previous concerns are satisfied
Experimental design: All my previous concerns are satisfied
Validity of the findings: All my previous concerns are satisfied
Additional comments: All my previous concerns are satisfied


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: SYMMETRY PREFERENCE IN SHAPES, FACES, FLOWERS AND LANDSCAPES
Review round: 2
Reviewer: 3

Output:
Basic reporting: Ok
Experimental design: Ok
Validity of the findings: Ok
Additional comments: The paper has been much improved in my view, and it is now  fit 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: IDENTIFICATION AND CHARACTERIZATION OF GYPSOPHILA PANICULATA COLOR MORPHS IN SLEEPING BEAR DUNES NATIONAL LAKESHORE, MI, USA
Review round: 1
Reviewer: 1

Output:
Basic reporting: The author’s report on identifying morphological variation found in invasive Gypsophila paniculata populations within the Michigan dune system. I commend the authors for clearly detailing the importance of the question and how different management systems are in use for the different colour morphs. The article is well written, however you should consider expanding the introduction with a brief overview of the taxonomy of Gypsophila and a discussion of the suitability of DNA barcoding to answer your question. The taxonomic overview could highlight why the green-yellow morph could only belong to Gypsophila paniculata and whether other members of the genus are invasive in different ecosystems.
Experimental design: DNA barcoding is well-suited to answer questions like this, however it does not work uniformly well across different plant genera. I believe it would be necessary to show that the chosen plant barcoding regions have the required resolving power to distinguish between a wider range of Gypsophila species. This is even more needed as you mention in lines 76-77 mention that other species of the genus are sold as part of wildflower seed mixes in the region, however you only include G. elegans in your analysis. GenBank and BOLD should be suitable to expand the sampling with a few more species.
The data analysis shows clear results, but you should consider using other barcoding analysis methods, apart from phylogenetic trees, that could further highlight the similarities between the colour morphs and show the difference to any other species (e.g. barcoding gap).
Validity of the findings: Gypsophila paniculata data included in the analysis is suitable to answer the question. However the results would be much more robust if you showed greater context of molecular variation within the genus.
Additional comments: The abstract reads somewhat confusing mentioning two colour morphs for G. paniculata in line 16 but refer to them as potentially different species in line 21. I think it would be clearer to frame the study question similarly as lines 80-81. (Are they the same species or not, rather than the other way round.)
The chloroplast DNA regions should be written as: rbcL, matK throughout the text.
Line 25 sequences were aligned to the reference instead of with the reference.
Line 79 traditional barcode genes needs a citation.
Line 90 Kit instead of Kits.
Line 96 Region instead of gene would be more suitable as ITS2 is not a gene.
Line 100 The citation for the rbcL primers should be the original one: Fay et al. 1997, but do check the primer sequences are the same.
Line 114 Did you mean with forward and reverse primers?
Line 152 As the G. paniculata reference is 16bp away from the samples, the results only suggests similarity between the colour morphs. Additional Gypsophila samples could show a clearer picture.
Line 179 matK is highly variable not conserved. The chloroplast DNA regions in any case most likely would fail at distinguishing cultivars, unless they originate from different maternal plants.
Line 189 may have derived instead of may be derived
Line 193 Whose personal observation was this?
Figure 2 Phylogenetic analysis instead of construction?
Figure 3 The size of the rectangles and the ovals are not readily comparable. It would help if they were all the same type. The figure should have a legend for the different sizes. As lines between the nodes are not dependent on length you could shrink the lines leading to the G. paniculata reference to get a more compact figure, while showing the same relationship.


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

Input:
Title: IDENTIFICATION AND CHARACTERIZATION OF GYPSOPHILA PANICULATA COLOR MORPHS IN SLEEPING BEAR DUNES NATIONAL LAKESHORE, MI, USA
Review round: 1
Reviewer: 2

Output:
Basic reporting: see below
Experimental design: see below
Validity of the findings: see below
Additional comments: This article is written in a clear language and the methodology used for DNA analysis is updated and correct. References are updated and pertinent.
Anyway I'm quite skeptical about suggesting to accept this manuscript for publication in its present form and with its actual finalities. Infact,  it sounds strange to my ears, to listen that the author decided to test if the two taxa belong to the same species, with DNA analysis, in order to plan management activities.
The two taxa (of the same or of different species) occupy the same habitat but have different habit and bahaviour. So, as reported by the same author, the manual procedures that have to be applied by the operators to eradicate the different plants are different.
So the results obtained by the authors and the conclusions reported are not consistent.
I suggest to the authors to present their research under a different light. The dna analysis can be justified to understand if the two taxa can be distinguished at specific or infraspecific level and to see if them coudl have been arrived from different sources, but not to decide the management activities.
Figure 1 includes only the upper part of the 2 taxa, it should include also the lower part, that is reported to be different.


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

Input:
Title: IDENTIFICATION AND CHARACTERIZATION OF GYPSOPHILA PANICULATA COLOR MORPHS IN SLEEPING BEAR DUNES NATIONAL LAKESHORE, MI, USA
Review round: 2
Reviewer: 1

Output:
Basic reporting: The paper has improved greatly with the revision of the molecular work and the seed experiment. I found the story of the paper straightforward and the text easy to read. I have a few comments below, but I am happy with the paper to be published.
Experimental design: OK
Validity of the findings: OK
Additional comments: I would suggest to include the author for each species at the first mention in the text.

rbcL and matK should be italicized throughout the text. ITS2 is correct as normal text.

Line 152: ITS2 is misspelled

Line 173: I think methods would be more suitable than models.

Line 204: G. elegans is missing the 's'

Line 292 & 300: I found these two sentences to be ambiguous. Do you recommend carrying on with the current management as cutting for the purple morph and glyphosphate for the green or cutting for both as they are the same species and seem to show the same taproot structure?

Figure 2-5: These figures need a line about the bootstrap support e.g. numbers at nodes indicate bootstrap support

Figure 6 : I would replace ovals with circles in the caption. I would recommend including the proportion of the colour morphs in the two red haplotype circles as a pie chart. This could show how much the two haplotypes are shared between the colour morphs. PopART should be able to do this.


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

Input:
Title: IDENTIFICATION AND CHARACTERIZATION OF GYPSOPHILA PANICULATA COLOR MORPHS IN SLEEPING BEAR DUNES NATIONAL LAKESHORE, MI, USA
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors have applied the requested improvements. Now, on my opinion, the article is presented under the correct perspective. As in the previous version the language used is clear and unambiguous. Figures and data associated are pertinent.
Experimental design: Now the research is well defined. The Methods used are appropriate and are described with sufficient detail.
Validity of the findings: Conclusions are well stated.
Additional comments: I recommend the acceptance of this MS as it is. No further revisions are required.


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: ITRAQ-BASED QUANTITATIVE PROTEOME ANALYSIS REVEALS METABOLIC CHANGES BETWEEN A CLEISTOGAMOUS WHEAT MUTANT AND ITS WILD-TYPE WHEAT COUNTERPART
Review round: 1
Reviewer: 1

Output:
Basic reporting: The article is generally well written and presented.  I have corrected some English and typographical errors as I found them but they are minimal.
Referencing appears appropriate.
Experimental design: No comment
Validity of the findings: I enjoyed reading the manuscript and found it followed a logical progression leading to the conclusions.
Additional comments: When presenting chemical names it may be advisable in future to get a professional chemist to check your naming.  You clearly have an issue with this in the manuscript that needs correcting.  The Figure 6 is  good though so please check that this has been produced by members of the team and does not require acknowledgement if produced by others.


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

Input:
Title: ITRAQ-BASED QUANTITATIVE PROTEOME ANALYSIS REVEALS METABOLIC CHANGES BETWEEN A CLEISTOGAMOUS WHEAT MUTANT AND ITS WILD-TYPE WHEAT COUNTERPART
Review round: 1
Reviewer: 2

Output:
Basic reporting: While this paper presents the results of important findings that should be of use to plant breeders, the use of English language is deficient throughout the paper and will require extensive editing.  In addition, there are numerous mistakes (e.g.FADs are referred to as FEDs in the diagrams and statistical results are not consistently interpreted correctly or are ignored).  The structure of the paper is sound and the results are meaningful, but there are many errors that detract from the paper and make it unpublishable in its current form.

