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

Input:
Title: THE EFFECT OF ELEVATIONAL GRADIENT ON ALPINE GINGERS (ROSCOEA ALPINA AND R. PURPUREA) IN THE HIMALAYAS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The article would benefit from professional English correction made by native speaker familiar with the research area.

Literature references are not sufficient in Introduction and Discussion sections. Citing papers dealing with herbaceous plants performance along an elevational gradient are required. Some references are suggested in General comments section.

The MS has professional article structure, figures and tables. Raw data is shared.

The MS is self-contained and include all results relevant to the hypothesis.
Experimental design: The MS presents primary research within aims and scope of the journal.

Research question is well defined, relevant and meaningful. However, Authors have not properly identified knowledge gap being investigated, because literature review they have made is not sufficient. According to Authors aim of the study is ‘to provide the primary information on how alpine herbs do respond with the potentially stressful environmental conditions associated with increasing elevation in Himalayas.’ (Lines 73-75). The most important question should be how do alpine herbs respond along an elevational gradient in mountains, not only in some particular region of Himalayan Mts. Therefore, aims of the MS should be placed in the wider context of available literature dealing with herbaceous plants responses along elevational gradients, also from other high mountain regions of the world.

The research was rigorously performed to a high technical and ethical standards.

Methods are described with sufficient detail and information to replicate.
Validity of the findings: The MS represents important and valuable contribution to knowledge on herbaceous plants responses to environmental conditions along an elevational gradients in mountains. Papers dealing with this topic are not numerous and there is urgent need to gather more evidences on how plants respond along elevational gradients. In the context of predicted future climate changes, knowledge of plant performance along elevational gradients may contribute to an enhanced prediction of plant responses under an altered climate.

Data is robust and properly analysed by statistical methods.

Conclusions are well stated and linked to original research question. In the General comments section I commented on unsupported by results discussion of optimum photosynthetic zone (Lines 251-253) and unsupported conclusion on optimum growth zones (Line 292-293). Other conclusions are supported by results.

Speculations are identified as such.
Additional comments: I found your manuscript to be interesting and valuable contribution to the research area. Beside the other investigated morphological traits, analysis of stomata density variation along an elevational gradient is particularly interesting. Here, I present detailed comments to the MS, that I believe may help you to improve the article. Because I am not native English speaker, please, treat all language corrections I have made as suggestions.

Title: How does the elevational climate affect ...
I suggest to change this title. I am not sure if there is something what can be called ‘elevational climate’. Climate parameters change along an altitudinal (elevational) gradient (elevational shift in climate), but there is no common ‘elevational climate’. Moreover, are Authors sure that only climate affect morphology of the studied plants along elevational gradient?
I suggest something like this: How does an elevational gradient affect... or Variation of ...along elevational gradient

Abstract
Line 26: ‘the elevational gradient’
Perhaps here should be ‘an elevational gradient’

Lines 28-29: ‘Despite this interest...’
I suggest to delete this sentence. I agree that field works in remote high mountains is a challenging task, however, it should not be mentioned in Abstract as rationale for publishing results of your research.

Line 32: after meters ‘m’ add ‘a.s.l.’

Line 32-33: ‘We measured...at five plots, along the elevational transect at an elevational gap of ca. 100 m’
This description is somewhat not clear. I suggest to write that both species were investigated at five plots distributed along two vertical transects of 400 m, R. purpurea at elevational range of 2174-2574 m a.s.l. in Daman (Mt.?), and R. alpina at 2675-3079 m in Ghorepani (Mt. ?).

Lines 37-39: ‘These findings provide (a) an insight ....(b) provide relevant information...’
This sentence is vague; what are these ‘insight’ and ‘relevant information’? I suggest to present some of your conclusion here.
And again, think of replacing phrase ‘elevational climate’

Line 41: ‘Key words: Climate change’
I suggest to delete ‘climate change’ here because in the MS there are no discussion on possible effects of climate change on the studied species.

Introduction
Lines 49-50: ‘...small changes in elevation can lead to large variation in temperature...’
I suppose it should be: ‘can lead to large shift in temperature’

Line 70: ‘along altitudinal gradient’
Please use the terms altitude and elevation consistently (not interchangeably) throughout the MS (see McVicar TR, Körner Ch (2013) On the use of elevation, altitude, and height in the ecological and climatological literature. Oecologia 171:335–337)
In most cases in the MS ‘elevational gradient’ or ‘elevation’ is appropriate, but when Authors refer to climatic or environmental conditions such as, for example, temperature or CO2 pressure then altitude or altitudinal gradient is adequate.

Lines 69-71: ‘Several studies on the growth and physiological responses of trees along altitudinal gradient...'
Why did Authors cite only literature concerning trees? There are also available studies on performance of herbaceous plants along an elevational gradient in mountains. I suggest to provide here an outline of morphological variation in vegetative and generative traits of herbaceous plants. Here I recommend some papers:
Kiełtyk P (2018) Variation of vegetative and floral traits in the alpine plant Solidago minuta: evidence for local optimum along an elevational gradient. Alp Bot 128:47–57
Seguí J et al (2018) Phenotypic and reproductive responses of an Andean violet to environmental variation across an elevational gradient. Alp Bot 128:59–69
Bastida JM et al (2015) Local adaptation to distinct elevational cores contributes to current elevational divergence of two Aquilegia vulgaris subspecies. J Plant Ecol 8:273–283
Maad J et al (2013) Floral size variation in Campanula rotundifolia (Campanulaceae) along altitudinal gradients: patterns and possible selective mechanisms. Nord J Bot 31:361–371
Hulshof CM et al (2013) Intra-specific and inter-specific variation in specific leaf area reveal the importance of abiotic and biotic drivers of species diversity across elevation and latitude. J Veg Sci 24:921–931
Alexander JM et al (2009) Establishment of parallel clines in traits of native and introduced forbs. Ecology 90:612–622
Gonzalo-Turpin H, Hazard L (2009) Local adaptation occurs along altitudinal gradient despite the existence of gene flow in the alpine plant species Festuca eskia. J Ecol 97:742–751
von Arx G et al (2006) Evidence for life history changes in high-altitude populations of three perennial forbs. Ecology 87:665–674

Lines 72-75: ‘...there is a paucity of available information...’. ‘...this study aims to provide the primary information...’

There are some studies on performance of alpine herbaceous plants along elevational gradient and you should cite some of them. This study do not provide the primary information. But, your study makes an important contribution to this research area and, especially, stomata density has been rarely investigated along elevational gradient in herbaceous plants.

