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

Input:
Title: EVALUATING THE OESTROGENIC ACTIVITIES OF AQUEOUS ROOT EXTRACT OF ASPARAGUS AFRICANUS LAM IN FEMALE SPRAGUE-DAWLEY RATS AND ITS PHYTOCHEMICAL SCREENING USING GAS CHROMATOGRAPHY-MASS SPECTROMETRY (GC/MS)
Review round: 2
Reviewer: 2

Output:
Basic reporting: In general, the article has improved, with the exception of some inconsistent use of acronyms.
Experimental design: Reporting of the experimental design has improved and is generally well reported.
Validity of the findings: I have some concern about text in the discussion that does not appear to match the figures provided. I have made comments in the pdf to support this statement, but an example would be "AEAA at a concentration of 200 mg/kgBW down-regulated the expression of calb3 gene in the absence of the oestrogen receptor antagonist." This statement should refer to Figure 5, which presents gene expression in the absence of ORA. In figure 5, there are no calbindin bars with an asterisk to denote statistical significance. The authors need to double check that the statements made in the discussion match those in their figures and in the body text.
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: PROXIMATE GRASSLAND AND SHRUB-ENCROACHED SITES SHOW DRAMATIC RESTRUCTURING OF SOIL BACTERIAL COMMUNITIES
Review round: 1
Reviewer: 1

Output:
Basic reporting: The authors investigated the effects of shrub-encroachment on the soil bacterial community structure by Illumina Miseq. Overall, the manuscript was written with professional standard English and easy to follow. The structure, figures and tables in the MS are sound and readable. However, the introduction seems lacking of few lines on the importance of microbes. One question is that why the authors did not investigate the shift in fungal community, which are more important in carbon cycles and close related to vegetation changes.
Personally, the text in result part could be more straightforward than by using sophisticated, but rather confusing sentences. e.g.
Experimental design: The aim of the MS was clear and the experimental design were sound and logical. Materials and methods section were well described in most of the parts, except in Statistical analysis, in which, lines of 136-143 were not well explained and clarified. What are the purposes to use NTI, betwNTI as well as Co-occurence network analysis. The interpretaton from these analysis in the result were somehow vague.
Validity of the findings: The conclusion seems not focusing the aim of the study. The listed 1),2) and 3) should be in the background information section rather than in the conclusion. Secondly, the suggestion (line 245) can be confirmed  easily by further analysis for community function based on the OTU taxonomy via computationally Predicted Functional Metagenomes (PFMs).

Line 198-199. Is it correct interpretation? Encroachment caused bacterial community shift, not vice versa.
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: PROXIMATE GRASSLAND AND SHRUB-ENCROACHED SITES SHOW DRAMATIC RESTRUCTURING OF SOIL BACTERIAL COMMUNITIES
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript “Proximate grassland and shrub-encroached sites show dramatic restructuring of soil bacterial communities” compared the bacterial community difference at the levels of composition and phylogenetics, aiming to understand the effects of shrub encroachment on soil bacterial community in grassland ecosystem. The results showed that shrub soil bacterial diversity was significantly higher than grassland, and their community structure substantially differred. SOC played a key role in driving the soil bacterial community difference in shrub and grassland, but pH did not. The study provided interesting findings, which contributes to understand the belowground response to shrub expansion.
Experimental design: No comments.
Validity of the findings: Here are my concerns:
1.	The introduction failed to appreciate previous studies about the association of shrub and grassland with soil microbial community.
2.	Phylogenetic diversity was an important index in the study, but was not mentioned the calculating method in the method section.
3.	Line 163-164 should go to the section of bacterial alpha-diversity.
4.	Line 169 "more relative abundance" should be a higher relative abundance.
5.	Line 182, I had difficulty in understanding the sentence, there was a language problem.
6.	SOC was demonstrated to be a key factor to differ the bacterial community in shrub and grassland soils, but authors did not carefully discuss this point. I'd encourage the authors discuss how SOC type and contents from shrub and grass affect bacterial communities.
Additional comments: See above.


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

Input:
Title: VISUAL COMPLEXITY MODELLING BASED ON IMAGE FEATURES FUSION OF MULTIPLE KERNELS
Review round: 1
Reviewer: 1

Output:
Basic reporting: In general the English is okay, but the writing could be improved at several places. For example, in l. 25, remove "the" in front of visual complexity; delete paragraph between l. 85/86; "influence of" is repeated in l. 144; change "have" to "has" in l. 385; l. 15, write number as 240; section number missing in l. 322; l. 156, it should say "5 seconds";

The abstract needs to be improved by i) mentioning the results of the study in much more details, ii) by rephrasing the first sentence which sounds too exaggerated, iii) by making it clear that this study is a re-analysis of data provided by Forsythe et al. 2011.

Throughout the manuscript there are incidences in which the names of authors are repeated in the text. For example, see l. 43, "Donderi Donderi (2006). The problem could be related to the reference software or the style template of PeerJ. In l. 33 the year is given once as 1932 and then as 1933. Please revise accordingly.

The beginning of the Discussion is too specific. I suggest that the authors repeat their main question and their main results in a few sentences before discussing details as color. The end of the Discussion would also profit from i) limitations of the current study, and ii) ideas for future research. Would it be possible to extend the Discussion and to discuss how the current results relate to perceptual/cognitive processes in more depth?

Research by Christopher Redies and Johan Wagemans on complexity could be added to the Introduction.

I could not open the figure files. In Figure 1 the year is missing after each reference, it seems. But this information would be useful to readers.
Experimental design: I suggest that the authors make it clear throughout the manuscript that this paper is based on a re-analysis of data collected in an earlier study. L. 124: This sentence reads as if the authors would have access to all data sets described in Figure 1, which is not the case. Please make it clear in the text that the authors' work - of course - also depended on the availability of data and that this also influenced the choice of the data set (besides the large N of stimuli etc.). Otherwise it is misleading.

Please do not mix information on the participants with information on the procedure (Section 0.3). L. 154, please provide more info on the definition of complexity (i.e., report it as it was given).

Figure 3: It is a bit unclear why the information about males/females is relevant for the current study. Please explain in the text.

L. 188: This sentences us unclear. What do the authors mean by "In order to design our experiments..."?

L. 205: I would not call the current study an experiment. Please explain why this term was used.

I like the rigorous approach to remove outliers. This section is important because it shows that we need to be careful because the results of the analysis may indeed change. I commend the authors on giving a detailed report.
Validity of the findings: Please provide more information in the Discussion on how the authors' results relate to human perception and cognition.
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: VISUAL COMPLEXITY MODELLING BASED ON IMAGE FEATURES FUSION OF MULTIPLE KERNELS
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1.Try to set the problem discussed in this paper in more clear, write more to define the problem. So the reader can understand it deeply.
2.The discussion is insufficiency the author should be focus more on this section so that the reader can understand the importance of your work.
3.The paper's English needs to be significantly improved, as it its current form requires a lot of effort to read it.
Experimental design: The experimental design can be shown by formula rather than a simple language description.The content of the experiment can be rich and diverse, not like a report
Validity of the findings: There are no mentions about the complexity of the algorithm, as well as its comparison with other results from the literature.
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: VISUAL COMPLEXITY MODELLING BASED ON IMAGE FEATURES FUSION OF MULTIPLE KERNELS
Review round: 1
Reviewer: 3

Output:
Basic reporting: There are some typo mistakes and I suggest authors to proof read the paper.
The paper is organized in a well manged format.
Experimental design: This authors proposed the application and validation of a set of different computational machine learning models for the evaluation of visual complexity. Six state-of-the-art Machine Learning regression algorithms were compared, with and without feature selection, and a ﬁnal statistical analysis step was performed in order to evaluate the results and select the most promising one. The main contribution of this work bit limited and I my suggestion is incorporate the following changes before acceptance (My suggestion is minor revision)

Training and test time should be reported in the form of tables of figures.
The optimization details/parameters selection about machine learning algorithms are missing at the moment. It must be added at the time of revision.
Validity of the findings: A solid model is selected for evaluation of proposed research  and main contribution of this work is bit limited (My suggestion is accepted marginally/minor revision) . My suggestions are to incorporate the following changes before formal acceptance.

1.	There are some typo mistakes and I suggest authors to proof read the paper.
2.	Training and test time should be reported in the form of tables of figures.
3.	The optimization details/parameters selection about machine learning algorithms are missing at the moment. It must be added at the time of revision.
4.	Some discussion about recent approaches based on deep-learning should be at least discussed in the paper and results should be compared even if they are less.
5.	Some recent citations/research based on traditional machine learning approaches like SVM//ANN/Feature extraction should be discussed/cited at the time of revision.

“Intelligent Image Classification-Based on Spatial Weighted Histograms of Concentric Circles.”
“Image classification by addition of spatial information based on histograms of orthogonal vectors”
“A Hybrid Geometric Spatial Image Representation for scene classification”
“A novel image retrieval based on visual words integration of SIFT and SURF”
“A Novel Discriminating and Relative Global Spatial Image Representation with Applications in CBIR”
" Image retrieval by addition of spatial information based on histograms of triangular regions"
Additional comments: Dear Editor

Thanks a lot for sending me this manuscript for review. My suggestion is minor revision before formal acceptance.