In the section "Formatting of Mathematical Components" (which is a totally inappropriate section heading, by the way), there seem to be some reporting errors.  The authors state that there are differences between YM18 and ZK001 from YAS to AS stages, but the results suggest that the differences lay between WAS and AS.  The extent of the difference cited by the authors is also questionable; they cite a difference of 39% for ZK001, but from Table 2 this difference appears to be 50% (and it's not clear where the "slight difference" for YM18 has been determined).

The formulae used for calculating starch and sugar levels are a bit difficult to follow because they extend beyond one line. More importantly, there seems to be something missing in the starch content equation: the last term ("of sample") is incomplete.  The authors also misuse "respectively", using it in places where it is not needed (e.g. line 289).

The specifications of the HPLC columns is vague; the first dimension in each case (1.9 um and 3 um, respectively) presumably refers to the particle size, but this is not spelled out (it's just inserted with no punctuation or clarifying information).  The authors have not used subscripts were needed in many places (e.g. C18 and H2O). The authors state that they slightly modified the extraction procedures for proteins published by Yang et al. (2013), but failed to say what those modifications were.

The figure captions are lacking in necessary detail.  In the caption for Figure 2, there should be a distinction between what is illustrated in panels A & C vs B & D.  Figure 6 presents a key below the figure that appears to be a heat map, but there's no indication what the bars represent (presumably the level of each of the proteins during the 4 flowering stages for YM18 and ZK001, but this is just a guess--the reader shouldn't have to guess!). The caption for Table 1 is also very deficient in details; presumably this table shows infection by Fusarium head blight by the four varieties, but this is not specified (the column heading should be Variety, not Species).
Experimental design: The experiments appear to have been well designed and adequately replicated.  Most of the data have been appropriately analyzed, but it appears that the starch and sugar levels during the YAS and AS stages were not analyzed (they may have been analyzed, but no statistical significance is mentioned in the results on this section, lines 279-284 and Fig. 2).
Validity of the findings: Data have not been properly interpreted in the section Accumulation patterns of DAPs and verification of DAPs of interest (note that I have reworded this title to be grammatically correct).  From lines 358-374, there are numerous instances of statements that are not true because statistical significance has apparently been overlooked.  For example, the authors state that the A0A1D5RR02 gene was downregulated during WAS and GAS for ZK001, but these differences are not significant (as indicated by the letters over the bars in Fig. 5C).  There are many similar misstatements in the following sentences.  The sentence regarding A0A1D5SYC3 and A0A1D5VE19 on lines 363-366 also makes no sense.
Additional comments: There seems to be a solid body of work that was conducted, but careless reporting and poor English make this paper look quite deficient.  I think the quality of the work was good, but the presentation is quite deficient.  I have not offered comments on all of the English grammar problems because they are too extensive.


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: ITRAQ-BASED QUANTITATIVE PROTEOME ANALYSIS REVEALS METABOLIC CHANGES BETWEEN A CLEISTOGAMOUS WHEAT MUTANT AND ITS WILD-TYPE WHEAT COUNTERPART
Review round: 2
Reviewer: 1

Output:
Basic reporting: .
Experimental design: .
Validity of the findings: .
Additional comments: The manuscript is much improved, but some additional minor editing is still required.
See attachment.


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: CHARACTERIZATION OF BIDIRECTIONAL GENE PAIRS IN THE CANCER GENOME ATLAS (TCGA) DATASET
Review round: 1
Reviewer: 1

Output:
Basic reporting: In the abstract, the authors should clearly specify the controls used. For example: (i) adding a sentence like "Gene pair on the opposite strand with transcription end site distance within 1 kb (tail-to-tail structure) were used as control", (ii) adding a phrase like "in tumor compared to adjacent normal" instead of just saying "up/down regulated" or "hypo/hyper methylated", etc. While the authors have already specified these in the "Materials and Methods" section, readers often look at the abstract separately, and omission of these crucial details can make the abstract difficult to understand.
Experimental design: Satisfactory
Validity of the findings: 1. In the methylation analysis, the authors used all the CpG probes annotated to a gene, i.e. the CpG probes located in the promoter region of a gene as well as the CpG probes located elsewhere in the gene. While hypo/hyper methylation of the former type of probes is easier to interpret in the context of expression, it is not always clear how to interpret hypo/hyper methylation of the later type of probes in the context of expression. So, the authors should check whether restricting the analysis to only the CpG probes located in the promoter region (say 1 kb up-stream) of a gene, i.e. excluding the CpG probes located elsewhere in the gene from analysis, produces results that are consistent with their current findings (and add a few sentences and possibly a supplementary figure on this).

2. TCGA has recently published a well-curated version of their pan-cancer clinical data (please see the tab "TCGA-CDR" in Table S1 of Liu et al, Cell 2018, PubMed 29625055), detailing not only overall survival (OS) but also disease-specific survival (DSS), disease-free interval (DFI), and progression-free interval (PFI). The authors should check whether analysis of these data produces results that are consistent with their current findings (and add a few sentences and possibly supplementary figures on this). These supporting results can considerably strengthen the claims made by the authors.
Additional comments: The result section "Characterization of up-/down-regulated bidirectional genes and gene pairs in multiple TCGA datasets" (line 287-319) incorrectly refers to Figure 3 (should refer to Figure 4 instead).


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: CHARACTERIZATION OF BIDIRECTIONAL GENE PAIRS IN THE CANCER GENOME ATLAS (TCGA) DATASET
Review round: 1
Reviewer: 2

Output:
Basic reporting: This manuscript is very well presented in language and provides thorough research background and well performed analysis result. Some crucial typos need to be revised before acceptance:
1. lines 294-lines 312, the figure mentioned should always be Fig.4
2. line 409, Fig.6E should be Fig.6D
Experimental design: How did the authors deal with batch effect when performing differential expression analysis?
Validity of the findings: The authors had a list of interesting findings when comparing bidirectional genes and control genes, especially the identified correlation with prognosis. However, the result interpretation is not thorough enough. For gene set enrichment, DAVID GO  analysis is far from enough. I would suggest the authors to try extra tools for interpretation like Enrichr (Kuleshov et al., 2016), which keeps a updated database of GO, pathways and etc.
Additional comments: The paper is well written. The finding is quite interesting. Better result interpretation and some validation experiments will elevate this paper a lot.


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: CHARACTERIZATION OF BIDIRECTIONAL GENE PAIRS IN THE CANCER GENOME ATLAS (TCGA) DATASET
Review round: 1
Reviewer: 3

Output:
Basic reporting: No Comment.
Experimental design: The analyses needs additional comparison with controls, or the authors might want to give a satisfactory explanation for why the control chosen by them proves to be sufficient in this study.

Line 109: Do the authors include both “blood” and “solid tissue” within the normal group? If so then only “solid tissue” normal must be considered as the RNA profile of a blood sample may vary greatly from that of normal tissue and hence a differential expression compared to the tumor may not be a sensitive predictor.survi

Line 120: When filtering for logFold how were the cut-offs +1.5 and -0.67 defined? Xiao et al 2018, does not give a reason for different cut-offs in the upper and lower limits for log fold change. I believe a different fold change cut-off might end up skewing the results. Further Xiao et al used multtest R package to compute FDR as against glmTreat native edgeR function by the authors. Are the authors confident that the two different packages predict similar FDR values?

Line 136: Similar concern for whether “blood normals” were also used in the normal group to identify differentially methylated genes.

Line 174: The number of controls is lower than the test set. What is the reason on tail to tail gene pairs are used as controls? For the control pairs, additional controls, like pair of genes in the same strand where the start of one gene is >1kb and <5kb from the stop of the other, can be used as such pairs might show co-transcriptional pattern. Further shuffling the location of all genes on the genome and then re-identifying the bidirectional pairs, is another random control which needs to be included in the study.
Validity of the findings: Results section is not often accompanied by a sound conclusion. However the nature of the study is as such that often a direct biological inference is difficult to obtain. Hence would recommend the authors to tone down their conclusion and omit or reduce statistics which do not have a clear biological hypothesis, thus making the message in the paper more clear and concise.