Line 74: ‘respond with’
- ‘respond to’

Lines 76-82: ‘The genus Roscoea, with 22 known species, is a Himalayan endemic alpine perennial herb...’. ‘Among the currently known seven Himalayan Roscoea species...’
Please, clarify here the number of Roscoea species: 22 or 7? These two sentences seem to be in contradiction.

Lines 87-89: ‘...we aimed to explore how do morphological (plant height and leaf length) and physiological variables (leaf area, SLA and stomata density)...’
and Lines 135-136: ‘Plant height and leaf length were measured to examine the morphological variables. Physiological variables included leaf area, specific leaf area (SLA) and stomata density.’

I am not sure if it is correct to classify leaf area and stomata density as physiological variables. I think these variables are morphological traits (stomata density may be regarded as micro-morphological trait). However, I agree that these traits are linked and related to physiological processes and can be used to assess and discuss plant physiology. Please, check classification of these traits in literature.

Materials and Methods
Study species
Lines 94-95: ‘Roscoea alpina is a common species with a wide distribution between the elevations of 2130-4270 m a.s.l. ...’
Lines 107-108: ‘Roscoea purpurea is the other most widespread members of the Himalayan Roscoea, distributed between the elevations of 1520-3100 m a.s.l. ...’

R. alpina was sampled from elevational range 2675-3069 m a.s.l. (404 m vertical range) what makes only 19% of the elevational range of this species. R. purpurea was sampled from elevational range 2174-2574 m a.s.l. (400 m vertical range) what makes 25% of the elevational range of this species. Therefore, both species were investigated only in central parts of their elevational distribution in Himalayan Mountains. Authors should be aware of this when interpreting pattern of elevational variation in the investigated species. If there are some reasons why these species were sampled only from 400 m vertical range explain it in Material and Methods section. And I suggest to give some restriction concerning this issue at the end of Discussion section.

Line 129: ‘We present the geographical details of the study sites in Table 1’
I suggest: Geographic coordinates and elevations of the study sites are presented in Table 1.

Line 135: ‘cc 100 m’
- ca. 100 m

Line 152: ‘...to access the variability.’
- to assess variability

Line 182: ‘... with low N.’
- with low sample size.

Results
Line 196: ‘Effect of altitude on physical traits ...’
- ‘physical’? Here it would be adequate the title from Line 209: ‘Variation of...’

Line 200: ‘...were not significantly affected by elevation...’
- were not significantly correlated with elevation

Line 203: ‘...we found no significant effect of elevation on either leaf length...’
- Suggestion: ...we found no significant relationship between elevation and leaf length... , as well as SLA’.
Generally, I suggest to avoid such definite statement as ‘effect’, ‘affected’ unless you have evidences on causal relationship.

Discussion
Line 210-214
Please, refer to some papers on elevational variation in morphology of herbaceous plants.

Line 219: ‘... as evidenced by the wider abundance of Zingiberaceae...’
- In my opinion this is not compelling argument.

Line 225: ‘...thus reflects...’
- thus may reflect

Line 249-: ‘Non-linear change of SLA of both species along the elevational gradient may indicate...’
In Results Authors stated: ‘we found no significant effect of elevation on ... SLA’ (Lines 203-204). Lack of significant effect in linear models do mean that there is significant non-linear change. On Fig 5 Authors presented mean values accompanied with +/- 1 standard deviation bars. The observed value can fall everywhere within range +/- 1sd with probability of 0.68. Because ranges of +/- 1sd for most of elevational samples for SLA overlapped considerably, what means high variation in this trait, and we cannot conclude if there is any significant change across elevation or merely random variation. Therefore, discussion of possible optimum photosynthetic zones (Lines 251-256) is not supported by the presented data. Moreover, analysed data was collected from 0.19-0.25 central fraction of elevational ranges of both species and may represent entirely optimal zones.

Line 261: ‘...have obtained mixed results.’
- different/contrasting results?

Lines276: ‘Our results indicate two major variations in ...’
- two major patterns in...?

Conclusions
Lines 292-293: ‘Thus, optimum growth zone of ...’
Results of this study (400 m vertical gradient which makes 0.2-0.25 fraction of elevational distributions of both species) do not support discussion of detailed elevations of optimum zones.

Figure 2: Please, indicate where did you collect R. alpina and R. purpurea.

Figure 3 and Figure 4: I suggest to put statistics directly on plots (at upper-right corners for example). Significant statistics may be distinguished with bold type.

Figure 5. Write (a), ... (b)... instead of (panel a), ... (panel b)

Raw data (csv file):
- add units to varaibles
- describe clearly which variables are for R. purpurea and which for R. alpina (Plant_height and Plant_height1 are not clear)


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

Input:
Title: THE EFFECT OF ELEVATIONAL GRADIENT ON ALPINE GINGERS (ROSCOEA ALPINA AND R. PURPUREA) IN THE HIMALAYAS
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: In my personal opinion, the study and the topics presented in this paper are very interesting, and the paper is enjoyable to read. The experiments appear well designed and the data properly analysed. I think that the results reported in the manuscript have important scientific significance.

I work in particular with seeds, so I would be curious to know if some variation is also reflected in the seeds, so I suggest to the authors (in the future) to add also the seed traits in their analysis.

I give some suggestions/comments:
I suggest to report the elevation in “m a.s.l.” instead to “m” along all the text.

In LL 93 and 211 “and” no italics.

LL 152-154 and LL 174-175: I suggest to move these information to the Results section.

About the Results section, I was surprised to see the text so short, but in accord with the choice of the authors, I think that the figures and the tables summarize in a pleasant way the great amount of results.

Review the formatting of table 2, in particular the column reporting the p values.


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

Input:
Title: THE EFFECT OF ELEVATIONAL GRADIENT ON ALPINE GINGERS (ROSCOEA ALPINA AND R. PURPUREA) IN THE HIMALAYAS
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The submitted version of the MS is thoroughly revised and significantly improved. Authors used clear and unambiguous, professional English throughout. In their rebuttal letter authors have given satisfactory responses to all my comments and suggestions. I recommend to accept the submitted manuscript for publication in PeerJ journal without need for additional revision.


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

Input:
Title: THE EFFECT OF ELEVATIONAL GRADIENT ON ALPINE GINGERS (ROSCOEA ALPINA AND R. PURPUREA) IN THE HIMALAYAS
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Dear Authors,

I thank you for taking my previous pieces of advice into consideration. I read with attention the final version and, for me, this version is improved respect the previous.

In my personal opinion, the paper now is OK and I suggest only other very small integration/corrections:

LL 96: Royle and Smith, no italics.