Regards

Nouman Ali, PhD


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: VISUAL COMPLEXITY MODELLING BASED ON IMAGE FEATURES FUSION OF MULTIPLE KERNELS
Review round: 2
Reviewer: 1

Output:
Basic reporting: l. 38, it should say "paperS"
l. 166, change "chosed" to "chose"
l. change to: access to "the" dataset used by Forsythe
l. 420, change to :  work by Forsythe

There are several incidences in the Introduction where the in-text references are repeated, e.g., l.44, l.61, l.114, l.116, l.129... please check the complete manuscript.

Figure 9, E, typo in "outliner2 5
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The authors have considerably improved their manuscript. I suggest that the authors read carefully through the manuscript to check for last language and formatting issues.


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: VISUAL COMPLEXITY MODELLING BASED ON IMAGE FEATURES FUSION OF MULTIPLE KERNELS
Review round: 2
Reviewer: 2

Output:
Basic reporting: The author propose the application and validation of a set of different computational Machine Learning 498 models for the evaluation of visual complexity. I think the article is innovative.The application direction is also relatively new.The paper’s English has been improved.
Experimental design: The author has add some discussion about recent approaches based on machine learning have been  discussed in the paper. SVM//ANN/Feature extraction have been added.The content of the experiment has been improved.
Validity of the findings: More info about models complexity and computing time has been added. The training and test time has be reported in the form of tables of figures.
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: VISUAL COMPLEXITY MODELLING BASED ON IMAGE FEATURES FUSION OF MULTIPLE KERNELS
Review round: 2
Reviewer: 3

Output:
Basic reporting: English used in the paper is standard. Results presented are significant. However, i will suggest 02 minor changes.
1. The quality of figures should be improved , at present, the figures are in low quality.
2. Following important relevant citation based on feature extraction are missing, I strongly suggest authors to cite the following papers.

'Multi-pyramid image spatial structure based on coarse-to-fine pyramid and scale space'
"Image retrieval by addition of spatial information based on histograms of triangular regions"
'Fast feature matching based on r-nearest k-means searching'
“Intelligent Image Classification-Based on Spatial Weighted Histograms of
Concentric Circles.”
'Fast object detection based on binary deep convolution neural networks'
“A Novel Discriminating and Relative Global Spatial Image Representation with
Applications in CBIR”
Experimental design: The authors have significantly improved this section.
Validity of the findings: The authors have significantly improved this section.
Additional comments: The quality of figures may please be improved and it is suggested to cite and discuss the following relevant citations.

'Multi-pyramid image spatial structure based on coarse-to-fine pyramid and scale space'
'Fast feature matching based on r-nearest k-means searching'
'Fast object detection based on binary deep convolution neural networks'
“Intelligent Image Classification-Based on Spatial Weighted Histograms of
Concentric Circles.”
“A Novel Discriminating and Relative Global Spatial Image Representation with
Applications in CBIR”
"Image retrieval by addition of spatial information based on histograms of triangular regions"


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: VISUAL COMPLEXITY MODELLING BASED ON IMAGE FEATURES FUSION OF MULTIPLE KERNELS
Review round: 3
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: I am happy with the latest version of the manuscript.


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

Input:
Title: VISUAL COMPLEXITY MODELLING BASED ON IMAGE FEATURES FUSION OF MULTIPLE KERNELS
Review round: 3
Reviewer: 2

Output:
Basic reporting: The authors have addressed my all suggestions and quality of this manuscript now very good. Therefore, I suggest the respected Editor to please accept this manuscript for publication.
Experimental design: Research question well defined, relevant & meaningful.
Validity of the findings: Data is robust, statistically sound, & controlled. Conclusions are well stated, linked to original research question & limited to supporting results.
Additional comments: I observed a typo error in the references section that I copied here as

'Wang, K., Zhu, N., Cheng, Y., Li, R., Zhou, T., and Long, X. (2018). Fast feature matching based on759 ¡i¿r¡/i¿-nearest ¡i¿k¡/i¿-means searching. CAAI Transactions on Intelligence Technology, 3:198–207(9).'

This is in the line 759-760 from references section. Respected Authors are suggested to please correct the above mentioned type error before sending the camera ready version 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: CHARACTERIZATION OF CARBONATE FRACTION OF THE ATLANTIC BLUEFIN TUNA FIN SPINE BONE MATRIX FOR STABLE ISOTOPE ANALYSIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The work is about the characterization of bluefin tuna spines using innovative techniques in the field such as electronic X-ray spectroscopy (XPS) and Raman spectroscopy. It consists of simple experiments without sacrificing the life of the specimen. With these techniques, data are obtained to provide information in a way that can be used to a better understanding of the protected species.
Despite its simplicity and low number of samples, it can provide a new path for the analysis of the environment in which these species develop. The structure and the presentation of figures and tables seems appropriate. The English used in the paper should be subject to a slight revision.
Experimental design: No comments.
Validity of the findings: In my opinion, the article presents the application of three spectroscopic techniques for the characterization of biological samples that are not ordinary in this field of science, therefore can contribute with an important advance for the field. With simple experiments to carry out, very promising results are obtained.
From my point of view, the only aspect to improve is the number of samples to be analyze. On the other hand, the authors are aware of the fact and highlight this issue in the text.
Additional comments: I will raise some questions about the work:
On line 207, when speaking about XPS techniques for nitrogen contents detection, the origin of nitrogen should be mentioned.
A comment on why P does not appear in two of the samples is missing.
In Figure 4, the 2p transition of phosphorus should be deconvoluted with two contributions, that is, as 2p1 / 2 and 2p3 / 2.
In Table 1, for a better understanding of the table contents it should be included some lines for dividing contents of the different samples
In Table 2, in the first column (assignment) the line must say v (C-H).


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 CARBONATE FRACTION OF THE ATLANTIC BLUEFIN TUNA FIN SPINE BONE MATRIX FOR STABLE ISOTOPE ANALYSIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: One of the main problems of this paper is the language. A review by an English native speaker would really make a difference and would allow for the paper to become more accurate and comprehendible. I have made a few suggestions within the manuscript (given below), but I strongly believe that a detailed review should be considered.

Line 43: “for a correct” should be replaced by “for the correct”