Line 263-273: Here the authors make certain observations, however there is a lack of inference, and whether the observations reflect the biology of cancer genomes, or the gene orientations in general.

Line 301: This is not necessary a cancer specific observation, but more prominent due to tissue specificity for Head to Head gene pairs, as also shown before in Fig 6 of a previously published paper (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2665707/). Hence I think the authors must be careful in their interpretation since, and use additional control sets to get a clear picture.

Line 311: Did not find Fig. 3 C, D and would recommend to refer to the previous publication
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2665707/
Which shows similar findings for Head to Head gene pairs.

Line 356-357: Here the text lacks any biological inference from the observation, hence it is not clear as to the purpose of the statistics. I would recommend to make concise all statistical observations (or even omit), which do not provide a clear biological hypothesis as it makes the core message in the manuscript a bit obscure.

Line 371-409: Bidirectional genes compared to any other category, tend to be significantly enriched in genes important for chromatin state, replication and repair. Hence their relation to prognosis might be reflective of their underlying function, and change in expression due to tumor state or treatment, and not because of the genes being important in that particular type of cancer. Hence the any conclusion drawn from such an analysis, needs to be toned down.
Additional comments: The current study looks at the role of bidirectional genes in head to head orientation with respect to cancer and gene expression and attempts to elucidate the expression patterns and functional role of such pairs in cancer. The study is well planned and technically sound and would be interesting for the community. However I strongly believe the authors must include or at least test their results with additional controls (same stranded gene pairs and random pairs) and get a more holistic picture as well as reduce the text about those statistics which do not provide a clear biological inference. Also an aspect of concern would be if the normals used in the study include blood normals, since in TCGA RNAseq the blood normals are not a good measure of normal tissue expression of a gene (https://tcga-data.nci.nih.gov/docs/publications/tcga/about.html). However the manuscript is fit for publication considering the authors address the issues and also include additional controls or give a satisfying explanation to why only tail to tail pairs were used as controls.


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: CHARACTERIZATION OF BIDIRECTIONAL GENE PAIRS IN THE CANCER GENOME ATLAS (TCGA) DATASET
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have satisfactorily implemented the changes I recommended in my last review.
Experimental design: no comment
Validity of the findings: The authors have satisfactorily implemented the changes I recommended in my last review.
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: CHARACTERIZATION OF BIDIRECTIONAL GENE PAIRS IN THE CANCER GENOME ATLAS (TCGA) DATASET
Review round: 2
Reviewer: 2

Output:
Basic reporting: Maybe add a supplementary text document with additional figures and text, thus cutting down on statistical information which is needed but tends to hide major points of the manuscript in the main text.

The Figures, specially in supplementary need minor corrections. Overall the structure of the manuscript is still slightly confusing with lot of information, which is at times too much for a reader, without deriving a clear stock message.

Minor things:
Figure S1A should be a table

Figure S3,S4, S6 not marked in A and B

Figure S5 E,F,G,H unavailable, or A, B, C, D incorrectly labelled
Experimental design: Strongly suggest to add as control gene pairs which are transcribed in the same direction and separated by 1 Kb to 10 Kb. It is important to show this group as a control along with tail to tail, and random pairs, as similar to the test dataset (head to head), genes transcribed in the same strand and lying close to each other, may often fall under a common transcriptional domain, thus showing a similar behavior as head to head group when it comes to differential normal/tumor expression as well as gene function and survival analysis.
Validity of the findings: The study adds to the field and is thus worth publishing. However the results need to be trimmed and explained in a concise manner without overbearing the reader (many could be biologists) with statistics.
Additional comments: The manuscript is descriptive and lot of information remains hidden unless carefully observed by the reader. Hence making it more concise, highlighting the principal findings with figures in the main text, and keeping the rest of the details, which is of course hard work of authors, in a supplementary text file would be suitable. Would recommend adding a control set of gene pairs transcribed in the same strand and lying close to each other (1-10 kb).


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

Input:
Title: A TROPOMYOSIN RECEPTOR KINASE FAMILY PROTEIN, NTRK2 IS A POTENTIAL PREDICTIVE BIOMARKER FOR LUNG ADENOCARCINOMA
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript entitled "NTRK2 is a potential predictive biomarker for lung
adenocarcinoma" by Yuanliang Yan et al. studies the data of expression profile of NTRK2 gene in lung adenocarcinoma.

Overall the manuscript is satisfactory. The findings are worth to report and will be useful in the studies mainly in understanding the NTKR2 expression profile. Literature is well referenced & relevant to the concept.
Experimental design: Not applicable.
Validity of the findings: Findings are valid, need additional controls for the study.
Additional comments: I have the following major concerns:
By considering NTRK tyrosine kinase family genes only and selecting only NTRK2 for further study appears as a biased approach. I would suggest that author should take control gene or set of genes that have regulated expression in lung adenocarcinoma, for the correlation based parallel studies and compare the data from normal and cancerous tissues.

Title of the manuscript is not a self-explanatory "NTRK2 is a potential predictive biomarker for lung adenocarcinoma". NTRK2 is a very specific abbreviated name of protein. Keeping in mind the broad audience of this (PeerJ) journal, author should expand or generalize the title, as for example “A tropomyosin receptor kinase (TRK) family protein, NTRK2 is a potential predictive biomarker for lung adenocarcinoma”.

“Neurotrophic receptor tyrosine kinase 2 (NTRK2), a member of the tropomyosin receptor kinase (TRK) family, has been found to be associated with the development of tumors.” This is passive voice statement and jargon. Active voice and direct sentences would be preferably used in all through the manuscript for e.g.
“Neurotrophic receptor tyrosine kinase 2 (NTRK2) is a member of the tropomyosin receptor kinase (TRK) family associated with the tumor development.”

Line 53-54: Currently, the relationship between NTRK2 and cancer has been found with continuous research. What is the meaning of this sentence?
Can be written as 'Recent research advancement in the field revealed the relationship between NTRK2 and cancer'

Line 63: researched to research

Line 65: ‘analyses of data’ to ‘data analysis’

Line 75: ‘methods’ to ‘tools’

Line 76: ‘analysis data’ to ‘data analysis’

Line 111-112: ‘Through the analysis of bioinformatics databases’ to ‘The bioinformatics analysis of databases’. Author is analyzing the database using bioinformatics tools and not studying the bioinformatics program itself.

Line112: “We evaluated the expression profiles of NTRK family members in LUAD.”
Authors need to explain first, which expression profile (mRNA/protein etc.) they are going to talk about.

Line 113-116: First, we used the Oncomine database to observe the expression of NTRK1, NTRK2 and NTRK3 in LUAD. The results showed that NTRK2 had significantly lower expression in LUAD through the comparison among nine datasets, whereas NTRK1 and NTRK3 showed no statistical significance (Figure 1A).”
Although, it is mentioned in figure legend, it would be appropriate that author could setup the reader accordingly. Specifically, author could have explained briefly why they are looking into Oncomine database? What does Oncomine database will provide (like RNA seq dataset, mRNA expression profile, tissues specific, cell type specific etc.,) and how oncomine database analysis will fit into their objective(s)? This journal (PeerJ) caters the broad audience and not very specific to lung cancer or kinase protein family, so author should structure their paper accordingly. This is one example I mentioned here, but the same can be done in all the section were author had used any database. Please provide the rationale, what you are looking for, and how the data analysis fit in your objective?

Texts in figures are hard to read, author should use ‘arial’ font throughout the figure.
Fig 1c: too large, please resize the figure.

Figure 2: Unreadable, please change the figure, text, axis, figure number.

Figure 4: Again same problem, please improve figure, text, axis labeling etc.,

Figure 6: This is direct use of flowchart from KEGG. It is not acceptable in current ‘as it is’ form. Author need to draw them in their own format and move the figure in supplementary section.

Combine all supplementary files in a single document.