LL 135-136 and after in L 140: When you report “…vegetation type … of Pinus, Rhododendron and Quercus”, is it possible to specify better the species of Pinus, Rhododendron and Quercus present? The complete information could help the readers in better understanding the vegetation of the sites without further analysis "outside" this paper.

L 146: 2574 m a.s.l. and 3079 m a.s.l.

Table 1: I suggest to complete the coordinate details adding “N” in latitude columns, and “E” in longitude columns.

Table 2 and Table 3: I suggest to explain in full SLA in the tables or, probably, you could simply report in the caption “SLA correspond to specific leaf area" or something similar.


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

Input:
Title: A NOVEL METHODOLOGY FOR OPTIMAL LAND ALLOCATION FOR AGRICULTURAL CROPS USING SOCIAL SPIDER ALGORITHM
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1.	The authors investigated the application of social spider algorithm in the optimal allocation of agricultural land. In general, the contribution is interesting. However, this paper is not well structured and written.
2.	At present, this manuscript is like an experimental report, rather than a common research paper.
3.	The extensive and methodical experimentation proves the effectiveness of the selected methods. However, the paper would gain in clarity if the achieved results were correlated with the characteristics of the algorithms.
4.	In this version, the section of discussion is missing. The author should make the best effort to modify this section. I want to see a considerable improvement in this regard.
5.	The introduction section can be revised and structured to improve the flow of ideas from presenting the context, identifying the problem and research gap and presenting the objectives.
6.	Need to justify why the social spider algorithm were used specifically.
7.	Intensive numeric calculation is involved in this work, but I do not know which platform is employed by the authors. R? Matlab?
8.	The research is well defined and relevant although the research gap was vaguely presented (possibly as a result of weak command of the English language).
9.	The unit is not included in Tables 4 and 5, which should be modified.
10.	The map in the Figure 1 is not complete, some basic elements (north arrow, scale bar and other legends) are missing.
11.	The figures are neither complete nor clear, some details of theses curves are lost.
Experimental design: no
Validity of the findings: no
Additional comments: No additional comments


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

Input:
Title: A NOVEL METHODOLOGY FOR OPTIMAL LAND ALLOCATION FOR AGRICULTURAL CROPS USING SOCIAL SPIDER ALGORITHM
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1. I suggest to rephrase the title as "A Novel Methodology for Optimal Land Allocation for Agricultural Crops using Social Spider Algorithm"
2. Line 32: Suggest rephrasing the word economy as revenue.
3. Equation 2: Inconsistency in using variable.  Equation 2 contains a variable, "Productivityi", whereas in description (Line No: 106) uses "proi".
4. Maintain consistency in claiming the scope of the variable.  For example, in Line No: 111 says seed cost whereas Line No: 114 says expense on seeds. I suggest use of variable names used in Equation 3 within brackets while describing them in Line Nos: 111 & 112 (if applicable).
5. Double check the citation format in Line No: 144.
Experimental design: Even though the authors have explained very well and established the social spider algorithm (SSA) to solve the problem, a couple of sentences required to be included to relate the chosen algorithm (SSA) to the problem applied.
Validity of the findings: No comment
Additional comments: Use of Figure 1: Does it have any relevance to the research?  I suggest you to remove Figure 1 if its of no relevance.


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

Input:
Title: A NOVEL METHODOLOGY FOR OPTIMAL LAND ALLOCATION FOR AGRICULTURAL CROPS USING SOCIAL SPIDER ALGORITHM
Review round: 2
Reviewer: 1

Output:
Basic reporting: As I can see from the previous review, you have corrected all of the reviwers comments. It is ready for publication.
Experimental design: -
Validity of the findings: -
Additional comments: -


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

Input:
Title: A NOVEL METHODOLOGY FOR OPTIMAL LAND ALLOCATION FOR AGRICULTURAL CROPS USING SOCIAL SPIDER ALGORITHM
Review round: 2
Reviewer: 2

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


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

Input:
Title: ESTIMATION OF MAIZE ABOVE-GROUND BIOMASS BASED ON STEM-LEAF SEPARATION STRATEGY INTEGRATED WITH LIDAR AND OPTICAL REMOTE SENSING DATA
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1) I noticed a few citations which follow statements that do not accurately reflect the findings of what is being cited. The two most noticeable examples of this are Line 108 with James and Robinson 2014 (Lidar is not mentioned in this paper, it focuses on Structure from Motion) and line 363 with Schlemmer et al. 2013 (AGB is not mentioned in this paper, and the paper concludes that nitrogen and chlorophyll content can be accurately measured using only multispectral data). I recommend you review your citations and ensure that the way they are being presented in your paper is accurate.

2) Several statements were made which should have been followed by citations backing up the claim. Examples can be found in lines 75, 117, 233-234, and 248-249.

3) The English language should be improved to ensure that an international audience will fully comprehend your work. Examples of areas which could use improvement can be found in lines 96-99, 147-148, 210, and 240.

4) As some readers, myself included, may not be familiar with NRMSE I recommend providing a citation which provides an explanation for the approach and how to interpret the results in line 249.
Experimental design: The paragraph from lines 227-237 needs more detail in order to make your experiment methodology clear and reproducible. I recommend providing more detail on which metrics were used, why you selected the method used here (citations if based on other studies), and how the SVI and LSP data were fused.
Validity of the findings: As this study generates a point cloud, results for the generated point cloud should be provided. These include the average point spacing, point density, and difference in Lidar derived elevation when compared to elevation values gathered via GPS. Most of this information is present throughout your paper but it would be more accessible if organized in a subsection of your results.
Additional comments: 1) I would recommend stating the SVIs/LSPs used in the model instead of the more general SVI/LSP language used in the methods/results/discussion sections.

2) The paragraph from lines 339-352 discusses an aspect of this study which is not clearly outlined earlier in the methodology and results. I recommend adding a subsection to both sections in order to (1) outline what was done to generate the image-based elevations and (2) present the results of this process. Otherwise, this paragraph seems out of place as it does not relate to what has been discussed so far.

3) The paragraph from lines 328-337 should also be discussed in the methodology. The results are appropriate here, but the steps taken to get these results need to be outlined as part of your methodology.

4) The paragraph from lines 364-376 should have a corresponding paragraph in the results section outlining the ANOVA results.

5) Overall, I though your research was interesting and your approach novel. I think your paper can best be improved by double checking citations to ensure they are cited correctly and where needed; ensuring that outcomes discussed have a corresponding component in the methodology/results sections; improve language clarity in some areas; and providing more detail to the previously noted paragraph in your methodology.