The abstract should be written in the third person and not the first, for example in Line 44: “We collected the fin spines from freshly caught bluefin tuna (Thunnus thynnus) ” should be replaced by “The fin spines of freshly caught bluefin tuna (Thunnus thynnus) were collected..”
Line 46: “Raman Spectroscopy” instead of “Raman”,  and “spectroscopy” instead of “spectroscopies”.
Line 47: “positions” instead of “areas”?
Line 50: “the C(1s)” instead of “C(1s)”
Line 51: Please rephrase the whole sentence…for example: In Line 52: “allowed for the study of the carbonate species “ instead of “detect the presence of carbonate ions”, in Line 52: “with the IR spectra being the most” instead of “being IR spectra more ”, in  Line 53: “the HA lattice” instead of “HA lattice”, etc…
Line 54: “The results of this study” instead of “Findings of this study”
Line 55: the word “certainly” should be deleted and lines 56-57 should be rephrased in order to be more comprehendible.
Line 63: “otoliths, fin spines” instead of “otolith, fin spine”
Line 65: “McMahon” instead of “Mcmahon” and “migration” instead of “movements”?
Line 67-69: Rephrase, it is a very confusing sentence
Line 71: “parameters” instead of “conditions”
Line 75: “isotopes in” instead of “isotopes on”
Line 80: “its” instead of “their”
Line 81: rephrase “a minimally invasive hard structure”
Line 83: Zymonas and McMahon, 2011 does not appear in Reference list, while “Zymonas, ND,McMahon TE. 2006” from reference list does not appear in text.
Line 85: what does “most structural components” mean?
Line 91: I do not understand the sentence” The hydroxyapatite (HA) ((Ca10(PO4)6(OH)2), is commonly used as a model for describing the inorganic components of bones and teeth due to its close chemical resemblance to apatite (LeGeros, 1981)”. Hydroxylapatite IS a type of apatite. Please rephrase. Once again, this has to do with the use of the language.
Line 93: “In biological tissues” instead of “As they occur in biological tissues”
Line 98: “bone conatins” instead of “bone can contain”
Line 117: “comprised of” instead of “comprised”
Line 121: “procedures” instead of “procedure”
Line 122: perhaps “removed” instead of “extracted”
Line 124-127: rephrase please
Line 128: “by placing” instead of “placing”
Line 129: delete “cleaning”
Line 130: I do not understand the last phrase.
Line 132: “in a class” instead of “in class”
Line 153: “with the maximum power at the sample being 150 mW” instead of “being the maximum power at the sample 150 mW”
Line 154: “detected by a Peltier cooled charge coupled device (CCD) detector with 576 × 384 pixels” instead of “the detection is done by a Peltier cooled charge coupled device (CCD) detector with 576 × 384 pixels”
Line 162: “were measured in” instead of “collected in this work have been carried out in” and “mode on” instead of “mode in”
Line 164: “Spectra were acquired over the range 4000 to 400 cm-1 while each spectrum represents the average of 40 scans at a resolution of 4 cm−1, in order to provide a good signal to noise ratio” instead of “Spectra were acquired from 4000 to 400 cm-1 with a spectral resolution of 4 cm-1 and accumulating 40 scans in each spectrum to get a good signal to noise ratio”
Line 166:” allows for the processing of the results” instead of  “allows processing the results”
Line 168: “Analysis of spectral data from Raman and FTIR Spectroscopy” instead of “Analysis of spectral data in Raman and FTIR”
Line 169: “The positions” instead of “The areas” and “of the amide” instead of “of amide”
Line 171: “from the IR” instead of “from IR”
Table 2 (in line 171) is mentioned in the text before Table 1 (line 194), please correct according to the journals instructions.
Line 172: what does “was linearly corrected using the same wavenumbers” mean? Please clarify.
Line 174: “from the ratio of the integrated” instead of “from the integrated”
Line 173-177: More detail must be provided concerning the specific integrated areas used (exact areas used for every ratio in wavenumbers, perhaps also a graph indicating the exact areas used).
Line 179: References
Line 189: “…peaks. The last three comprise the compositional elements of” instead of “…peaks, comprising the three later compositional elements of”
Line 190: “sample BFT2”instead of “BFT2 sample”
Line 191: “, with its atomic concentration being around 1.4%” instead of “, being its atomic concentration around 1.4%.”
Line 191-192: “Phosphorous” instead of “Regarding to phosphorous, it” and “the” instead of “these”.
Line 194: “are given” instead of “resumed”
Line 195: The brackets should be deleted.
Line 201: “from sample” instead of “in sample”
Line 208: “overall” can be deleted, as it used again in the next sentence
Line 212: “probably due” instead of “due very likely”
Line 216: “low” instead of “small”
Line 217: “in Table” instead of “in the Table”
Line 218: “Based on the concentrations” instead of “Analyzing the concentrations”
Line 218-219: “the stoichiometric correspondence to synthetic HA Ca5(PO4)3(OH) can be concluded” instead of “it can be readily seen the stoichiometric correspondence to synthetic HA Ca5(PO4)3(OH).”
Line 219 onwards: usually the Ca/P ratio is calculated and not the opposite, as mentioned here. Even Lu et al, 2000 which is cited here, uses the Ca/P ratio and not the opposite. I suggest the correction of this ratio. It would be then comparable to relevant studies from the bibliography.
Line 220: “close” instead of “closed” and “ of 0.6 for hydroxyapatite“ instead of “0.6 in hydroxyapatite”
Line 222-223: “is a basic component of bone” instead of “is one such constituent regularly observed in bone”
Line 224-228: Please rephrase..
Line 230: “in order to eliminate” instead of “eliminating hence”
Line 236: “spectroscopy” instead of “spectroscopies”
Line 243: delete “bands”
From this point onwards I will not point out the language problems, as they are too many. Please ask an English native speaker to go through the manuscript with you.
Line 279-280: Which peak are the authors referring to? Which peak in IR spectra combines all types of carbonates?
Line 287: Could the authors explain this ? What do they mean by more mature mineral?
Line 288: Since crystallinity is measured by Raman, it could also be measured by FTIR according to Weiner & Yosef, 1990. This way it could comparable to other relevant studies.
Line 289: I don’t see how the crystallinity measurements help this study. Perhaps if they were compared to other similar measurements (other studies) or to fresh bone. Here they only prove the obvious, that the samples are all of similar maturity.
I am not sure if Figures 4-7 are necessary. Does fig.3 combined with Table 1 not provide the necessary data?
 Figures 6, 7, 8, 9 are not mentioned within the manuscript. On the contrary, Figurer 3a, 3b, 3c, 3d etc are mentioned within the manuscript but are not given as figures. They obviously correspond to figures 6-9.  Also, in Figure 8, a human bone sample is used for comparison. This is not mentioned/described within the material chapter. Please include the necessary information.
Table 3: “Parameters” instead of “paremeters”, “crystallinity” instead of “crystallinity”. Also, how are the mineral/Matrix (areas) and mineral/matrix (intensities) used in this paper? How do they help? I cannot find any discussion/conclusions relevant to these ratios.
The specific peaks used for the calculation of the Mineral/Matrix and Carbonate/Phosphate ratios should be given in detail (here and within the text)
References
1)	In text citations should be double checked, as they do not fully comply with the journals indications. For example, for three or fewer authors, all author names should be listed:
For example:
Line 66: “Acosta-Pachón, Ortega-García & Graham, 2015” instead of “Acosta-Pachón et al., 2015”
Line 97: “Figueiredo, Gamelas & Martins, 2012” instead of “ Figueiredo et al., 2012”
Please check the rest of the text.
Also “Walther and Limburg, 2012” should be written “Walther & Limburg, 2012”
2)	 377-378: “LeGeros” instead of “Legeros”.
3)	Line 388: insert space between references
4)	Line 447 onwards: change text size from 11 to 12, to comply with the rest of the manuscript.
5)	In the reference section, the format should be checked and corrected according to the journal’s instructions. For example:
Landi E, Celotti G, Logroscino G, Tampieri A. 2003. Carbonated hydroxyapatite as bone substitute. Journal of the European Ceramic Society 23(15): 2931–2937 doi.org/10.1016/S0955-2219(03)00304-2.
Instead of:
Landi, E., Celotti, G., Logroscino, G., and Tampieri, A. (2003). Carbonated hydroxyapatite as bone substitute. J. Eur. Ceram. Soc. 23(15), 2931–2937. https://doi.org/10.1016/S0955-2219(03)00304-2.
Please check the rest of the text.
6)	The following references are listed in the References chapter but cannot be found within the text:
 Farlay, D., Panczer, G., Rey, C., Delmas, P. D., & Boivin, G. (2010). Mineral maturity and crystallinity index are distinct characteristics of bone mineral. Journal of Bone and Mineral Metabolism. 28(4), 433–445. https://doi.org/10.1007/s00774-009-0146-7.

Gamsjaeger, S., Masic, A., Roschger, P., Kazanci, M., Dunlop, J. W. C., Klaushofer, K., et al. (2010). Cortical bone composition and orientation as a function of animal and tissue age in mice by Raman spectroscopy. Bone. 47(2), 392-399.
Experimental design: This paper describes the study of fin spine bones of fresh blue tuna, by Raman and IR Spectroscopy combined with XPS. The objective of this work is to prove that fin spine bones can be used for isotopic analyses of tissues of such fish, especially when they include endangered species, instead of other parts of the fish (otoliths etc) that can only be taken from killed individuals.
In the abstract but also throughout the manuscript, it appears that the main result of this study is the verification of the fin spine bones composition. I am afraid that this is not something new, as the composition of the bio-mineral of bone (fish bone as well) is well known. I believe it should be rewritten in order to better indicate the scope of this study, perhaps as it is analyzed in the last paragraph of the introduction.
In general, I do not see how this paper contributes to our general knowledge of bone bio-apatite, further than simply verifying what we already know in general, for the specific kind of bone (fin spine bone). Obviously, the authors want to suggest the use of this kind of bone instead of other parts of the fish usually used for isotopic analysis. I am afraid that this is not stressed enough in the text. I believe that the objectives of this paper should be explained in a different manner, so that its importance for fisheries studies is stressed.
Validity of the findings: The presentation of the results could be improved, in order to better serve the original scope of this paper, as suggested in the first part of this 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: AN AMBIGUOUS N-TERMINUS DRIVES THE DUAL TARGETING OF AN ANTIOXIDANT PROTEIN THIOREDOXIN PEROXIDASE (TGTPX1/2) TO ENDOSYMBIOTIC ORGANELLES IN TOXOPLASMA GONDII
Review round: 1
Reviewer: 1

Output:
Basic reporting: This manuscript by Mastud and Patankar is written in good English and generally makes use of the appropriate litterature references. That being said, I think the manuscript could be reorganised slightly and benefit from presenting the results in a more logical way (see my detailed comments to the authors).
Experimental design: The research question is well defined and most of the experimental approaches used seem valid and well controlled. However, a couple of strategies seemed a bit odd to me and would need to be clarified further (see my comments below).
Validity of the findings: Conclusions of the paper are generally well supported by the findings.
Additional comments: Main targeting signals for mitochondria, plastids and the ER share in their N-terminal position an α-helical structural element and subsequent removal from the core protein by intra-organellar cleavage. Dual targeting to endosymbiotic organelles through ambiguous targeting signals has been observed in plants previously (proteins localised to both mitochondria and chloroplasts) and is an interesting feature, especially from an evolutionary perspective. In Toxoplasma, there are some rare examples of dual protein localisations, like with antioxidant enzymes TgSOD2 and thioredoxin-dependent peroxidase TgTPX1/2 (Pino et al 2007). However, while the determinants of TgSOD2 targeting have been investigated by Pino et al, no precise determination of the sequence elements regulating organelle targeting has been performed for TgTPX1/2. The present manuscript aims at providing a detailed investigation of these sequence elements and is thus potentially interesting.

Main comments.

As a general comment on the organization of the manuscript, I feel that some the results are not presented in a logical way. Bioinformatic prediction of the signals should be used more clearly as a base for justifying subsequent experimental validation. For instance, for the part starting at l.234, it should be mentioned beforehand that a SignalIP analysis indicates a potential signal peptide with a putative cleavage site at AA 30. Mitoprot analysis should be also detailed, and not only the score but also the putative position of the potential cleavage site should be mentioned. Whether or not this fits with the observed molecular mass of the mature TgTPx1/2 as observed by western blot should be also discussed. Because as the manuscript currently stands, the rationale for choosing to express the first 50AA of the protein is unclear. If the Mitoprot analysis suggests this fragment could be encompassing the mitochondrial targeting signal, this should be more clearly mentioned.

I was very surprised when running the latest version of Mitoprot (Mitoprot II- v1.101) with TgTPx1/2, to see a much higher score (0.9762) and a putative cleavage site at AA100. Afterwards, I realized it is briefly mentioned in the ‘Materials & Methods’ section (l. 184-l.187). Any idea as to why there is such a discrepancy when using the latest algorithm of Mitoprot II? In any case, I thus do not think the authors can discuss extensively the relatively ‘low’ mitochondrial probability score by comparison with other known mitochondrial proteins (l. 519-521).