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

Input:
Title: A TROPOMYOSIN RECEPTOR KINASE FAMILY PROTEIN, NTRK2 IS A POTENTIAL PREDICTIVE BIOMARKER FOR LUNG ADENOCARCINOMA
Review round: 1
Reviewer: 2

Output:
Basic reporting: Manuscript should be redrafted for English.
References should be appropriately cited.
Experimental design: The article “NTRK2 is a potential predictive biomarker for lung adenocarcinoma” described the investigation on NTRK2 down regulatory function in lung adenocarcinoma cancer using bioinformatics analysis of literature database. However, claim of this study as mentioned in introduction is to evaluate the role and mechanism of NTRK2 in human LUAD is unclear, and stronger arguments/controls connecting the results and conclusions are needed.
In general, the figures are difficult to read (especially figure 2, 5B, and 6 with low resolutions), requires appropriate citations, and figure legends as well as the relationship between the data and the proposed model is poorly explained. Additionally, it possesses significant lack of positive and negative controls or communication of controls with the appropriate data set.
I think the paper requires a major revision.

Major Concerns:
1.	Kyoto Encyclopedia of Genes and Genomics (KEGG) should be cited with appropriate reference (doi:10.1093/nar/28.1.27) and all bioinformatics techniques used should be cited with appropriate reference or web link.
2.	Line: 76 and 77: Mention detailed bioinformatics web resources used for this study.
3.	Figure 1 and Table 1 uses reported nine datasets which should be included in reference list.
4.	Figure 1: Figure legends should be changed with appropriate designation. [May use LUAD Vs. Normal Lung (Beer et al, Nat Med 2002)].
5.	Data and proposed model is overstated, author should explain overall and detail results of bioinformatics analysis.
6.	In Table 1 and 2: “Age at initial pathogenic diagnosis.”  Entry “80” should be stated appropriately.
7.	In a running text full form for OS, PPS, and RFS should be added. (Ex. OS: Overall Survival).
8.	Result section needs to be redrafted with appropriate and additional details on outcome of bioinformatics analysis (Especially, for Figure 4 and 5 provide detailed explanation of analysis).
9.	What are the positive and negative controls were used for data analysis of Figure 3?
10.	 Figure 4A: author should provide “n” values for the samples used.
11.	Conclusion should be redrafted appropriately.

Overall this study deals with the data acquisition and analysis using different bioinformatics methods with initial comparison of nine datasets as shown in Figure 1. In addition to this, if author can provide additional bioinformatics analysis for recently reported dataset/s, it will be extremely important and useful for strengthening the field as well as further understanding the therapeutic role of NTK2 in LAUD.
Validity of the findings: No comments
Additional comments: No additional comments


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

Input:
Title: A TROPOMYOSIN RECEPTOR KINASE FAMILY PROTEIN, NTRK2 IS A POTENTIAL PREDICTIVE BIOMARKER FOR LUNG ADENOCARCINOMA
Review round: 2
Reviewer: 1

Output:
Basic reporting: Overall the manuscript seems get better. The findings are worth to report and will be useful in the studies of NTKR2 expression profile. Literature is well referenced & relevant to the concept. But still here are few minor concerns that need to address before acceptace.
Experimental design: No comments
Validity of the findings: No Comments
Additional comments: It is unusual to have no space between word and reference bracket??
eg. Line: 48  treatment(Zheng et al. 2018),
Line 52: schizophrenia(Spalek et al. 2016)  and many more throughout the manuscript. Author requested to check and confirm with journal guidelines.

The author should use appropriate tense to write the part of discussion in which they are discussing their current work, for eg.
Line193-195: “This study was the first to give comprehensive evidence through bioinformatics analysis of different public datasets that NTRK2 was identified as anti-oncogene in LUAD and could be used as a potential biomarker.” This is not the past work and it is been presented for the first time. It could be written as ‘This is the first study which presents comprehensive bioinformatic analysis of different public datasets that NTRK2 was identified as anti-oncogene in LUAD.’

Line 200: ‘cancer.NTRK2’ space is missing after sentence.
In ‘Acknowledgement’ section author could mention funding details. ‘Polishing’ is not an appropriate word for language editing services by commercial provider.
Present designation of the author is not required to be mentioned. Present author affiliation can be mentioned in ‘authorship and affiliation section’.


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

Input:
Title: A TROPOMYOSIN RECEPTOR KINASE FAMILY PROTEIN, NTRK2 IS A POTENTIAL PREDICTIVE BIOMARKER FOR LUNG ADENOCARCINOMA
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comments
Experimental design: no comments
Validity of the findings: no comment
Additional comments: The manuscript: A tropomyosin receptor kinase (TRK) family protein, NTRK2 is a potential predictive biomarker for lung adenocarcinoma.
The authors have corrected the manuscript according to the suggestions of both the reviewers. They have responded well on suggestions and corrections. Therefore, I suggested this manuscript could be accepted for the publication in this journal.


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

Input:
Title: A TROPOMYOSIN RECEPTOR KINASE FAMILY PROTEIN, NTRK2 IS A POTENTIAL PREDICTIVE BIOMARKER FOR LUNG ADENOCARCINOMA
Review round: 3
Reviewer: 1

Output:
Basic reporting: The manuscript entitled " A tropomyosin receptor kinase (TRK) family protein, NTRK2 is a potential predictive biomarker for lung adenocarcinoma" by Xiang Wang et al. studies the expression profile data of NTRK2 gene in lung adenocarcinoma.

Overall the manuscript has improved after revision and can be accepted for publication.
Experimental design: NA
Validity of the findings: NA
Additional comments: Acceptable for publication.


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

Input:
Title: PHD7FASTER 2.0: PREDICTING CLONES PROPAGATING FASTER FROM THE PH.D.-7 PHAGE DISPLAY LIBRARY BY COUPLING PSEAAC AND TRIPEPTIDE COMPOSITION
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript “PhD7Faster 2.0: predicting clones propagating faster from the Ph.D.-7 phage display library by coupling PseAAC and tripeptide composition” by Bifang He, Heng Chen, Jian Huang proposed an SVM-based method to identify fast-growing peptides from the Ph.D.-7 phage display library, which is a update of the PhD7Fater tool. The SVM-based predictive model was further implemented into a web server, and a standalone version of the tool was also developed, which can predict propagation-related target-unreltaed peptides in small-scale biopanning data as well as large-scale phage display data. Minor revisions are required before publication.
Experimental design: The topic is important. Experimental design is correct.
Validity of the findings: The method is novel. Good results were obtained.
Additional comments: The manuscript “PhD7Faster 2.0: predicting clones propagating faster from the Ph.D.-7 phage display library by coupling PseAAC and tripeptide composition” by Bifang He, Heng Chen, Jian Huang proposed an SVM-based method to identify fast-growing peptides from the Ph.D.-7 phage display library, which is a update of the PhD7Fater tool. The SVM-based predictive model was further implemented into a web server, and a standalone version of the tool was also developed, which can predict propagation-related target-unreltaed peptides in small-scale biopanning data as well as large-scale phage display data. Minor revisions are required before publication.

I have a few small concerns:
1. The CD-HIT program is a widely used to remove redundant sequences. Did authors try to use it to remove redundant sequences in the benchmark dataset?

2. Page 6, "PseAAC and tripeptide composition have been widely used in protein prediction related research", authors should cite related references here.
Page 9, "Feature selection, thus, is a critical step to reduce feature dimensionality and build a highly effective prediction model", authors should also cite related references here.

3. I have noticed a few selected grammar/writing style errors (I assume someone else will proofread and edit the manuscript before publishing it).


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: PHD7FASTER 2.0: PREDICTING CLONES PROPAGATING FASTER FROM THE PH.D.-7 PHAGE DISPLAY LIBRARY BY COUPLING PSEAAC AND TRIPEPTIDE COMPOSITION
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript, PhD7Faster 2.0: predicting clones propagating faster from the Ph.D.-7 phage display library by coupling PseAAC and tripeptide composition, by B. He, H. Chen, and J. Huang, reports a new web tool to predict propagation-related target-unrelated peptides (Pr-TUPs) from the Ph.D.-7 phage-displayed peptide library from New England Biolabs. The authors have made improvements compared to their previous version of the web tool (PhD7Faster 1.0) by employing the copy numbers of clones from the naïve library as well as the amplified library, by considering tripeptide composition, and by equipping the tool to handle large data sets such as next-generation sequencing data. PhD7Faster 2.0 is a valuable tool for researchers who perform phage display and/or are interested in target-unrelated peptides.