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: ESTIMATION OF MAIZE ABOVE-GROUND BIOMASS BASED ON STEM-LEAF SEPARATION STRATEGY INTEGRATED WITH LIDAR AND OPTICAL REMOTE SENSING DATA
Review round: 1
Reviewer: 2

Output:
Basic reporting: This paper described how to estimate maize above-ground biomass based on stem-leaf separation strategy integrated with LiDAR and optical remote sensing data. This is an interesting topic, dealing with a still unresolved methodology suitable for the different conditions of the plants at a global scale: to get AGB from drone remote sensing. The writing is clear. Overall, the manuscript is publishable, but, needs a critical revision first. There are some minor grammatical and structural mistakes in the manuscript and I am not able to highlight/revise all of them. So, please revise the manuscript and improve it considerably in terms of grammatical and structural mistakes.
The literature is suitable for the purpose of the article.
Experimental design: 1) Within aims and scope of the PeerJ journal.
2) Research question well defined, relevant & meaningful. It is stated how the research fills an identified knowledge gap.
3) Structure conforms to technical & ethical standard.
4) Methods described with sufficient detail & information to replicate.
Validity of the findings: 1) Data is robust, statistically sound, & controlled.
2) Conclusions are well stated, linked to original research question & limited to supporting results.
3) Speculation is welcome.
Additional comments: 1.	The research is well defined and relevant although the research gap was vaguely presented (possibly as a result of weak command of the English language). The authors should modify the introduction.
2.	Line 151: Did the authors consider different growth cycles? It may result in some uncertainty. The authors should consider different periods to reduce the uncertainty and improve the accuracy of the predictions.
3.	Line 149: The sample size should be added in the context, more than the site map. In addition, a total 30 samples are relatively small, which used to construct models. Did the authors divide them into calibration set and validation set? These contents should be clear.
4.	Line 210: Why did select these spectral indices? Please add the declarations.
5.	Why the RPD (ratio of performance to deviation) and/or RPIQ (ratio of performance to inter-quartile distance) was not used in this study? In common, these parameters are more effective than R2 and NRMSE.
6.	The authors should redesign Table 1, the description (full name of indices) and references should be separated.
7.	There is no need for Table 3. The involved content is common knowledge.
8.	In all the numbers with decimals, please consider to homogenize to only two decimals.
9.	better to mention how large is the plot is.
10.	I would suggest that the authors more clearly describe the processing of drone images, in particular, the parameters that have been used in the atmospheric correction such as the atmospheric mode and aerosol model that should be mentioned.
11.	Reflectance spectra curves of soils with different AGB should be added in the revision.
12.	The clear descriptions of spatial resolutions of UAV Multispectral Data and LiDAR data should be included. How do you deal with this specific issue, were they resampled？
13.	I recommend the authors add a clear flight path in this study.
14.	Why MLR was used in this study? In fact, this method is too old. PLSR is a commonly used approach to build a multi-variable model, with the advantages of dimension reduction and handling of collinearities among various independent variables. PLSR is better than MLR. In addition, the application of MLR has a strict requirement (normal distribution).
15.	The detailed threshold of p-value of 0.01 and 0.05 should be added.
16.	The introduction of confidence level is welcome, however, they should be displayed in the Figure. (Refer to Quantitative estimation of soil salinity by means of different modeling methods and visible-near infrared (VIS–NIR) spectroscopy, Ebinur Lake Wetland, Northwest China[J]. PeerJ, 2018, 6: e4703.)
17.	The pretreatments of drone data could improve the sensitivity between the dependent and independent variables in the spectral analysis. The authors should consider them.
18.	At present, the section of discussion is too weak. The relevant contents of estimation mechanism, introduction of machine learning algorithms, comparisons between of different models… are welcome.


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: ESTIMATION OF MAIZE ABOVE-GROUND BIOMASS BASED ON STEM-LEAF SEPARATION STRATEGY INTEGRATED WITH LIDAR AND OPTICAL REMOTE SENSING DATA
Review round: 2
Reviewer: 1

Output:
Basic reporting: As I can see from the previous review, you have corrected all of the reviwers comments. It is ready for publication.
Experimental design: no
Validity of the findings: no
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: COLLISIONAL MECHANICS OF THE DIAGONAL GAITS OF HORSES OVER A RANGE OF SPEEDS
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comments
Validity of the findings: While underlying data have been provided - documentation of the definitions of data in a few columns are unclear - e.g., Import QQ sheet, B/F?
Where applicable - include units in underlying data
Additional comments: Figure 1 – consider putting parts a, b, and c on the same horizontal time axis – so that the differences in stance time are evident

Figure 1 – consider adding footfall sequence for each movement – e.g., piaffe – the fore and hind would overlap some

Line 239 – affected


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: COLLISIONAL MECHANICS OF THE DIAGONAL GAITS OF HORSES OVER A RANGE OF SPEEDS
Review round: 1
Reviewer: 2

Output:
Basic reporting: This report describes a small but well conducted study of the specifics of three somewhat related equine dressage competition gaits that are somewhat related; the passage, piaffe and trot. In particular the objective was to assess these gaits in terms of collision dynamics, an emerging perspective on gait movement strategy and its energetic consequences. Although only a small number of animals were assessed, these were highly trained animals well schooled in these gaits. The relatively small variability of the data indicate that the results are indicative of the general behaviour of these animals and the results can be depended upon. Table 1 and Fig. 2 alone justify publication of this paper, but the question addressed is provided within a solid background of theory and relevant literature. This paper will be of substantial interest to a relatively small group of individuals who focus on equine gaits, particularly those of dressage. This paper provides a novel perspective and introduces a new, and in my opinion valuable, method of comparing the dynamics of these gaits.
Experimental design: Line 89 re: data recording rate - is this 960 Hz in total or per channel? This should be specified.

Line 90 What is meant by the term ‘consistently’? Did you measure horizontal acceleration? Was speed consistent stride to stride?
Validity of the findings: The findings are valid and the authors do a good job in describing the value of the findings and their implications.
Additional comments: Lines 245-248 I don’t understand this statement. At the trot the system should act like a spring-mass combo, not an inverted pendulum. If you are invoking the more complex spring-loaded inverted pendulum (SLIP) model, the statement would make some sense, but this should be clearly described and specified.


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: COLLISIONAL MECHANICS OF THE DIAGONAL GAITS OF HORSES OVER A RANGE OF SPEEDS
Review round: 1
Reviewer: 3

Output:
Basic reporting: Overall, I do not feel the literature is entirely well referenced. There is opportunity here to compare these data to other gaits and other species. This should be considered: Usherwood, J. R. (2019). An extension to the collisional model of the energetic cost of support qualitatively explains trotting and the trot–canter transition. Journal of Experimental Zoology Part A: Ecological and Integrative Physiology.
Experimental design: No Comment
Validity of the findings: No comment
Additional comments: This paper makes use of a series of force plates and a motion capture system to calculate the mechanical cost and associated collision variables in ridden equine trot, passage and piaffe. It would appear these data have been collected to a high technical and ethical standard and the methods are described sufficiently to allow replication. The underlying data have been provided as values extracted from the raw data files which is sufficient given the nature of this type of data.