It is somewhat confusing that a dual Mitoprot/SignalIP analysis is conducted in parallel on the first 30AA (Fig. 4 and corresponding results). Although, this region can be both important for targeting to the apicoplast and to the mitochondrion, the sequence determinants for targeting to the mitochondrion likely involve a larger region, which is supported by experiments described in the current manuscript.

Related to this, the l. 67-76 part in the introduction should be written more clearly to distinguish between the typical apicoplast bipartite signal with ER type N-terminal secretory signal peptide followed by a plant-like transit peptide, and the mitochondrial targeting signal involving a single targeting signal, and which is generally independent from trafficking through the ER. In fact, many mitochondrial proteins even lack N-terminal pre-sequences and instead harbor internal signals to direct them to the mitochondrion. For the same reason, the authors should rephrase l. 300-301, ‘As the first steps of targeting for the apicoplast and mitochondrion are recognition by the ER and the mitochondrial translocons’, which is confusing.

A transit peptide has been potentially identified (l. 475), but it hasn’t been formerly experimentally verified to act as such. Thus, the authors shouldn’t write ‘This is the first report that shows a clear delineation of amino acids residues in the transit peptide…’ l. 477-478. Besides it seems to me some transit peptides have been identified previously, at least in Plasmodium. Again, the position of the transit peptide and the cleavage of the protein should be discussed further as the mature apicoplast and mitochondrial forms seem to have the same apparent molecular mass.

L. 398. I do not understand why the authors generated a double mutant for both E15 and R24. According to the results of the bioinformatics screen shown on Fig. 4E, a single E15A mutation is enough to increase both Mitoprot and SignalIP scores, while a single mutation R24A raises the SignalIP but decreases the Mitoprot score (and clearly affects mitochondrial targeting as seen on Fig. 5C). Although the authors claim prediction for the double mutant also show an increase in the Mitoscore score, I do not see the advantage of using this mutant over the simple E15A mutant. Given the apparent importance of the R24 residue for mitochondrial targeting, it might be wiser not to alter it in the context of this experiment. Instead, the authors should evaluate the localization of the single E15A mutation and see if it supports their hypothesis that increasing the two prediction scores favours apicoplast targeting over mitochondrial targeting.

Fig. 4 A-D could easily be merged to Fig. 7 and discussed altogether for the sake of simplification. Moreover, there is no proof the region containing the ambiguous signal is restricted to AA15-30. Fig. 4E identifies potential AA mutations affecting the Mitoprot score in region AA1-14.

Minor comments.

L. 206-207: ‘for tagging proteins in T. gondii, GFP fusions show altered organellar localization while small epitope tags do not’. This seems like a general statement, but it obviously depends on the protein. If the authors want to refer to TgTPx1/2 in particular, this should be mentioned precisely.

There is no denying the in silico alanine scanning strategy yielded interesting results and led to the identification and experimental validation of key residues. However, alanine has a short hydrophobic side chain (some signal peptides are actually quite alanine-rich), although it is clearly not the most hydrophobic AA. Thus, the rationale for using an in silico alanine scanning strategy to identify potential important residues for targeting in the first 30AA should thus be explained more carefully in the manuscript. As charge is also likely to be affected and the authors suspect part of the signal peptide is also involved in an aliphatic chain important for mitochondrial targeting, this should be mentioned.

Fig. 4B. Residues 19-20 do not seem to fit in the representation of the helix model.

The authors have to describe precisely in the ‘Materials & methods’ section how the Pearson’s correlation coefficient has been calculated. Some of the currently values are awkward. For example, for the signals displayed on Fig. 1B and Fig 2C, the PCC is unusually high (given that although 100% of the pixels of the apicoplast signal may be co-localising with the TgTPx1/2 signal, but the latter contains many additional pixels not colocalised with the apicoplast signal). In addition, when considering large field images with Mitotracker labelling (Fig. 3D,F, Fig. 5C,D, Fig. 6C) the very low values  sometimes displayed suggest there could have processing of the whole field (including signal from the Mitotracker-labelled host mitochondria). A region of interest has to be defined for this type of analysis and background subtraction by thresholding may also be used.

An accession number for TgTPX1/2 should be mentioned in the “Materials & Methods” 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: AN AMBIGUOUS N-TERMINUS DRIVES THE DUAL TARGETING OF AN ANTIOXIDANT PROTEIN THIOREDOXIN PEROXIDASE (TGTPX1/2) TO ENDOSYMBIOTIC ORGANELLES IN TOXOPLASMA GONDII
Review round: 1
Reviewer: 2

Output:
Basic reporting: Use of references should be extensively revised. Some specific examples for places where the used references are not suitable: the red algal origin of the apicoplast (line 63) and the references for SP/TP/MTS cleavage (line 68/69, 219). Likewise, missing key references e.g. Sheiner et al; Traffic 2015, lists most of the known targeting signals of apicoplast proteins in Toxoplasma (relevant for line 73/74), likewise, several reviews describe the metabolic roles of the two organelles (line 64).
Experimental design: Research question (basis of Tpx1/2 bimodal targeting) is well defined and experiments well designed and relevant to explore the features of the trafficking signal.

This is with one exception: I don’t see the value in the in silico result paragraph (296-333), I would suggest to remove it.
Validity of the findings: The findings are valid, however, conclusions should be drawn more carefully – examples: the mentioning of receptor competition (line 98-100) is an extrapolation; likewise claiming that 1-40-GFP fusion “escaped the ER” (line 258); stating 17% without providing statistically relevant approach and while not showing the data (line 283). There are more examples, proposed to revise conclusions throughout.
Additional comments: This is an interesting topic and one that is understudied and deserve more attention. In addition to the above comments, which are important to improve the clarity, depth and accuracy of the work, a more in-depth discussion in the intro about what we do and do not know about signals for dual targeted protein is needed (currently in lines 77-86).
For example, there are more described cases of dual targeting in Plasmodium (e.g. https://www.ncbi.nlm.nih.gov/pubmed/22889167 ) and in Toxoplasma (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3767399/). Likewise, this intro should discuss the different proposed mechanisms (alternative start sites, alternative splicing, overlapping amino-acid signals etc) and highlight work done to address those options (e.g. the Pino paper that is mentioned in other context and other work in Plasmodium). Finally, it is important to discuss criticism for previous work – e.g. SOD2 dual targeting was only seen under non-native promoter (and in fact the current study also expresses TPx1/2 under Tub promoter) – this should be highlighted.


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: DEPRESSION AND SEXUAL RISK BEHAVIOR AMONG LONG-DISTANCE TRUCK DRIVERS AT ROADSIDE WELLNESS CLINICS IN KENYA
Review round: 1
Reviewer: 1

Output:
Basic reporting: Romo et al estimated prevalence of depression among the among a specific group in Kenya, long-distance truck drivers at roadside wellness clinics, and evaluated the association of depression with sexual risk behavior. The topic is very interesting and the data are valuable. The authors talked the story with a clear 'storyline’, the language is clear and unambiguous.

The format of the manuscript, including tables, needs improving greatly. A well formatted manuscript makes reviewers more enjoyable.
Experimental design: The study are well designed and the research question are well defined.
Validity of the findings: Data is robust, statistically sound, & controlled.
Additional comments: Romo et al estimated prevalence of depression among the among a specific group in Kenya, long-distance truck drivers at roadside wellness clinics, and evaluated the association of depression with sexual risk behavior. The topic is very interesting and the data are valuable. The authors talked the story with a clear 'storyline’, the language is clear and unambiguous.

However, the format of the manuscript, including tables, need improving greatly. A well formatted manuscript will make reviewers more enjoyable while reading.

Major REVISIONS:
The main concern is that the dual direction of the causality between depression and sexual risk behavior. Furthermore, both depression and sexual risk behavior might be influenced by same factors, ie occupational stressor. If depression and sexual risk behavior share one ancestor, they are also associated statistically. The authors may explain the association from an etiological perspective.

The second issue is that the authors might focus more on the association between depression and health risk behavior (with multiple condomless partners). The results and the discussion of the confounding factors distract the core subjects .

The third issue is that the authors discussed the association superficially, did not explain the potential reasons behind the association, using existing theories or hypotheses, if any.

The fourth issue is that is the authors used two much space to discuss the implication part. Of course, the implication is important, especially for policy makers. As mentioned before, they should discuss more on the potential mechanisms that can explain the associations. If there are not reasonable mechanisms to support the association, then we can infer the reverse conclusion, that depress is caused by sexual risk behavior.

Minor REVISIONS:

Line 72  it’s associated with both… does not necessarily mean that it mediate the relationship, because if it is a confounding factor or ancestor(cause) of the exposure and outcome, it’s also associated with both.
Line 114 The authors may use a table or figure to show that what measured in baseline interview and what measured in follow-up interview.
Line 130 Two periods after the word “man”.
Line 131 …those with more than multiple condomless partners. In this sentence, “more than” should be removed??
Line 204  What’s the difference between crude model and multivariable model? According to the note of Table 2, multivariable model adjusted clinic location additionally. The crude model adjusted other covariables, why still be call “Crude” model?
Line 214 Mediation analysis could be inspired by the paper ”Mediation analysis in epidemiology: methods, interpretation and bias” https://doi.org/10.1093/ije/dyt127
Line 291 Logistic regression? The authors should describe clearly where they used poisson regression and where where they used Logistic regression. In Table 2, all the indicators are aPR, so I guess they were from poisson models.