The manuscript is assembled in a professional manner, with sections appropriate for the content of the study. It is self-contained, and the amount of content is appropriate for one publication unit. It is organized, generally well-written, and conforms to professional standards of the scientific literature. Below I have made a few specific suggestions about wordings and grammar. I have also included here some small corrections to the citing of the literature (I have more significant comments about the relationship of this report to the literature in a section below).

Lines 39-40  Probably should have one or more references for the inorganic, organic, and biological materials at the end of the first sentence.

Lines 46-48  Does the Martins et al. reference apply to the whole sentence? If so, it should be at the end of the sentence. If not, there should be specific references for “materials science, biotechnology, pharmacology, cell biology and diagnostics.”

Line 49  The term “phage display screen” is a bit awkward. How about “The phage display methodology is notorious…”

Line 52 Not “which can fall” but rather “and can fall” because “which” is supposed to refer to the noun right before the comma (target of interest), but if you use “and” it’s clear that you are referring to the same subject of the sentence, the false positive TUPs.

Line 52  PrTUPs, as coined in Thomas et al., stands for “propagation-related” not “proliferation-related.”

Lines 52-53  Displayed peptides do not normally “react” with anything (this word implies a chemical reaction). They bind to targets and/or components of the screening system. (Reacting is a chemical process; binding is a physical process. Sorry  I teach general chemistry! ;)

Line 56  lower growth rates

Lines 68-69 “archives special for” is unclear; I’m not sure what you’re trying to say here. Also, it’s not clear what you mean about “many different screening experiments” and “lack of target specificity” – are these two different ways that the TUPs are identified or are they the same/connected?

Lines 96-97  Better to write, “…to characterize both the naïve Ph.D.-7 phage display library and the same library after one round of amplification.”

Lines 173-174  “These measures were expressed as follows” implies that all five measures are going to be elaborated upon, but only the first four are actually defined. Either separate the AUC from the others in the list (line 172) or elaborate on it also.
Experimental design: Here I must insert the disclaimer that I am not a computational researcher, not do I know a lot about computational methods. I hope that the other reviewer was able to critically analyze the methods used in the study. For my part, I can say that the experimental design was clearly described and seemed to follow the conventions of the particular practices employed (at least based on the things I looked up while I was reading). It also appeared to me that the experiments were described in enough detail that they could be reproduced by another researcher who has a knowledge of computational methods. Additionally, the investigation was performed with high technical and ethical standards. In terms of the knowledge gap that is filled by this contribution, I would say that there is always more room for better methods to identify both selection-related and propagation-related TUPs. Phage display is a widely-used technique that can be complicated by the presence of target-unrelated peptides, which crop up in panning data and can be mistaken for actual target-binding peptides.
Validity of the findings: My primary concern about this article is the strong implication that propagation-related TUPs (Pr-TUPs) arise as a consequence of the displayed peptide, which I assume is the reason the authors employed peptide composition to develop the classifier (Lines 81-83, 110-144, 236-244). In fact, our three publications (Brammer et al., Nguyen et al., and Zygiel et al.) have demonstrated that numerous Pr-TUPS possess mutations in the 5’-untranslated region of gene II in M13 phage. Zygiel et al. describes the likelihood that these mutations compensate for the replication defect afforded by the lacZ insert present in the M13mp-based vector upon which the Ph.D.-7 (and Ph.D.-12) library was based. Thus, the particular peptide displayed (e.g., HAIYPRH, GKPMPPM, AKIDART, …) is merely a stowaway on a clone that propagates fast due to its gene II 5’-UTR mutation. In these clones, the peptide itself is completely arbitrary; it just happens to be the peptide displayed on a clone that picked up a mutation prior to or during library construction (see Zygiel et al., p.17, 3rd paragraph). When Matochko et al. showed sequence bias in both the naïve and amplified libraries, they did not suggest that the cause of clone enrichment was the displayed peptide. In fact, they clearly stated, “Our report uncovers ‘parasites,’ which do not have a specific amino acid sequence. Their high abundance cannot be predicted from positional abundance of amino acids … The biological mechanism that makes some sequences ‘parasitic’ is already known: they emerge due to mutations in the regulatory region of the phage genome” (and here they referenced Brammer et al). It was soon after this publication that we reported (Nguyen et al.) several more gene II 5’-UTR mutants that overlapped with the parasites Matochko et al. identified.

The authors support the use of tripeptide composition to develop predictors by citing how it was successful in predicting the subcellular localization of mycobacterial proteins (Zhu et al.) and apoptosis protein subcellular location (Liao et al.) (Lines 240-242). But these events are clearly dependent on the amino acid composition of the proteins involved, while the evidence around Pr-TUPS points to mutations that have nothing to do with the displayed peptide sequence.

Now, it is possible that PART OF the peptide sequence bias in the naïve Ph.D.-7 library is due to biological factors that make some displayed peptides more stable than others. It is likely that certain amino acids are censored because they create less stable peptides. But this censorship is a completely different phenomenon from that of displayed peptides making the phage faster, particularly after the library has been constructed and already contains the subset of peptides that are stable enough to have survived library construction. I would recommend that the authors focus on the biological basis for the amino acid censoring that may lead to the original sequence bias in phage display libraries, but avoid any implication that once the library is constructed, certain clones are enriched during amplification due to the properties of the displayed peptide (unless they can find actual experimental data that supports this). If the authors do intend to state that after the library is constructed, some displayed peptides confer to the phage properties that enhance infection, assembly, secretion (or whatever else), which consequently allow the phage to propagate faster, then they should (i) be clear about how in particular the displayed peptide affects these properties and (ii) back it up with some evidence from the literature.
Additional comments: Well-constructed paper overall, and very useful web-tool. Please flesh out your discussion of how/why propagation-related TUPs arise (gene II 5’-UTR mutations) and address the problematic linking of Pr-TUP enrichment to the displayed peptide sequence.


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: PHD7FASTER 2.0: PREDICTING CLONES PROPAGATING FASTER FROM THE PH.D.-7 PHAGE DISPLAY LIBRARY BY COUPLING PSEAAC AND TRIPEPTIDE COMPOSITION
Review round: 2
Reviewer: 1

Output:
Basic reporting: Authors have made revisions according to my suggestion. I think the paper should be accepted.
Experimental design: The experimental design is correct.
Validity of the findings: The results are reliable.
Additional comments: The paper should be accepted.


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: PHD7FASTER 2.0: PREDICTING CLONES PROPAGATING FASTER FROM THE PH.D.-7 PHAGE DISPLAY LIBRARY BY COUPLING PSEAAC AND TRIPEPTIDE COMPOSITION
Review round: 2
Reviewer: 2

Output:
Basic reporting: My general comments are the same as in my first review, and all the small edits that I recommended have been made. Good work!

One strange thing:  This passage was mentioned in the author’s rebuttal, but I do not see it in the manuscript: “For example, the peptide HAIYPRH (a typical PrTUP) has been identified by 23 completely different targets according to results of searching the BDB database. The phage displayed the peptide was later verified to have a propagation advantage owing to mutations in the regulatory region of the phage genome (Brammer et al. 2008). HWGMWSY (a SrTUP) has been isolated by 10 completely different targets according to records in the BDB database. The peptide was proved to be a plastic binder (Vodnik et al. 2012), accounting for the target-unrelated selection of this peptide in multiple reported panning experiments.”
Experimental design: My comments are the same as in my first review.
Validity of the findings: The authors have added the key content that I requested, and I appreciate this improvement. However, I still have a few concerns about writing style and organization:

1.	The new paragraphs are inserted at the very start of the discussion, making for a very abrupt start to the section. It also does not connect and integrate the new content to the pre-existing content. For example, the part about the use of tripeptide composition to develop predictors in the cases of the subcellular localization of mycobacterial proteins (Zhu et al.) and apoptosis protein subcellular location (Liao et al.) (Lines 262-265) is, in part, what warranted some discussion of whether peptide composition is the determinant of the property being analyzed (in those cases it is; in the case Pr-related TUPs, it is not). But the new content is not connected to the pre-existing content, which is 2-3 paragraphs later. Also, how does the censorship of peptides play into how this new method works? How does the presences of arbitrary peptides coincident with gene II 5’-UTR mutations play into how the new method works? I would recommend that the authors considered the organization of the first four paragraphs of the discussion with an eye for flow and cohesion.
2.	The first sentence of each new paragraph is unclear. “Some peptides are likely to be censored...” The reader may think this means censored from the analysis, when it really means censored from being displayed on the phage in the first place. “The opposite, PrTUPs are biased towards during amplification.” This sentence is not grammatically correct. Also, “opposite” is a very specific word, which I wouldn’t use here. I think the authors may be struggling with these opening sentences, in part, because of my first point above about how the new content should be integrated with the pre-existing content.
Additional comments: N/A


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

Input:
Title: COMPREHENSIVE ANALYSIS OF AN LNCRNA-MIRNA-MRNA COMPETING ENDOGENOUS RNA NETWORK IN PULPITIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1. I suggest detailing further on non-coding RNAs modulating immune responses via post-transcriptional regulation. For example, miR-146a and miR-155 work in a feedback manner to maintain immunological homeostasis. It would be a plus if the authors could relate the state of the art of postranscriptional regulation of microRNAs, specifying on miR-455. This would round up the whole idea introductory idea.