I believe this manuscript is a valuable and worthwhile contribution to the literature though work is needed to make this clear to the reader and to maximise its usefulness. I actually think the authors sell themselves short and the manuscript would really benefit from a more thorough introduction and discussion. In particular, I feel that there needs to be more context and more reference to the literature. I also feel strongly that the figures are somewhat thin on the ground and, again, much more could be made of this dataset (I am unsure whether the authors were limited by the journal in this or whether they have plans for further manuscripts using the same data).

This work is interesting and I would be very happy to see it published but I think the authors need to revise their introduction and discussion to make this clear to all readers.

Abstract:
I understand there is a word limit here but I think there needs to be a sentence at the end to hit home the relevance, given this is what readers will use to decide whether they read the rest of the paper.

Introduction:
The introduction currently reads like an excerpt from a biomechanics textbook, it gives a very specific introduction to collisions but lacks broader context. I appreciate the authors have a tricky job here but this paper is actually interesting and useful to both pure biomechanists and equine specialists - this is very valuable impact-wise but a juggling act to please all parties. Specifically, I think the introduction would very much benefit from an early paragraph discussing broadly the cost of locomotion and its importance followed by how manipulating gait (ie. gaits used in horse riding that don’t exist in nature) can provide valuable insight into underlying mechanics and mechanical cost. As this is not a specialist equine journal, I do think it is necessary to fully describe and give background to the passage and piaffe gaits (similarly, working, medium and extended trot would need to be explained to a non-equine audience). I hope the authors do not take this as too heavy a criticism as it is not intended to be, I simply think this interesting dataset could be better framed and be more impactful as a result.

Methods:
The methods are clearly described and repeatable. I have a few questions that I did not pick up answers to when reading and apologise if I missed them:
-	horse and rider weight are given to 1 decimal place but I did not see mention of the weight of the tack, was this considered?
-	were the data checked for acceleration/deceleration within trials. I fully appreciate that perfect steady state is impossible to achieve but this would make a difference to the data, a sentence mentioning how this was controlled for/checked would be useful.
-	the range of speeds in the piaffe especially is very small, do you have any metric for how accurately this was measured eg. over what distance/time?

Results:
As above, I think more could be made of the data with the use of figures.
-	In figure 1C, it would be nice to see separate forelimb and hindlimb traces either in addition to the summed or instead of. Also, it would make sense to show a mean in all panels (+/- std) for a mid-range speed or at least one from either end of the speed range to give an idea of variability rather than a single step.
-	As you discuss energy exchange a bit later on, would it be possible to show PE, KE etc. throughout a stride for each gait? This would be of great interest to those studying cost of locomotion and would add significant weight to the discussion points. These points come across as largely speculative but you have the data available to support these statements.
Overall, I can fully appreciate that these will have been challenging data to collect so showing it off with greater use of figures would make sense.

Discussion:
As with the introduction, I believe this can be extended to be more impactful.
-	208: you mention the effect of the rider, is it possible to suggest how the results might be affected by the rider? I appreciate these data could not be collected without a rider but some discussion/quantification would be beneficial for the reader.
-	211: at what point of dissociation is it no longer a trot? Is there any study into the limb phasing here?
-	Reference to other gaits is limited, there is a wealth of literature on equine gait, I feel comparison with other gaits would really help contextualise these data for the reader. For example, how does the cost of passage compare to a canter or tolt?
-	Overall, I do not feel the literature is entirely well referenced. There is opportunity here to compare these data to other gaits and other species. This should be considered: Usherwood, J. R. (2019). An extension to the collisional model of the energetic cost of support qualitatively explains trotting and the trot–canter transition. Journal of Experimental Zoology Part A: Ecological and Integrative Physiology.

Minor points:
Line 16: maybe “loss” would read better than “losses”?
Line 37: “from place to place”… “in relation to the environment”?
Line 39: need a more basic reference here.
Line 40: not really a goal, more worthwhile to say there is benefit to minimising energy expenditure for given movement.
Line 41: ‘Method of achieving’ sounds a bit like there’s some conscious choice, also worth mentioning that these are highly reductionist etc.
Line 53: need some references here as a number of equine gaits are shown to be relatively inexpensive.
line 55: true in gallop, maybe walk but is this shown in trotting gaits given they are the focus of this paper? Otherwise need to specify.
Lin 62: wording here is a little ambiguous, a speed exists where cost is minimised, moving further away from this speed increases cost.
Line 53/64: one of the theories.
Line 111: comma after data.
Line 129: maybe an editing point but the bars in the equation have come out as capital i which is confusing.


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

Input:
Title: THE CO-EXPRESSION NETWORKS OF DIFFERENTIALLY EXPRESSED RBPS WITH TFS AND LNCRNAS RELATED TO CLINICAL TNM STAGES OF CANCERS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The reviewer thanks the authors for their work and interesting principles and methods described in the manuscript. However, there are issues throughout the papers which must be addressed: Please find below my comments with the line in the text at which they are associated:

Generally, English needs to be improved throughout the text. There are few grammatic errors which must be changed. Please use an online grammar checker or simply your text editor.

Line 62: Please add a more recent review which describes the role of RBPs in disease.

Line 62: Please add a more recent review which describes the role of RBPs in disease.

Lines 63-75. The authors broadly described the role of RBPs pointedly adding references. However, the papers cited in any specific case only suggest a role, not demonstrate a role. The reviewer suggests the authors to shift the sentences to a more ‘suggested’, ‘potential’ role of the RBPs. Ex: Instead of using: X plays, X and Y block, Z inhibits, It would be better to switch in: X is suggested, Y has been proposed and so on.

Lines 82-88. I suggest the authors describe RBPs as dysregulated not specifying cases in which they have only been found downregulated. It is broadly accepted that RBPs are dysregulated in cancer,  it is not necessary to specify only the cases of downregulation.

Lines 95-102. The authors need to reference more this passage and add some more specific published notions.

Lines 103-109: Add references and be more specific, citing well-described cases.

Lines 117-118: unclear

Lines 124: Please write the two types of cancer in long format and then acronyms

Line 409: The largest majority of RBPs studies show dysregulation not only downregulation. Please reformulate or add more details to this concept.