If the authors used logistic regression here, ordinal logistic regression should be used as the outcome was recoded as none, one, two or more.

Maybe I am wrong, the authors should state these in methods part more clearly.


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: DEPRESSION AND SEXUAL RISK BEHAVIOR AMONG LONG-DISTANCE TRUCK DRIVERS AT ROADSIDE WELLNESS CLINICS IN KENYA
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1. The English language of this manuscript need to be revised and polished due to many errors that make the readers confused.
2. The references included in the paper and the raw data are sufficient.
3. Multivariable logistic regression model was used. For table 2, I'm not sure which model was used, logistic or Posson regression model?
Experimental design: 1. The study was derived from a randomized controlled trial (about HIV), but this study aims to conduct a cross-sectional study, and estiamte the prevalence of depression in the special population. The rigorous inclusion criteria could introduce large selection bias. So the estimates of prevalence may have a weak comparability.

2. Only two clinics were enrolled. It is hard to say the representativeness of the aim population.

3. The measure of sexual risk behavior lack reference.

4. Measures were actually taken from two interviews that were six
months apart, which may introduce large information or recall bias.
Validity of the findings: Due to the limitation of the study design, I am afriad the data could not state the conclusions, such as the high prevalence of depression.
Additional comments: I do not think the initial study design of the RCT could derive the cross-sectional study. A scientific epidemioloical survey is needed for this topic.


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: DEPRESSION AND SEXUAL RISK BEHAVIOR AMONG LONG-DISTANCE TRUCK DRIVERS AT ROADSIDE WELLNESS CLINICS IN KENYA
Review round: 1
Reviewer: 3

Output:
Basic reporting: .
Experimental design: .
Validity of the findings: .
Additional comments: This is an interesting article on an important subject but there are methodological issues..the study was  designed for a different research question.  If it had been designed for this question, then the PHQ would have been administered at Time 1 instead of or as well as at Time 2.

The sample frame appears to be all truck drivers who visited the two clinics between October and Dec 2015 for services other than HIV treatment, The results should report the size of the sample frame, how many were eligible, and how many agred to take part, thus giving a response rate. The external validity of the study is influenced by this response rate.

The authors argue in the discussion that depression status would be unlikely to change over 6 months if untreated, but in fact this is not the case. Follow up studies show that around 50% of cases improve over 6 months.

The sample was drawn from truck drivers seeking services at roadside wellness clinics in Kenya...thus the sample is a primary care clinic based sample rather than a community based sample. Such PHC samples generally have higher rates of depression than community samples. Furthermore, truck drivers have a younger age distribution than the general adult population.

Thus line 341 in the discussion needs changing -this study suggests that truck drivers attending wellness clinics have a higher rate of depression than the general population, but this is not surprising as such studies of
clinic attenders always find a higher prevalence than in community populations. It would have been good to compare truck driver rates with a control group of non truck drivers also attending the wellness clinics., as this would have given
more of an indication of whether being a truck driver was the operative variable .This issue is subsequently raised at lines 374-376, but it is fundamental to the earlier sections of the discussion and should be made explicit there.

It's not clear why the PHQ was not conducted at the first interview.

line 388-there has also been an RCT of primary care training about mental health in Kenya, as well as roll out to around half of primary health  care workers.
(Jenkins et al. International Journal of Mental Health Systems December 2013, 7:25)

In general, I suggest a revision to clarify these methodological issues of sample frame, response rate,  the fact that appropriate comparisons of prevalence rates are with clinic based samples, not general population samples, and the fact that depression rates would change over a  six months period.


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: DEPRESSION AND SEXUAL RISK BEHAVIOR AMONG LONG-DISTANCE TRUCK DRIVERS AT ROADSIDE WELLNESS CLINICS IN KENYA
Review round: 2
Reviewer: 1

Output:
Basic reporting: Romo et al have improved the revised manuscript greatly in many ways according to the comments raised by three reviewers.
Experimental design: The study is well designed and the research question are well defined, it should be noted that the exposure was measured after the exposure.
Validity of the findings: Data is robust, statistically sound, & controlled.
Additional comments: I reviewed the revised manuscript, and there are my comments as listed below. I did not recognize these in the first round because there were more important issues to be mentioned at that time.

Line 97: “which is likely due to (no reference)” sounds hypothetical, should more likely to appear in discussion, not introduction.

Line 156: “and asked about HIV testing in the past six months” means that HIV status in the 2nd survey might affect the association the authors want to evaluate (HIV causes depression measured in the 2nd survey), which should be acknowledged in discussion part.

Line 241-246: I knew that the authors used a fully conditional method for imputation, however the authors should explain to the readers that why they used FCS, not MVN.

This specification may be necessary if you are imputing a variable that must only take on specific values such as a binary outcome for a logistic model or count variable for a Poisson model.
https://stats.idre.ucla.edu/sas/seminars/multiple-imputation-in-sas/mi_new_1/

Besides, is there any evidence to support your statement “The multivariable model had about 25% missing data, so we ran 25 imputations”? I am not good at Mi, just a little curious(25% missing data, so  25 imputations?).

Line 344: The nature direct effect was statistically nonsignificant, which means self-efficacy is an intermediate variable between exposure and outcome, then what? The authors stopped here abruptly and did not finished the story. Are there any evidence to support the finding? What does the finding mean, what’s the possible reasons underlying, and what’s the potential implication for intervention? Without addressing these topics, there seems no need to do the mediation analysis.

Line 382-383: “…our exposure and outcome was not undertaken simultaneously.” should be rephased. No, they were not undertaken simultaneously, but the statement is less accurate because the exposure was measured later than outcomes.


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: DEPRESSION AND SEXUAL RISK BEHAVIOR AMONG LONG-DISTANCE TRUCK DRIVERS AT ROADSIDE WELLNESS CLINICS IN KENYA
Review round: 2
Reviewer: 2

Output:
Basic reporting: In the limitation part, the first limitation, the authors said "the prevalence of depression among truck drivers in general may in fact be lower, as clinic-based sampling can result in selection bias for mental health disorders ". I did not find any evidence showing that depression among truck drivers in general may in fact be lower. Please clarify this.
Experimental design: Thank the authors for considering my concerns and revised the manuscript accordingly.

The title is for the "long-distance" truck drivers at roadside. So there are also short-distance truck drivers. But in the Eligibility criteria, the authors stated that persons "employed as a truck driver" were employed. Details on the "long-distance" may be necessary.
Validity of the findings: no comment
Additional comments: Thank the authors for considering my concerns and revised the manuscript accordingly. Additional concerns as follows:

1. In the limitation part, the first limitation, the authors said "the prevalence of depression among truck drivers in general may in fact be lower, as clinic-based sampling can result in selection bias for mental health disorders ". I did not find any evidence showing that depression among truck drivers in general may in fact be lower. Please clarify this.

2. The title is for the "long-distance" truck drivers at roadside. So there are also short-distance truck drivers. But in the Eligibility criteria, the authors stated that persons "employed as a truck driver" were employed. Details on the "long-distance" may be necessary.


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: DEPRESSION AND SEXUAL RISK BEHAVIOR AMONG LONG-DISTANCE TRUCK DRIVERS AT ROADSIDE WELLNESS CLINICS IN KENYA
Review round: 3
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Romo et al have improved the revised manuscript according to the comments raised by two reviewers in the second round. The manuscript, in my opinion, meets the PeerJ criteria and should be accepted as is.


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: DEPRESSION AND SEXUAL RISK BEHAVIOR AMONG LONG-DISTANCE TRUCK DRIVERS AT ROADSIDE WELLNESS CLINICS IN KENYA
Review round: 3
Reviewer: 2

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: Thank the authors for considering all my concerns and revised the manuscript accordingly.


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

Input:
Title: ASSESSING SOIL COMPACTION AND MICRO-TOPOGRAPHY IMPACTS OF ALTERNATIVE HEATHER CUTTING AS COMPARED TO BURNING AS PART OF GROUSE MOOR MANAGEMENT ON BLANKET BOG
Review round: 1
Reviewer: 1

Output:
Basic reporting: Comments to the author Manuscript Number: 29086
Comments to: “Assessing soil compaction and micro-topography impacts of alternative heather cutting as compared to burning as part of grouse moor management on blanket bog”
The study addresses an interesting issue related to how avoid compaction in organic soils. The theoretical basis of this paper looks solid. However, I recommend a major revision of the manuscript.
Experimental design: The experimental design is so complex! Your data could be represented by four factors:
-	5 Treatments (FI, DN, LB, BR, LB+Sp, BR+Sp)
-	3 Sites (N, M, W)
-	4 Sampling Depths
-	2 Before-After control impact
It is very difficult to understand the results. I suggest you:
1.	Add a picture of your experimental design  for each of your figures (Probably, it will be supplementary files)
2.	Determine the statistical analysis for each experimental design, e.g. three-way ANOVA, two-way ANOVA, one-way ANOVA, Kruskal-Wallis, etc, indicating how did you validated your analysis (data normality or the equality of error variances (Levene’s test))
3.	Indicate how did you validated your regression analysis (outlier presence, normality of unstandarised residuals, etc)
4.	Please explain what kind of software was used.
Validity of the findings: Other comments are:
Introduction
Lines 118-120: Please add a reference to support  this sentence.
Lines 121-123: Please add a reference to support  this sentence.
Lines 136-139: Please define clearly the main objectives of your study. This phrase is too ambiguous.
Lines 159-183: I suggest you to do summary this information.
Line 193: What are the treatments?
Lines 216-219: Please explain the meaning of DN, LB, BR, etc, in the main text, not just in the legend of figures.
Line 288: I suggest you to add a Statistical analysis section, explaining it.