2. A more detailed figure description and including labels from the main steps on Figure 1, as in “Step 1, Step 2... etc.”, can make it easier to the reader to analyze the diagram without recurring to reading the whole document.

3. Figure 2: On Figure 2-A, adding a label that read “Regulation:” before the symbol list from “Down, Not, Up” could help for better understanding by only looking at the figure.

4. Name on Figure 3 seems misleading.
It reads "Top 10 processes revealed in GO enrichment analysis of clusters of the upregulated DEGs." but it apparently show top 10 processes of 3 categories, so maybe stating it as "Top 10 processes revealed in GO enrichment to influence biological process (BP), molecular function (MF), and cellular component (CC).” could guide readers to better analyze the information.

6. Line 359 Has a red mark on a reference
Experimental design: No comment
Validity of the findings: The study and its findings are very interesting. I advise the authors to describe a little bit further about the relation of noncoding RNAs in immunological modulation. This will nicely round up your discussion and conclusions.
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: COMPREHENSIVE ANALYSIS OF AN LNCRNA-MIRNA-MRNA COMPETING ENDOGENOUS RNA NETWORK IN PULPITIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1.The manuscript is clearly written in professional english.
2. The format of the manuscript is meet the standard of Peer J. They provided sufficient background and literates.
3.Figures quality is OK.
Experimental design: 1.The experiment design is only based on bio-informatics analysis. It is strongly suggested to add some experimental data to validate your conclusion.
2. The original research meets the Aims and Scope of the journal.
3.Research question is well defined and meaningful. However, it is hard to say whether the conclusion of the manuscript is right or not and whether the hypothesis fills an identified knowledge gap.
4. Methods provide the enough information to replicate.
5.Routine technology is used for the investigate.
Validity of the findings: It is very hard to evaluate the novelty of the manuscript now. The conclusion is not very  convincing. The authors selected 10 hub genes including IL6, IL8, PTPRC, IL1B, TLR2, ITGAM, CCL2, PIK3CG, ICAM1, and PIK3CD. However, they built a ceRNA network is In the PVT1-hsa-miR-455-5p-SOCS3 and PLXNC1. There is no hub gene at all. In addition, it is strongly suggested the authors do some experiments to evaluate your conclusion in pulptitis tissues and dental pulp cells.
Additional comments: In Figure 2 B, please replace “code name” by real gene name.


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: COMPREHENSIVE ANALYSIS OF AN LNCRNA-MIRNA-MRNA COMPETING ENDOGENOUS RNA NETWORK IN PULPITIS
Review round: 2
Reviewer: 1

Output:
Basic reporting: Redaction is unambiguous and professional. Correction from authors on previous comments has brought the previously needed references and background to support their statements and findings.
Experimental design: The experimental design was improved with rtqPCR validation.

I agree with authors that in vitro establishment of an in vitro pulpitis model could differ highly from in vivo sampling since there are so many variables involved in the process it is impossible to model it accurately enough.

This study describes with sufficient detail the bioinformatic approach to analyze this inflammatory state and could serve as a platform to develop future studies involving cell culture and specific gene validations of the proposed regulatory hub.
Validity of the findings: Conclusions are well stated, highly improved discussion and conclusions with corrections from reviewers comments.

The information contained in this article will benefit literature and hopefully encourage the scientific community to continue the study of regulation networks relating to non-coding RNAs in different inflammatory states, this is helpful to design and develop more effective therapies.
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: COLEOPTERA GENOME AND TRANSCRIPTOME SEQUENCES REVEAL NUMEROUS DIFFERENCES IN NEUROPEPTIDE SIGNALING BETWEEN SPECIES
Review round: 1
Reviewer: 1

Output:
Basic reporting: No issues
Experimental design: Meets all the criteria
Validity of the findings: No issues
Additional comments: Manuscript: Coleoptera genome and transcriptome sequences reveal numerous differences in neuropeptide signaling between species

General comments:
This manuscript describes the identification of neuropeptides/neurohormones from multiple coleopterans in publicly available genomic and transcriptomic datasets. I commend the author on such a comprehensive analysis which even takes in to account neuropeptides (eg Hansolin) that have been recently discovered. I don’t have any major issues with the manuscript – just some typos, queries and suggestions to improve the figures.

Introduction:

Lines 45-46: Gain and loss of neuropeptides are two phenomena in the broad sense. But what about the modification of existing neuropeptides/receptors? This type of peptide evolution is later described for PDF and NPF.

Line 74: delete “there are”

Line 81: replace “in stead” with “instead”

Material and methods:

Lines 98-99: which precursor is this?

Lines 225-227: These are in vitro studies and should be treated with caution. Has it been shown that CCHa1 can activate CCHa2 receptor in vivo and vice versa? As far as I am aware, only the Drosophila PDF receptor has been shown to be activated by DH31 in vivo. I understand the point that the author is trying to make but perhaps these 2 examples of sNPF/NPF and CCHa-1/2 might not be the best ones.

Results:
Lines 270-275: This part is a bit confusing and can be elaborated.

“none of Leptinotarsa neuropeptide genes predict the same structure as the allatotropin ortholog from Locusta migratoria” – does this mean that Locusta allatotropin ortholog was not identified here?
“while this species has a proctolin gene, it does not predict an [Ala1]-proctolin” – what is the significance of [Ala1]-proctolin?
“Two other peptides that were reportedly isolated...” – what are these peptides?

Line 323: Figure 4 is for Elevenin and Figure 5 is for calcitonin. Swap the two.

Calcitonin B: Any speculation on how such a precursor could evolve? What is the similarity between DH31 and the calcitonin peptide without the disulfide bridge?

Line 392: Replace Figure S7 with Figure S8.

Lines 394-395: How can you be sure that a gene is no longer “functional”? I assume based on the alignment that it is missing the bits encoding the N-terminal fragment and hence doesn’t get translated. Please clarify.

Line 407: Figure 7 is AKH alignment not elevenin.

Line 445: Replace figure S12 (bursicon) with figure S11 (RFLa).

Line 460: Should be Figure S12 and S13. Make sure all the figure references are accurate.

Line 470: Is this calcitonin A?
Line 474: Although this gene is duplicated, the second gene produces a non-amidated peptide and might thus be non-functional. This is mentioned in the figure caption but should be elaborated in text.

Also, as mentioned above, CCHa1 has its own specific receptor and CCHa2 has its own. That is also the conclusion of Hansen et al that deorphanized these receptors. So I don’t see how the duplication of CCHa1 receptors is related to CCHa2.

Line 476: add “are” before “more similar”

Line 567: delete the extra “one to”

Line 584: Figure 12 refers to DH37 and DH47 genes.

DH31 transcripts appear to be quite interesting. Do you predict the other neuropeptides to have a separate receptor? Are there any extra Family B GPCRs in the species which produce these extra peptides? Also, Rhodnius DH31 also has an interesting splicing pattern (see Ons et al 2011). Any similarities between Rhodnius and Coleoptera splicing?

RYamide: Any comments on the conservation of Cys residues in the C-terminus of the precursor? Seems similar to the conservation seen for AKH precursors.