Line 437: The final statement must be reformulated. Demonstration implies experimental validations which have been not performed in this study.
Experimental design: The reviewer finds a lack or ambiguous description of the developed methods. The authors must clarify in the supplementary the source of the dataset. The reviewer finds not clear whether the authors downloaded only the raw gene expression data or the bam files and then run the RNA quantification. Moreover, in the case, they downloaded the raw counts they must address whether particular filtering of data took place to remove the noise of differential spectrum of expression, which may derive from characteristics of the samples and data processing to RNA quantification.

Line 137: How did the authors define the 2 groups of genes? How did they assign the genes to the 2 groups? This must be addressed in the methods.

Line 137: The authors did not clarify if they treated the differential genes in a cancer-specific way or a more general cancer vs normal (both pooled)

Line 171: The authors use a dubious terminology “The TNM system is simple to use and the prognostic accuracy between the different stages is quite good”  which confer approximation the clinical annotation used. The reviewer suggests rephrasing the sentences in this paragraph adding more context and references of successful studies.

Line 177: What does WGCNA mean, please clarify?

Please add a figure which shows the computational workflow recapitulating the crucial points of the analysis.
Validity of the findings: Line 246: The final statement suggests that RBP genes in different cancers occurring at related tissues have similar expression patterns. Please add a figure showing that the gene clusters enrich for same pathways.
Line 263: Please add a reference to this statement.
Line 274: It is not clear how did the authors define and classify the role of genes in Mand N stages. Please clarify.
Line 299: please reformulate the statement adding some uncertainty or demonstrate what stated with experimental validations.
Fig 5. Figure not clear. The referee suggests summarising the data in bar plots per cluster.
Line 390, 399-400. Please reformulate or eliminate this very general and approximate sentence.
Line 401-404: The authors must provide more data showing which genes are potentially downregulated housekeeping or tissue-specific and which can be potentially linked to oncogenesis.
Additional comments: No additional comments


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

Input:
Title: THE CO-EXPRESSION NETWORKS OF DIFFERENTIALLY EXPRESSED RBPS WITH TFS AND LNCRNAS RELATED TO CLINICAL TNM STAGES OF CANCERS
Review round: 1
Reviewer: 2

Output:
Basic reporting: This manuscript by Shuai bin Lian and colleagues, titled “The co-expression networks of differentially expressed RBPs with TFs and LncRNAs related to clinical TNM stages of cancers”, presents a comprehensive analysis of the expression of RNA-binding proteins (RBPs), transcription factors (TFs) and lncRNAs in cancer. Authors take advantage of publicly available dataset from the TCGA studies to evaluate deregulated RBPs and subsequently analyze their association with information about the TNM classification of tumors. They identify in this way modules of RBPs associated with TNM specifically in two cancer types, CHOL (Cholangiocarcinoma) and KICH (Kidney renal clear cell carcinoma).
Next, they evaluate correlation of these RBPs with TFs and lncRNAs, identifying relevant networks related to clinical TNM classification in these cancer types.
Generally, the study is well conducted and described. Its relevance is mainly related to CHOL and KICH cancer types.
I include a few comments below for manuscript improvement.

Major points:
Page 9, lanes 132-135: In this description of the generation of list of differentially expressed genes it is not sufficiently clear whether you selected only genes differentially expressed (Tumor vs Normal) pervasively in all cancers and then you next analyzed their expression in the various types OR if you started from DEGs from each subtype. Please specify clearly.

In Figure 3A-B it not clearly described what raws represent. Please revise explaining more clearly what modules represent and how did you create these.

Line 277 page 14: …in each sub-network, one RBP interacts with almost all non-RBPs, suggesting that particular RBPs are key regulators for each model: please include description of what do you mean for “interact”. A clearer description of the relation between RBP and non-RBPs in each module needs to be also better described. Please just mention the method  (WGCNA?) you used for correlation analysis and please mention if you also considered traditional correlation analysis for specific modules.
Generally, the point is that the text should be easily readable by scientists from all disciplines and I think you could try to better specify the analysis method applied at the beginning of each data/results section description (just mention the method), even if all analysis are well described in the Materials section.

Data presented in Figure 6 do not add much information in my opinion and this general analysis of RBP commonly deregulated in all cancer types could be moved to supplementary material and presented in the results text immediately after the first step of the analysis which led to the identification of these commonly DE RBPs.

Minor:
Speculations on the basis of the obtained results should be preferably included in the Discussion, while authors frequently include these in the results, Please revise.

Wang et al., 2018 is mentioned but not listed in the references
Experimental design: nc
Validity of the findings: nc
Additional comments: nc


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

Input:
Title: THE CO-EXPRESSION NETWORKS OF DIFFERENTIALLY EXPRESSED RBPS WITH TFS AND LNCRNAS RELATED TO CLINICAL TNM STAGES OF CANCERS
Review round: 1
Reviewer: 3