Results and discussion section need to be modified, in agree with the changes suggested.
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: ASSESSING SOIL COMPACTION AND MICRO-TOPOGRAPHY IMPACTS OF ALTERNATIVE HEATHER CUTTING AS COMPARED TO BURNING AS PART OF GROUSE MOOR MANAGEMENT ON BLANKET BOG
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript presented is generally well written and the language is professional, but currently the introduction promotes cutting as a means of reducing the dominance of heather where there is no evidence. The text does mention that there is a lack of evidence on the implications of cutting but the current benefit of cutting promoted here is speculative.
Heather dominated blanket bog is degraded and rather than continuing to promote dominance of Calluna these ecosystems need rewetting to promote diversity of floral/bryophyte assemblages and reduce the dominance of Calluna. In lines 97-100 the authors state:
“Moreover, alternative heather management methods could also be used to reduce ‘over’-domination of heather in favour of more diverse and ‘active’ peatland forming vegetation, especially with regard to the restoration of peat hydrological function and Sphagnum growth.”
This statement needs supporting with evidence or must be recognised as speculation. I am not aware of any published evidence that demonstrates that cutting vegetation reduces heather dominance. This is important context here because grouse moor management aims to retain a dominance of heather, so it is unclear why a management technique that might reduce heather would be supported by land managers. It is key that the authors highlight to the reader that heather cutting might still promote heather dominance as publications such as this could be used by a range of stakeholders and feed into future management strategies. In lines 128-129 the authors refer to a report by DARDNI (2011) that states cutting should not be used on blanket bog as the machinery could damage the vegetation. It is surprising therefore that no assessment of vegetation was made in this study. This is crucial and indeed the author submission states:
“Notably, peat could compact under the weight, thus affecting eco-hydrological processes and ultimately carbon cycling and storage, but could also affect water cycling by altering micro-topography, vegetation composition and thus runoff rates.”
The wider impact of cutting machinery on blanket bog must also be highlighted in a publication such as this. Adoption of cutting will require repeatedly getting heavy machinery to the area marked for cutting but in so doing will undoubtedly affect surrounding areas. This will likely involve the use of tracks or routes which damage blanket bog (see e.g. Lindsay et al., 2016; IUCN briefing note 12) and continued use of the same route may alter the hydrology, perhaps even create new surface flow and promote drainage. If managing a central area of blanket bog by cutting results in drainage of the edges of the bog then cutting may not be an appropriate management technique. This must be highlighted in the introduction and discussion.
In addition, the authors note that ‘rebound’ occurs (line 240) when a machine moved over the peat, but the damage is far more significant where the vehicle stops and/or turns. It is of concern therefore that the authors did not assess this (line 387) although they do point out that it needs to be assessed in future (line 389). The manuscript must address the wider impact of cutting machinery.
Figures
It is very difficult to understand the study sites and experimental design from Figures 2-3. The experimental design text from line 185 onwards could neatly be supported with a schematic diagram and additionally an overview figure that presents the sites in aerial photography and the location of the plots with contour lines (or some presentation of elevation to show any variation in topography) would be extremely informative. Grips and gullies are mentioned and the locations of all these features need to be presented – these features will affect the data and need to be included in analysis and interpretation.
Figure 3 shows some heather being burned but with no context. The burning on Mossdale appears to be on an area of relatively high slope, something that is uncharacteristic of blanket bog. Is this area one of the experimental plots? Line 243-244 states “slopes at plot locations were <5 degrees”. Also line 251 states that there was a “step in topography”. These observations raise questions as to whether all plots are comparable topographically.
For Figure 3 it might be more informative to show the same plot before and after burning or mowing.
Raw data
The data are presented clearly and well annotated though there is a minor inconsistent labelling (the microtopography spreadsheet uses ‘M’ where graphs, text and other data spreadsheets report ‘LB’)
Experimental design: The design of the experiment provides a good number of treatments and replications with which statistically robust analysis could be undertaken. However, there are a number of serious concerns about the data and analyses presented.
Different catchments
At all sites the burn treatment is in a different catchment to all the mow treatments and it must be demonstrated that peat depth, topography (both micro- and meso-) and vegetation is comparable between catchments prior to the experimental manipulation. These data may exist and ought to be presented.
Microtopography
The approach of measuring surface variation at 45 points in each plot appears to provide a good number of data for analysis. However, the approach does not provide a before and after data for comparison – the authors note on line 351 that “The micro-topography assessment did not include a pre-management change measurement”. This would be the most robust measurement for comparison of change.
The use of data from uncut plots may provide some insight, but as noted above with the catchment descriptions, a full description of all plots needs to be provided to demonstrate that the uncut plots are comparable to the manipulated plots prior to the experiment. It is crucial that the microtopography is described as erosion features, ditches, and gullies are mentioned in the site descriptions but not accounted for in analysis. A full vegetation survey including indicators such as the particular species of Sphagnum present is also needed as this provides insight to the microtopography. Figure 5 shows significant variation in topography between the mown sites with brash where Sphagnum was and was not applied. It is unlikely that the addition of Sphagnum propagules would have any affect and suggests that the plots were not comparable prior to the experiment. This indicates that the variation recorded between replications cannot be interpreted without actual pre-treatment measurements.
Can you also explain how the microtopography of the mown plots with brash left was measured? Was the brash removed prior to measurement? Or is the brash included? What impact does this have on the data?
Peat depth and bulk density
There is no description in the method of precisely where samples were taken/measured. For both these measurements it needs to be demonstrated that the ground where samples were measured/collected were in fact impacted by the machine(s). There is a gap between the wheels (under the tractor). Does the mower have a roller that runs along the surface? Some areas will be impacted by both front and rear tyres, while some will be impacted by just rear tyres. There needs to be a lot more detail in the description of the footprint of the mower and tractor and a clear description of where samples were taken and why.
Ideally the experiment needs to collect measurements from all combinations of machine impact as this will vary and might provide insight as to the optimal vehicle setup to cause the least damage. See also note above about not assessing where vehicles stop/turn.
Peat depth
Unless I have misunderstood, one peat depth measurement was taken in each plot. Peat depth varies spatially over very short distances, often following changes in the underlying substrate, and therefore significantly more than one measurement of depth is needed to provide a representative record for an area of 25 m2. The mean peat depth data do not provide a comparison of before and after, they simply provide two sets of randomly located peat measurements. No interpretation or conclusions can be drawn from these data. If RTK DGNSS was used to take measurements at exactly (+/- 2 cm) the same location this needs to be stated as it may increase the validity of the observations.
Bulk density
In Figure 8, the data for Mossdale show that for 11 of the 12 samples BD increased after management intervention including in uncut areas. The authors note on lines 371-373 “Therefore, the observed change in BD was evidently unrelated to management and reflected a natural process of BD change.”
Lines 374-385 indicate that no interpretation can be drawn from these data: “That this underlying natural cause explained the observed change in BD was supported by a larger apparent increase in BD at the surface for both Nidderdale and Mossdale (Figure 8), which could be related to rainfall and thus peat moisture. Whereas pre-management samples were taken after a particularly wet summer period in 2012, leading to expansion and thus lower peat bulk density, post-management samples were taken after a particularly dry spring in 2014 (Heinemeyer et al., unpublished climate data), leading to peat shrinkage and thus higher BD. Moreover, the remarkably similar BD at Whitendale (between pre- and post-management assessments) could be linked to similar moisture conditions in relation to high rainfall events (~60 mm) in mid-March 2014 (two weeks before post-management sampling), and a further ~30 mm of rain during the week of sampling (all Heinemeyer et al., unpublished climate data), causing peat expansion and thus a decrease in BD.”
In order to interpret these data there must be some inclusion and analysis of moisture content. If soil moisture was not recorded in the field, it might be possible to estimate PET and net moisture from temperature and rainfall data. This could potentially be used to quantify/model the change in moisture to enable comparison of the data.
Validity of the findings: The conclusions drawn are not supported by the data or analyses presented.
For peat depth there are insufficient data and for BD, in lines 371-373 the authors state;
“Therefore, the observed change in BD was evidently unrelated to management and reflected a natural process of BD change.”
It is therefore not possible for the authors to conclude that:
“…there was no lasting effect of management treatment on either peat depth or bulk density” (lines 394-395). Natural variation as a result of changing moisture needs quantifying.
There may be potential in the microtopography data – see comments in previous section.
It may be that more data exist which can be used to undertake major revisions of the analyses presented here.
Additional comments: My comments may overlap slightly across the review sections but I hope that the comments are clear and constructive. The study is timely as burning of vegetation on blanket peat/bog is being phased out and cutting does appear to be being adopted as an alternative management strategy. This study therefore has the potential to provide some crucial insight into the impacts of cutting on peat compaction and microtopography. The overall design of the experimental plots appears to provide a good number of treatments and replicates, however the conclusions regarding the impact on peat depth and bulk density are not supported by the data or analysis presented. It may be that more data exist with which to add confidence to the analysis and therefore need including to allow conclusions to be drawn.
There is potential in the microtopography data but far greater detail is needed. There are no data presenting the vegetation or microtopography in the plots. These data are needed to demonstrate that the plots were comparable prior to experimental manipulation. The site description simply mentions Sphagnum species – it is important here to identify the species as they occupy different microtopographic zones. Microtopography is more than hummocks and hollows. In line 133 the authors state “hummocks (e.g. protruding clumps of sedges)…” – this sounds like a description of a ‘tussock’ not a hummock. Revegetating erosion features may appear to be ‘hollows’ but the vegetation is required to classify the microtopography and identify whether it is a hollow or an erosion feature. Erosion features may revegetate with Sphagnum spp. but the presence of species such as deer grass (Trichophorum cespitosum) are clear indicators that this would not be a hollow. Without this information it is not possible to understand if the sites or plots are comparable.
The wider impact of cutting machinery on blanket bog must also be highlighted and the manuscript requires major revisions prior to consideration for acceptance. As these revisions relate primarily to the data and analyses I have not commented as much on the written component.