Discussion:

Line 691: add “it” after “possible that”

Line 705: “neuropeptide structures” – are you referring to the primary structure, secondary structure or both? Do you think this is the reason why insulin structures are quite variable?

Line 721: What about the possibility that the evolution of peptides is what facilitates complete metamorphosis? Difficult to tell what is the causative factor.

Line 738: “accidental elimination” – what do you mean by this?

Figures:
For most of the alignments in supplementary files, it is difficult to spot the mature peptide sequences and count the variability when paracopies are present. Perhaps you could underline or bold them to make them standout.

FigS2: Indicate the baratin mature peptide sequence
FigS3: Indicate the myosuppressin mature peptide sequence
FigS9: Indicate the sNPF mature peptide sequences
FigS10: Indicate the proctolin mature peptide sequences
FigS11: Indicate the RFLa mature peptide sequences
FigS18: Indicate the mature peptide sequences
FigS20: Indicate the vasopressin mature peptide sequences
FigS22: Indicate the allatotropin mature peptide sequences
FigS26: Indicate the DH47 mature peptide sequences
FigS27: Indicate the DH37 mature peptide sequences
FigS28: Indicate the ETH mature peptide sequences
FigS29: Indicate the RYa mature peptide sequences
FigS30: Indicate the sulfakinin mature peptide sequences. Do this for the remaining alignments where the location and number of peptides are fairly constant.

Figure 4: Pogonus elevenin sequence is missing.

Figure 10: Instead of using red (which is usually used to indicate losses in this manuscript), use green or another color to indicate an increase in paracopies. Also, the Tenebrio gene appears to be translucent (not shaded). Can this be made more pronounced?
Typo in caption: “hat” should be “that”

Figure 12- Typo in caption: “hat” should be “that”

Figure 13- Typo in caption: “hat” should be “that”


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: COLEOPTERA GENOME AND TRANSCRIPTOME SEQUENCES REVEAL NUMEROUS DIFFERENCES IN NEUROPEPTIDE SIGNALING BETWEEN SPECIES
Review round: 1
Reviewer: 2

Output:
Basic reporting: This manuscript titled “Coleoptera genome and transcriptome sequences reveal numerous differences in neuropeptide signaling between species” examines the neuropeptide composition of 17 beetle species. Overall this is an in depth and compressive look at neuropeptides in a diverse range of Coleoptera. It provides useful insights into the flexibility and necessity of these peptides in insects and provides some interesting findings on those neuropeptides that are essential versus those that can be lost, the level of conservation in the primary structure of these neuropeptides, and which ones have undergone duplication.
Basic Reporting: I only have few minor concerns on this topic.

The manuscript is well written, provides relevant and up to date references and succinctly covers this topic as much as possible considering the scope of this study. There are a few confusing issues regarding figure labeling. Specifically, on lines 323 and 326 it appears that the figures have been switched. Similarly, I believe that in line 407 the author is actually referring to Figure 4. There are also a few simple typos throughout that need to be identified and corrected.
Experimental design: This work, while important, is largely descriptive and as such does not have a traditional experimental design. As such I have no concerns with this section.
Validity of the findings: The author has done a good job interpreting the findings and providing possible explanations for the loss, duplication and amino acid changes in the characterized neuropeptides. Obviously at this stage the discussion is largely speculation and additional work will need to be done to verify the biological activity of these peptides. I am not certain that I buy into the idea that loss of a neuropeptide signaling system is typically led by changes to or deletion of the receptor, but it is valid speculation. Importantly, this work lays the groundwork for these future studies.
Additional comments: Overall all this is a very thorough body of work that expands our understanding of neuropeptides, not only in beetles, but in invertebrates in general.


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: COLEOPTERA GENOME AND TRANSCRIPTOME SEQUENCES REVEAL NUMEROUS DIFFERENCES IN NEUROPEPTIDE SIGNALING BETWEEN SPECIES
Review round: 1
Reviewer: 3

Output:
Basic reporting: 1. Have there been studies that compared neuropeptidomes in other insect orders, or other groups of organisms? I did not find a reference to this in the introduction. In the discussion you say that it is now the first time that neuropeptidomes were compared for species that are not closely related. This seems to imply that such studies exist for more closely related insect species. So, please add the references.

2. line 733: You state that several neuropeptides were also missing in other insect species, but it is not indicated which insect species/orders and the statement does not contain any references.

3. Figures:

- The purple branch in Figure 1 and 2 is not the same. Is there a difference?

- Figure 2: It is not stated what the asterisk stands for.

- Figure 8c: unclear what the dots mean. Not explained what the asterisk indicates.
Experimental design: 4. How representative are the Polyphaga for the order of Coleoptera? In this analysis, there is only one species included for which a genome is available, that is not part of this sub-order. Even if this contains most of the current Coleoptera species, you might still miss important information from an evolutionary point of view.

5. There is no comparison at a level lower than infraorder. It is stated the studied species were not closely related (line 661). Is there a reason that closely related species were not included? It seems to be interesting to see whether there are significant neuropeptidome differences between these closely related species. Is it known on what time-scale gain and loss of neuropeptides occurs?

6. Loss of a receptor gene can equally affect neuropeptide signaling as loss of the neuropeptide. Why did you not look at loss of receptors?
Validity of the findings: 7. It is stated that the variability in neuropeptidome composition between insect species from the same insect order may be as large as the one that exists between species from different orders. How is this variability quantified? Was this variability compared or is it a hypothesis? How about variability within an order? Can this statement be generalized from one order to another?
Additional comments: 8. Minor comments:
- Conclusion: “insect species from from the same insect order”
- Line 677: “it might be become obsolete"


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: COLEOPTERA GENOME AND TRANSCRIPTOME SEQUENCES REVEAL NUMEROUS DIFFERENCES IN NEUROPEPTIDE SIGNALING BETWEEN SPECIES
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: I only have one comment for the author (for clarification) which he can choose to address if he wishes to.  It is a response to the comment I made earlier (below).

Lines 225-227: These are in vitro studies and should be treated with caution. Has it been shown that CCHa1 can activate CCHa2 receptor in vivo and vice versa? As far as I am aware, only the Drosophila PDF receptor has been shown to be activated by DH31 in vivo. I understand the point that the author is trying to make but perhaps these 2 examples of sNPF/NPF and CCHa-1/2 might not be the best ones.

-------------It is very difficult to to do these things in vivo in insects. The reviewer states that the only example is DH31 acting on the PDF receptor in Drosophila. I described the myosuppressin receptor in Bombyx. Although I did not describe all the details (but they are in the references cited), the FMRFamide neurons innervate directly the prothoracic gland and all evidence suggests that in vivo FMRFamide acts on the myosuppressin receptor. Why else would neurons producing a peptide that is active on the receptor present in the prothoracic gland synapse onto these glands.? Given the very high affinity of this receptor for myosuppressin and the release of myosuppressin into the hemolymph one has to assume that it similarly activates this receptor in vivo. I described this example rather than that of DH31/PDF, because of the sequence similarities between FMRFamide and myosuppressin and because there are several other examples of structurally related peptides that in vitro can activate each others receptors. This suggests that this is likely a very general phenomenon. I disagree with the implicit argument of the reviewer that since it has not been demonstrated in vivo it remains to be seen whether this actually happens. The vertebrate example of CCK using the gastrin receptor in the brain is very telling in my opinion in that it illustrates the consequences of having a sensitive neuropeptide receptor in the periphery (the CCK receptor) that can not be used within the CNS, simply because it is too sensitive. When a neuropeptide is released into the hemolymph its concentrations are very low, but when it is released inside the brain, locally its concentrations will be much higher. It is for this reason that DH31 can activate the PDF receptor in the Drosophila brain and that FMRFamide, when it is released locally within the prothoracic gland in Bombyx mori can still activate the myosuppressin receptor even though its affinity for this receptor is a thousand-fold lower than that of myosuppressin. Neuropeptides are not only used at the periphery, but also within the CNS.