Output:
Basic reporting: In this study, Lian and co-authors explored the dysregulation of human RBPs across 20 cancer types compare to normal tissues. RBPs result differentially dysregulated between cancerous and normal tissues and RBPs belonging to the same system showed similar expression profile in different cancer types. The clinical TNM stage was related to pattern of RBPs expression in CHOL and KICH cancer types. Moreover, authors assess the co-expression networks of RBPs with Transcription factors and lncRNA in CHOL and KICH. Finally gene ontology analysis among 801 deregulated RBPs shared by 16 cancer types revealed that these genes are involved in some common biological processes and were enriched in some common biological functions.
RBPs are known to finely control gene expression and their dysregulation has been extensively reported to promote tumorigenesis. Thus, the evaluation of RBPs dysregulation and co-expression networks may help to elucidate the underlying biological mechanisms involved in tumorigenesis across cancers.
Despite the relevance of the topic, this manuscript is too descriptive and conclusions need to be expanded with additional information concerning the relevance of the obtained results. In Materials and methods section, the references of data analyses should be added. Moreover, some topics are unrelated, thus it is not easy to follow the rationale of the analyses. Finally, the authors' entire emphasis hinges on Figures 1, 3, 4 and 5 in the manuscript, which I cannot make any sense of. Text presents numerous contradictory statements and legends of these figures have to be more precise. All elements presented in the figures should be described in the legend or at least the text.
Experimental design: 1. Figure 4. The link between Figure 3 and Figure 4 is not obvious. The link has to be clarified in the text.
2. Materials and methods section must be rewritten to be more detailed and accurate. References for data analyses are missing.
Validity of the findings: 1. To corroborate the conclusions from Figure 1, the authors should assess whether the differences in each panel are significant. Thus, if applicable, statistical analysis should be added.
2. Lane 193-195: “In particular, RBPs show severely dysregulated expression in eight types of cancers, .... For these cancers, the corresponding relative proportions of dysregulation relative to normal tissues are....”. To reinforce the conclusion, the “relative proportions” of RBPs dysregulation should be shown for all cancer types (ie bar graph).
3. Figure 1B: To me, the rationale refers to this panel is not obvious. Up- and down-regulated RBPs are differentially expressed genes in tumors compare to normal tissues. While for normal tissues, what are they referring to? Moreover, how you can explain that the expression level of up-regulated RBPs in cancer is lower than in normal tissues? And vice versa. Please, indicate what the data refers to.
4. The main text concerning Figure 3 presents some contradictory statements that make it incomprehensible:
	Lane 265: “…two RBPs (PANBP17 and HNRNPA1) in the green module”. In Table S3 these RBPs are in the royal blue module. Please verify.
	Lane 265: “…and two RBPs (DCPS and C2orf15) in the royal blue module”. In Table S3 these RBPs are in the green module. Please verify.
	Lane 270: “These included four RBPs (TNRC6A, MECP2, ZCCHC14, and POLR2F) in the green model…”. In Table S3 these RBPs are in the royal blue module. Please verify.
	 Lane 271: “….three RBPs (TDRD1, TDRD9, and CELF4) in the royal blue model”. In Table S3 these RBPs are in the green module. Please verify.
	Lane 279: “Third, we found that among ten key (regulator) RBPs, six were down-regulated genes”. In Figure 3C, eight RBPs are resented as block (down-regulated genes). Please verify.
5. Figure 3, panels C and D. What are the different colors for? Please, indicate in the legend of panels C and D what the colors refer to. For example, in panel C, RBPs from Royal Blue module (RANBP17 and HNRNPA1) are indicated in light blue. RBPs from green (DCPS and C2orf15) and red (PUS7, PPARGC1a….) modules are indicated in red and green colors, respectively. This last representation is quite misleading. Please, if applicable, the green and red color in panel C should be inverted. Similarly, in panel D, RBPs from royal blue module are reported in green; while RBPs from green modules are indicated in red.
6. Figure 4 and 5: What are the different colors (green and red) for? Please, indicate in the legend also the meaning of circular, triangular and square dots.
7. To further strengthen the relevance of deregulated RBPs in CHOL and KICH pathogenesis, the prognostic impact (ie survival for patients) of co-expression networks should be addressed.
8. Please correct PANBP17 with RANBP17 in the main text
9. Lane 270 and 272: change “model” with “module”
10. Figure 1A, Panel a-d): To help reading comprehension, the title in the axis of abscissa should be added (ie cancer type).
Additional comments: No additional comments


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

Input:
Title: THE CO-EXPRESSION NETWORKS OF DIFFERENTIALLY EXPRESSED RBPS WITH TFS AND LNCRNAS RELATED TO CLINICAL TNM STAGES OF CANCERS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The reviewer would like to thank the authors for having submitted a new version of the manuscript totally revised. The reviewer appreciates the changes applied and do not rise any more concerns regarding language, references used, sentence formulation and final statements.
Experimental design: The reviewer appreciates the changes applied in the second version of the paper which strongly clarify the validity of the approach used. No more concerns are raised after reading the rebuttal and the second version of the paper regarding the computational workflow, which fulfils the technical standards in computational biology.
Validity of the findings: The authors clarified and changed what requested properly. The reviewer considers the conclusion and findings in line with the description and properly linked with the literature.
Additional comments: No further comments.


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

Input:
Title: THE CO-EXPRESSION NETWORKS OF DIFFERENTIALLY EXPRESSED RBPS WITH TFS AND LNCRNAS RELATED TO CLINICAL TNM STAGES OF CANCERS
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors improve the main text and Material and Methods sections. Figure legends are more precise.
Experimental design: All my comments concerning the experimental design have been answered.
Contradictory statements have been amended.
Validity of the findings: I believe that this work is now more solid.
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: MYB TRANSCRIPTION FACTORS IN ALFALFA (MEDICAGO SATIVA): GENOME-WIDE IDENTIFICATION AND EXPRESSION ANALYSIS UNDER ABIOTIC STRESSES
Review round: 1
Reviewer: 1

Output:
Basic reporting: #1 Abstract:
The qPCR experiment is not mentioned. Please include it. It is the only experimental result in this manuscript. It comprises conclusive and inconclusive results as compared to a previous RNAseq project and should be discussed as well in more depth (see additional comments at “validity of findings”).
#2
Line 103-105: This goes beyond the depth of this study. The authors should tune down this sentence e.g. to “Results from this study will provide a resource for potential applications of these genes for the improvement of alfalfa tolerance against abiotic stresses.”

Words / nomenclature and sentence structure / readability
#3
Please write out the abbreviation MYB once.
#4
Line 475-482: Please consider using Arabidopsis thaliana (or A. thaliana) instead of Arabidopsis.
#5
Line 392-395. Is renaming required?  Apart of this, these lines should appear earlier in the manuscript.
#6
Line 20 and 46: Please consider to split the sentence in two, e.g.:   ”… in the world. Its survival…” and e.g.:   ”… of the country. Freezing …”
#7
Line 64: The sentence is incomplete. Maybe add “determination” before the bracket.
#8
Line 252ff: I do not find how the presented data contributed to “further understand” the nature of the MsMYB proteins as it is not discussed further.
#9
Line 288 and 482: Please avoid using meanwhile. Do you want to point to another publication dividing the 37 MtMYB proteins in 12 subgroups while their study was ongoing? If so, the citation is missing. Please also delete it in line 482.
#10
Line 374: replace “genes that interact” with “candidate genes that putatively interact”
#11
Line 378ff: Without experimental evidence, please avoid to write about protein-protein and protein-DNA or genetic interactions without unambiguously naming them predicted. Some suggestions: “predicted” instead of “obtained”, “predicted protein-protein interactions”, “predicted protein-DNA interactions”, “its putative interactor’s genes”
#12
Line 476: Adjust “genes that interact“ in a way as suggested before (see related results part).
#13
Line 409: Delete “All”.
#14
Line 426: Insert “conserved” between “six” and “Trp”.
#15
Line 479: MYB15 is the homologous gene for MsMYB184? Please support the reader and unambiguously link the homologous genes/proteins.
#16
Line 487-490: These lines read as if the direct activation of a promoter by a MYB factor can be concluded from the mere presence of a MYB-core motif in this promoter. Please rephrase.