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: ASSESSING SOIL COMPACTION AND MICRO-TOPOGRAPHY IMPACTS OF ALTERNATIVE HEATHER CUTTING AS COMPARED TO BURNING AS PART OF GROUSE MOOR MANAGEMENT ON BLANKET BOG
Review round: 2
Reviewer: 1

Output:
Basic reporting: As this is a review of a revised manuscript I have submitted a separate document containing my comments
Experimental design: As this is a review of a revised manuscript I have submitted a separate document containing my comments
Validity of the findings: As this is a review of a revised manuscript I have submitted a separate document containing my comments
Additional comments: As this is a review of a revised manuscript I have submitted a separate document containing my 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: KININOGEN-1 AS A PROTEIN BIOMARKER FOR SCHIZOPHRENIA THROUGH MASS SPECTROMETRY AND GENETIC ASSOCIATION ANALYSES
Review round: 1
Reviewer: 1

Output:
Basic reporting: The paper needs reorganization in some sections to make it clearer and easy to understand.
Experimental design: There are some additional experiments that need to be added in this manuscript.
Validity of the findings: The finding is meaning but needs to be further confirmed.
Additional comments: General Comments: Schizophrenia is a mental illness, which serious influence the life of human beings. However, there is still no effective testing methods for its clinical diagnosis. In this manuscript, the authors have explored the application of using Kininogen-1 as a protein biomarker for schizophrenia diagnosis, which is meaningful for the early diagnosis of schizophrenia and its prediction. However, there are some big problems in this manuscript, and it can be acceptable after major revision. The details have been listed below:

1, The authors claimed that it was the first study that explored if Kininogen-1 could be a protein biomarker for the diagnosis of schizophrenia. However, there are some studies have already reported that Kininogen-1 was significantly reduced in SCZ patients. The novelty of this study should be further described.

2, As a new testing method for the clinical diagnosis of schizophrenia, it should be compared with the traditional method to investigate its reliability. I understand that the receiver operating characteristic has been used to confirm this. But more experiments need to be carried out including the double-blind experiments.

3, Figure 1 showed that the expression level of Kininogen-1 was obvious decreased in schizophrenia patients compared with healthy controls. Would the expression level of Kininogen-1 different from each other for the schizophrenia patients under severe, moderate and mild level? These data should be provided in this manuscript.

4, In this study, the authors explored the association between three tagSNPs in CST9 and SCZ. The results should be further confirmed by real-time PCR method.

5, The resolution of Figure 1 is too low. Both these figures should be made by Excel, origin or other software but not directly take a photo and put in the manuscript.

6, There are some language errors in this manuscript. The whole manuscript needs to be edited by an English-speaking author. The paper needs reorganization in some sections.


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: KININOGEN-1 AS A PROTEIN BIOMARKER FOR SCHIZOPHRENIA THROUGH MASS SPECTROMETRY AND GENETIC ASSOCIATION ANALYSES
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: This is a pretty interesting and meaningful study exploring whether Kininogen-1 protein is a biomarker for schizophrenia diagnosis and the relationship between protein-coding gene (CST9) and schizophrenia. However, some issues should be solved. My detailed comments are as follows:

Title: - The title is not very precise.
Abstract:
-In this part, the authors should clearly tell us what the specific protein-coding gene is?
-The results are not perfectly summarized.
-The conclusion is a bit simple.

Introduction:
-Line 40.  Reference 3 is out of date. Authors should cite the latest literatures published in the Lancet or its sub-journals.
-The authors should introduce the latest schizophrenia-associated proteomics and genomic studies to highlight the importance of current research.
-Page 67.  “we first adopted MALDI-TOF/MS to profile serum protein patterns in SCZ patients and control subjects”.  This sentence is an inappropriate expression.  Please correct it.
-Page 69-70.  The authors should use “protein-coding gene” rather than “candidate genes”.Method:-Page 94-95. Both the subheadings of Ethics statement and subheadings of Subjects mentioned participants from their study wrote informed consent. Please delete one of them.
-Page 111. Please provide the full names of CST 9, when it appears in the Abstract or mai text for the first time.
-Page 135-136. Authors mentioned that P values were adjusted for age and gender in Table 1 and Table 2. However, in the statistic section, authors mentioned that the allele frequency and genotype distribution of SNPs were analyzed by χ 2 tests. As far as I know, Chi-square test cannot implement controlling variables. Please introduce the rationale clearly.-Page 138. please add references about AIC.

Results：
-Page 146-147. There are not any statistical figures and graphics to prove this result. Please provide more information.
-Page 162. Due to the fact that CST 9 appears in the main text for the second time, an abbreviated form will be more concise than the full name.

Discussions：
-Page 241-249.  A complete paragraph about research limitations important for readers. Thus, it is not common to combine conclusions with limitations.-Gene CST 9 interacted with other genetic (or environmental) factors, may play a role in the occurrence of SCZ. Please add this limitation.

Conclusions:-Further research content can be appropriately described and forecast in this 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: KININOGEN-1 AS A PROTEIN BIOMARKER FOR SCHIZOPHRENIA THROUGH MASS SPECTROMETRY AND GENETIC ASSOCIATION ANALYSES
Review round: 2
Reviewer: 1

Output:
Basic reporting: Good.
Experimental design: The experiments are well-designed.
Validity of the findings: The finding is well investigated.
Additional comments: General Comments: The authors have developed a novel method, which uses Kininogen-1 as a protein biomarker for schizophrenia diagnosis. Authors have addressed some concerns but there are still some problems. Therefore, the manuscript can be acceptable after minor revision. The details have been listed below:

1, The resolution of Figure 1 is still too low to recognize. Authors should describe the X,Y axes in Figure 1A. And the sensitivity and specificity are very unclear in Fig. 1B. So it is highly recommended to increase the quality of this figure because it is one of the most important figure in this work.

2, In the response, authors said that “the mRNA expression level of CST9 gene was too low to reach the detection threshold, so we failed to detect its mRNA expression level and failed to compare the different of mRNA expression level between patients and controls”. I understand that mRNA is in low level and unstable, but after reversed to cDNA and combined with qPCR, the difference of mRNA expression level between patients and controls maybe detectable.


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: KININOGEN-1 AS A PROTEIN BIOMARKER FOR SCHIZOPHRENIA THROUGH MASS SPECTROMETRY AND GENETIC ASSOCIATION ANALYSES
Review round: 2
Reviewer: 2

Output:
Basic reporting: pass
Experimental design: pass
Validity of the findings: pass
Additional comments: The authors have made good responses to the comments/suggestions of this reviewer. No more revision is needed.


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

Input:
Title: COMPARATIVE PROTEOMICS ANALYSIS REVEALS THE DIFFERENCE DURING ANTLER REGENERATION STAGE BETWEEN RED DEER AND SIKA DEER
Review round: 1
Reviewer: 1

Output:
Basic reporting: See the attached pdf file
Experimental design: See the attached pdf file
Validity of the findings: See the attached pdf file
Additional comments: See the attached pdf file


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

Input:
Title: COMPARATIVE PROTEOMICS ANALYSIS REVEALS THE DIFFERENCE DURING ANTLER REGENERATION STAGE BETWEEN RED DEER AND SIKA DEER
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1. English has to be improved in terms of grammar, syntax and typos and the language has to be edited by a professional/native speaker.

2. Introduction and background are relatively poorly covered. Some key references for antler gene expression are missing (Borsy et al, Mol Genet Genomics (2009) 281:301–313; Molnár et al, Mol Genet Genomics (2007) 277:237–248; Stéger et al, Mol Genet Genomics (2010) 284:273–287). A more detailed description of the antler development, as per the missing references, would be required. I think that the miRNA part of the introduction is irrelevant and should be omitted.

3. Structure of the manuscript complies with the criteria of Peer J, although acknowledgement is missing.

4. Figures are fine, but my comment on the language is also valid for them.

5. Raw data were supplied.
Experimental design: 1. The study is within the aim and scope of the journal

2. The research question is not very well defined, it is a sort of much generalised statement about “distinctive differences of antler regeneration between sika deer and red deer” (line 62) and “to study the antler systems biology” (line 63). Antler system (?) biology is a very broad area and the authors did not provide any particular details what they would like to know by the study.

3. The investigation is not very well designed. Antler regeneration is a long, about 110-120 days, and we only know that sampling was done on day 80. We even do not know, because the precise description of the sampling is not given, that which exact part of the developing antler was sampled. I could only guess that samples were taken from a calcified and ossificated section of the antler. Since previous gene expression studies for both red deer (see missing references) and sika deer (Yao et al.) used the growing tip of the antler for sampling, the authors should define that which part of the antler they sampled and why.