Response: I am not disagreeing about the conclusion of the author based on the findings of Yamanaka et al 2005. 2006. My point was that in vitro assays used to deorphanize GPCRs are usually correct but sometimes they do give a response for other ligands (depending on the type of assay used in deorphanization). Hence Hansen et al 2011 see some response (at very high doses) when CCHamide1 is applied to CCHa2 receptor and CCHamide2 is applied to CCHa1 receptor. However, it is highly unlikely that these high doses will ever be encountered in vivo. That is why I advise the author to treat those deorphanization studies which show that multiple ligands can activate a single receptor with caution.


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: COLEOPTERA GENOME AND TRANSCRIPTOME SEQUENCES REVEAL NUMEROUS DIFFERENCES IN NEUROPEPTIDE SIGNALING BETWEEN SPECIES
Review round: 2
Reviewer: 2

Output:
Basic reporting: The few minor errors I pointed out were corrected,
Experimental design: No concerns in original review.
Validity of the findings: no comment
Additional comments: Excellent summary of beetle nueropeptides that will be useful to the community. I again recommend to accept.


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: COLEOPTERA GENOME AND TRANSCRIPTOME SEQUENCES REVEAL NUMEROUS DIFFERENCES IN NEUROPEPTIDE SIGNALING BETWEEN SPECIES
Review round: 2
Reviewer: 3

Output:
Basic reporting: The revised version is much improved and has carefully taken into account the reviewer's comments.
Experimental design: Ok
Validity of the findings: ok
Additional comments: ok


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: TARGETED METABOLOMICS STUDY OF SERUM BILE ACID PROFILE IN PATIENTS WITH END-STAGE RENAL DISEASE UNDERGOING HEMODIALYSIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Several errors in punctuation and spelling need to be addressed. Specifically:
—Line 50: use a semi-colon or period before ‘…on the other hand…’
—Line 59: change it to ‘…are secreted’
—Line 91: change ‘[(’ to ‘[’; change ‘))’ at the end to ‘ )]’
—Line 96: change ‘Reagent’ to ‘Reagents’
—Line 97: change ‘...was the product of…’ to ‘…was purchased from…’
—Line 106: change ‘…curves samples’ to ‘…curves’
—Line 140: uncapitalize ‘…Extensive…’
—Line 166: use the correct plural form of ‘index’
—Line 166: ‘…baseline data and laboratory indexes…’ This is not an accurate description of the contents in Table 1.  Just incorporate ‘Demographic and clinical characteristics’ into this sentence.
—Line 180: use capital T in ‘(table 2)’ as you previously did in Table 1. Add space in front of the parentheses.
—Line 184: what does ‘positively detected’ even mean? Change to a phrase that makes statistical sense.
—Line 215: ‘…had different phenotypes.’ This conclusion seem to be derived from the model parameters; however, these values cannot be considered as conclusive evidence that there is statistically significant differences between two phenotypes. Consider rephrasing, or provide evidence that these values indicate actual phenotype-level differences.
—Line 224: use capital T in ‘(table2)’.
—Line 262: change ‘amido’ to ‘amino’
—Line 280-283: This sentence is too long to comprehend in one read. For clarity sake, consider rephrasing or separating into multiple sentences.
—Line 310: ‘… showing an U-curve relationship)’ – what is the definition of a U-curve relationship?  Where in your data is this shown?
—Line 312: ‘Bile acid is the main approach for cholesterol metabolism’.  This sentence does not make logical sense with the previous sentence. Did you mean to start a separate paragraph here?  Also, I do not understand what this sentence even means.

There are other problems with sentence constructions throughout the manuscript, makes it uneasy to comprehend. Overall English language revision of the total manuscript is desired. Authors should be more careful while preparing the manuscript. Besides typographical and English language mistakes, there are several mistakes on data presentation, as described below.

In this study, the authors addresses the relationship between changes in bile acid profiles and prognosis of End Stage Renal Disease (ESRD) in patients undergoing hemodialysis. Although the authors have briefly discussed importance of gut microbial metabolites in kidney disorders, they have not sufficiently justified why they are specifically interested in bile acids in their targeted metabolomics study.

Line 70 - ‘Previous studies have seldom focused on the expression of bile acid metabolic profile in chronic kidney disease.’ is not at all appropriate. There have been studies describing the role of bile acids in kidney diseases:
1.	Marečková, O., Skala, I., Mareček, Z., Malý, J., Kočandrle, V., Schück, O. & Prat, V. (1990). Bile composition in patients with chronic renal insufficiency. Nephrology Dialysis Transplantation, 5(6), 423-425.
2.	Jimenez, F., Monte, M. J., El-Mir, M. Y., Pascual, M. J., & Marin, J. J. G. (2002). Chronic renal failure-induced changes in serum and urine bile acid profiles. Digestive diseases and sciences, 47(11), 2398-2406.
3.	Chu, L., Zhang, K., Zhang, Y., Jin, X., & Jiang, H. (2015). Mechanism underlying an elevated serum bile acid level in chronic renal failure patients. International urology and nephrology, 47(2), 345-351.

In the discussion section, authors do mention some of these studies, but state that the results of these studies are controversial (line 253). Is ‘controversial’ the correct term here? — Controversial according to whom?

Tables:
—What is superscript ‘a’ in Table 1?
—Abbreviations are missing for CA, CDCA, DCA, TCA,THCA,TalphaMCA, etc. in Table 3.
—p-values are shown as 0.000 in Table 4. Technically, this is incorrect based on how I presume the analysis was done; rather, put ‘<10^-3’.
—In the footnote of Table 4, correct the spelling error for ‘aculated’.

Figures:
—Correct the title of Figure 1.  ‘Metabonomic’ should be ‘Metabolomic’ to be consistent with the contents of the manuscript.
—In Figure 1A, B and C, correctly label the names of the axes.
—What is R2, Q2 in figure 1, and also described in line 217?  For those unfamiliar with OPLS-DA modeling, provide a brief explanation.
—I have no idea how to interpret Fig. 1C.  Do the red and blue dots at X=1 indicate outliers?  Make this more self-explanatory, as it’s hard to understand the main message of this figure.
—There are no legends in Figure 2.  Please describe.
—It’s hard to understand what message authors want to convey by looking at Figure
3. The three groups are not clearly distinguishable. What are the differentially abundant bile acids?
—In the y-axis of Figure 3, change ‘Fold of Change’ to ‘Fold-change’; correct the spelling mistake of ‘heathey’.
—Make the bars in Figure 3 into color.
—If the fold-changes are relative to the control cohorts (as described in the first sentence of the legend), then the bar for ‘control’ is not necessary, as its height should always be 1.
Experimental design: – Methodology section does not provide sufficient information. For example, authors defined total bile acid (TBA) as a sum of 26 BAs (line 141). But it is not clear what are those BAs that the authors target.
– Why was multiple hypothesis correction not performed when comparing the groups?
– Authors should provide sufficient justification for using different types of statistical tests.
– Please explain parameters R2X, R2Y and Q2Y in OPLS-DA.
– Authors should clearly explain the subgroup analysis in a separate head in methodology section.
Validity of the findings: – In this manuscript, through a targeted metabolomics approach, the authors performed a comparative analysis of bile acid profiles of ESRD patients and healthy samples. Authors have identified six bile acids as biomarkers that can distinguish ESRD patients from healthy samples. However, as the authors did not perform any analysis on the gut microbiome composition of ESRD and healthy samples, repeatedly associating the observed differences in bile acid profiles to gut microbiome seems far-fetched.  Consider truncating the speculative discussion on gut microbes.
– Line 315: ‘On the contrary, our study found that the FXR activity of ESRD patients was inhibited due to the decrease of FXR agonists…’ – where in your manuscript results have you checked FXR activity?
– It’s hard to find any clinical significance in distinguishing ESRD patients from healthy samples based on six bile acids biomarkers as proposed by authors. Also, the difference in bile acid profile may be due to the consequences of ESRD condition, which is needed to be verified. A more intriguing observation would have been to study whether these bile acids biomarkers can detect early stages of renal failure.
– Why the ROC analysis and subgroup analysis yield different sets of bile acids? The authors claim six bile acids as potential biomarkers to differentiate healthy and ESRD patients, but when they compare in three groups, they found different bile acids. Does that mean these biomarkers have no role in disease prognosis?  Please clearly explain, as this pertains to one of the main messages of the manuscript.
Additional comments: No additional comments