4. Materials and methods. The precise tissue sampling (lines 75-84) should be described. Line 91: which lysates were combined? Line 93: full name for BCA should be given. Lines 128-130: settings for the BLAST search and GO analyses should be given. Lines 136-138: I am not certain why the authors used the Fisher’s exact test for the enrichment analysis. Enrichment tests typically require a list of gene terms (name or similar), which is then compared to a background list, while Fisher’s exact test requires numerical data. The authors should give a detailed explanation on this and/or repeat the analysis by the GSEA module of BLAST2GO or BINGO or similar.
Validity of the findings: The authors have identified a number of differentially expressed proteins in their study. A number of these (47) were common between red and sika deer, while others (19 and 114, respectively) were unique for the two species.

Regarding their results.

1. Samples are not paired, i.e. animal RD2-1 is not the pair of SD1-1. Therefore, calculating the log2 ratio between these two individuals, and between the other animals too, is meaningless, and thus the showing the heat map in Figure2 is unnecessary. RD2-1 could be paired with either SD1-2 or SD1-3 too and then log2 expression ratio would be different. Theoretically, nine combinations could be generated, each resulting in different ratios. I suggest to eliminate Figure2. The averages and their ratio as shown in Table S2 is sufficient.

2. In Table S2, the animal IDs are missing in columns J, K, and N, O. 3.

3. In total, the authors identified 1060 proteins were identified (line 143), but for 82 of these there is no LFQ intensity in TableS1, and I could not figure out why those were listed. Regarding this, column headings need a short definition to show for example in a separate additional file listing all Supplementary tables. So, 978 proteins had LFQ intensity and it needs an explanation how this number was reduced to 578+114+19 = 711. I can guess that for example a protein which was detected only in one red deer was not considered as unique, but still it should be describe either in the method or in text. Moreover in Table S3, there are 861 protein are listed on the “enrichment” sheet, but the sum of the protein in the three ontology group is 864. There is a discrepancy in these numbers and the authors should investigate, what went wrong. So how this 861 number came?

4. I am curious, why the enrichment study was performed on all genes, whatever number. Enrichment analysis on all genes has no meaning in term of the differences between the two species. I suggest to redo the analysis on the 578 common and the 114 and 19 unique genes in order to see what sort of functional differences are between red and sika deer.

5. The enrichment data looks strange. If you perform an analysis whose output is both P-value and FDR, then if the analysis was sound, the P-value is smaller than FDR by about 1, 2 or more order of magnitude. For example, a P-value of 1.40E-09 should correspond to and FDR of 1.00E-07, 0.025 to 0.055 and 0.78 to 0.85 (not significant). But a 0.002557 P-value with a 0.731 FDR indicates that something is wrong with the analysis. Also, different P-values should not have the same FDR. Regarding this, what is the “rich factor” in Figure 4 and Table S3?

6. Talking about up- and downregulation can be tricky. The authors identified 30 proteins upregulated in sika (obviously downregulated in red deer) and 17 proteins downregulated in sika (upregulated in red deer). In the text (lines 154-160) they are mentioning the ratios from the viewpoint of sika (“30 up-regulated, 17 down-regulated in sika deer group”). However in Table S2 they show the red/sika ratio. I think it would be better to show the sika/red ratio, which would reflect better to their preferred way of talking about ratios relative to sika. In Table S2 it would be also nice to show the log2 ratio of the expression ratio, which would very clearly show the 2-fold change threshold (+1 for 2-fold upregulation, -1 for 2-fold downregulation), this is the most common way of displaying expression ratios anyway.

7. In the discussion, the authors cite a number of publications related to bone development and discuss the differentially expressed they identified in that contest. They also use physiological result from two publications (Chen et al. 2008 and Pattappa et al. 2011) as their focal point for their discussion. These two publications are about human mesenchymal stem cells. But they do not use the Li et al. 2012a publication and the above mentioned red deer studies to discuss their results. One major source of the problems with the discussion that we do not precisely that which portion of the antler was used for sampling. If the site of sampling was a fully differentiated bone part of the antler, then all of the bone development literatures are not very valid. For me it seems to be that the authors mined for literatures which describes those genes that they identified and are mentioned in relationship with bones, but the two is not necessary means causality. It is difficult to explain anyway that why a gene related to bone development in differentially expressed between tow deer species in an established tissue, if so. In the case of the authors if would reconsider the discussion strategy and mainly focus on antler development studies.
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: COMPARATIVE PROTEOMICS ANALYSIS REVEALS THE DIFFERENCE DURING ANTLER REGENERATION STAGE BETWEEN RED DEER AND SIKA DEER
Review round: 2
Reviewer: 1

Output:
Basic reporting: English still needs a serious editing. I do not think that it was checked properly. For example, the words proteome, proteomic and proteomics are used in an incorrect way. The articles “The” and “a” are used incorrectly. Proteomics is the science, proteome is the sum of proteins. The authors sometimes changed the good word to bad, and Many other syntax and wording errors. A typical example: “more kinds of proteins…”. This should be “more proteins” or “more type of proteins. Typing errors are present too.

If a reviewer ask for some information, it not only means that the authors should answer it, but it also should go into the revised text. A typical example is the question from Reviewer 1 “What software was used for the diﬀerential analysis? What kind of test was used (ANOVA, t-test... )? Were protein abundances normalized? The answer was not added to the manuscript. It should be.
Experimental design: Line 95: sagittal means: “situated in the direction of the sagittal suture; said of an anteroposterior plane or section parallel to the median plane of the body.” (From the Free Dictionary). From this it is difficult to find out how the antler tip was sectioned. I guess that the authors made cross section of the antler. Why don’t you say so?

In the methods section, line 104, it is still not clear that what the combination of lysates are. Be more precise.

Sampling. A 5 cm piece of the antler tip was removed and then sectioned into 5 to 8 mm sections. That means 6 to 10 sections. They were combined for protein extraction or extracted separately and then extractions were combined?
Validity of the findings: I still have serious problems with data presentation.
1. Despite that the corresponding author wrote so, the headings for some of the intensities in Table S2 were not corrected (animal ID number).

2. There is no legends for the supplementary tables. Thus it is difficult to understand them. The authors should briefly but precisely describe what the headings are. What is “coverage” for example, and how it was calculated. Let see an example: In the case of protein A0A220IGB1, there are four protein in the group. Then the 17 peptides were identified in all four proteins, or just the 8 unique peptides were identified in all four proteins? 17 peptides were identified in each sample or they were identified in the six samples? The peak intensity is the sum of the intensity of all peptides in a sample? I am not sure that the peptide numbers should be given in any table.

3. DO NOT show those proteins in Table S1 that were eliminated from further analysis. There is no reason to show them. But precisely describe in the main text what data were eliminated and why, but do not show them!

4. Have a legend for all supplementary tables.

5. In line 163 the authors say that “In total, 4721 unique peptides and 1060 proteins were identified”. But in Table S1, the total number of unique peptides is 4439. Why is that? The total number of peptides on the other hand is 5459.

6. In Table S1, the peptide numbers are a bit confusing. If there are “2” in Peptide cell, it does not mean that two peptides were present in the sample, it means that two different peptide sequences were identified, their absolute number is nor know. From the intensity data we just can predict that
2-fold higher intensity (2-fold upregulation) in one species that the absolute number of peptides were roughly 2-fold higher in one samples then in the other, but it can be 2 and 4 or 25 and 50. This is another reason to provide Table legends.

7. Figure 2 is still problematic. The scale looks for me log2. So it must be expression ration, because the intensity values in Table S1 cannot produce negative numbers. But there are six columns on the figure the same number as the samples. If you calculated rations and paired the samples then you should have 3 columns or 9 if you pair each samples. Moreover, you did not provided log2 values for the ratios in Table S1, so where the numbers for colour scale for the Heatmap was obtained from? I cannot accept your answer for this suggestion. If I suggested: remove it, then I meant REMOVE it. You could not defend, why the figure should stay.

8. For Table S3 give the log2 values of the ratios, and explain what the “t test p values” is. If it is for the averages then that column should go before the ratio column.

9. You did the enrichment analysis on the 180 DEGs. That is fine, but I suggested that do it (and I mean do it), on the common and the specific genes, because this is the only way to find species specific and common functions. So use the 114, 19 and the 47 genes, and the last group would be even better to separate into up- and down regulated groups. Your answer regarding this talking about the background gene list construction, did not answer my comment.
You used only the 1060 protein identified in you experiments as a background list for the enrichment analysis. This is ridiculously low number and, therefore, your analysis is very limited and far from acceptable. In an enrichment analysis, you have to use the widest possible gene set of the organism as a background, not only an organ specific or limited gene set. The UniProt database has 21,509 Cervus protein entries, so for the new analysis, which I require, you have to download this entries and use those proteins as background that have a GO annotation.
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: COMPARATIVE PROTEOMICS ANALYSIS REVEALS THE DIFFERENCE DURING ANTLER REGENERATION STAGE BETWEEN RED DEER AND SIKA DEER
Review round: 3
Reviewer: 1

Output:
Basic reporting: cf General comments
Experimental design: cf General comments
Validity of the findings: cf General comments
Additional comments: The authors addressed all the comments in my opinion.


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

Input:
Title: COMPARATIVE PROTEOMICS ANALYSIS REVEALS THE DIFFERENCE DURING ANTLER REGENERATION STAGE BETWEEN RED DEER AND SIKA DEER
Review round: 3
Reviewer: 2

Output:
Basic reporting: Questions were answered
Experimental design: Questions were answered
Validity of the findings: Questions were answered
Additional comments: No additional comments