References, Background, introduction
#17
E.g. in line 112: Please cite for the used BLASTs in this manuscript. Here, for example, the named website www. alfalfatoolbox.org refers to Altschul et al., 1990. Please also identify the correct citations for other parts of this study using BLAST including local BLAST.
#18
General: Used web-resources and webtools.
It is required that the authors check all used resources again carefully for potentially overseen required citations and correct for this.
To give an example: e.g. line 156-164 and 316-318: Please read the terms of use as provided by a statement preamble prior to access to the respective website of the Nobel Research Institute and ensure that all required measures allowing you to use and publish the respective data were met. The providers of this web resource request “Prior to publication of the analyses, to accurately and completely cite the pre-publication data, including any applicable analysis as “The Alfalfa Breeder’s Toolbox and Gene Expression Atlas, Maria J. Monteros, Patrick Zhao, Xinbin Dai and Chunlin He. Annotation with CADL Genome v1.0. https://www.alfalfatoolbox.org””
Some of the available datasets are unpublished and it is requested to follow specific terms of usage. I assume that you used only the data set on B47 from the CALD-Gene Expression Atlas here which is published, but not cited, in the current version of your manuscript (O’Rourke et al., 2015).
This also holds true for all used (web)tools. Naming the respective website is not sufficient. Please add the required citations.
#19
Line 60: Please chose a more appropriate reference for the general classification of the MYB family which occurred long before the mentioned paper.
#20
Line 61-62: Please add a citation for this comparison.
#21
In line 73, “lowest level” and “most stable” seem to be out of context here. Please improve this sentence.
#22
Line 65: In the following the authors provide examples for processes for which data is analyzed in this manuscript and qPCR experiments are conducted. At this point of the manuscript, the choice appears arbitrary. The authors could bridge here with the following or something similar and continue with the chosen examples adding an examples for ABA: “MYB transcription factors act for examples in response to cold temperatures, drought, salt stress and ABA.”
#23
Line 68-75: Please adjust these lines together with lines 76-81. In these lines, examples for MYB factors and their function in abiotic stress responses are given and interrupted by the sentence “In fact, …” in line 76. Restructuring by conditions and providing some recent examples per condition might improve this part. Moreover, I do not see that the use of “however”, “moreover” and “in addition” is needed in these lines.
#24
Line 432-447: These examples and those in the following are rather introductory or suited for a review and should be selected for their fit for this study. It would be beneficial if the authors could report on homologous genes which were under investigation here.

Figures
#25
Figure 1: Describe A-N in the legend.
#26
Figure 2: Exchange “asterisks” and “triangles” in the legend.
#27
Figure 3: Describe what is represented by each red dot.
#28
Figure 4: Describe A-H here and in the following figures in a way avoiding the reader might think A in this figure groups the same MYBs as in one of the following figures.
#29
Figure 5: Define “CK” here and in following and supplemental figures.
#30
Figure 7: Consider changing “the cis-element number of MsMYBs.” to “the number of cis-elements in the analyzed promoter region of the indicated MsMYBs.”

Data
#31
Availability of used data.
Section in this manuscript: Line 167-172 and declarations:
The mentioned data for SRR7091780~7091794 is not accessible on Mai, 15th, 2019. Moreover, in the materials and methods section, it is described that the results of the related sequencing projects are deposited under the aforementioned SRR IDs and that these are results of previous studies. Therefore, the data should not be linked in the declarations with this article. The reference in the materials and methods section should remain but authors need to ensure that these resources are accessible which they should be as the previous studies were published with a reference to the same repository and SRR ID. Previous research: Luo et al., 2019a; Luo et al., 2019b; Zhou et al., 2018 with SRR7091780~7091794 and SRR7160313~7160357.
Experimental design: #32
Line 126, 166, 177: It is required to unambiguously differentiate between in silico analysis and experimental analysis. Towards this end, please include “in silico” in the respective headers in the Materials & Methods section and also in the other sections.
#33
Line 466. Please change from: “during multiple abiotic stresses” to “determined in previous studies during multiple abiotic stresses was analyzed.” or similar.
#34
Line 482-483: As the authors used various data of previous studies it is important to specify which finding was experimentally achieved in this study and which finding is a result from analyzing data from previous studies. This occurs repeatedly in the manuscript and should be specified. In the indicated lines, for example, the authors did not “find” that MYB184 is “inhibited during cold” as a results of this study but extracted the results from previously conducted studies. MsMYB184 was also not among the MsMYB factors tested in the qPCR experiment on cold treatment conducted in this study.
#35
Figure legends: Please give information about the respective underlying data for each figure wherever not given so far.
qPCR experiment
#36
Line 364: How did you chose the 14 MYBs? Later, in the discussion, you write that they were randomly selected. How was this done? Can you specify in line 363 what “these” means? Are the 14 tested MYBs a subset of “these” and does “these” relate to the MYB factors anlysed for cis-regulatory elements and therefore belonging to the set of stress responsive MsMYB gene promoter?
#37
E.g. line 209 and 203-206: In this study whole seedlings and root tips were used. Can you motivate the use of the root tips beyond the comparability with the previous RNAseq project of the lab? Please also improve the description and specify for which experiment shown in this study which material was used (seedling or root tips).
#38
Line 218-222: For the primer design: Did you use a genome/transcriptome to avoid off-targets? If so, can you please state which one was used (your lab’s data using Zhongmu No. 1 or e.g. B47 data). As different splicing forms might occur under different treatments, it would also be interesting to know if the used data originated from a control experiment or specific treatments. How did you test the primers? Did you control for primer dimers in the actual experiments using melt curves and did this for all treatment conditions results? Were efficiency tests conducted?
#39
Did you test - or please cite if already published – if one reference gene is sufficient and that the chosen reference gene is not changed under the experimental conditions and therefore qualifies as a reference gene for all conditions.
#40
Line 367-368: Is the induction significant for all genes? See also line 469: What does “markedly” mean in terms of statistics. The significant difference of treatment versus controls could be marked in the figure to visualize which statements can be drawn from both studies (RNAseq and qPCR).
#41
Figure 8: Which sample type was used for which experiment (root tips or whole seedling)? Why did the authors not test MsMYB184 for the cold treatment? The MsMYB184 homolog from A. thaliana and the homolog of its predicted interaction partner are the only pair obtained from the predicted interaction approach for which experimental data from previous studies is discussed by the authors in response to cold.
Validity of the findings: #42 Line 475-482: This is really an interesting finding and interesting background from previous studies on homologous genes and proteins in A. thaliana. Is this the only case for similar data from homologous genes or proteins?
#43 Line 401-406: These are results. Which two studies do the authors relate to here? It can not be this study and Dong et al., 2018 as the manuscript under review does not contain a sequencing experiment.
#44
Line 447-449: Please rephrase. This is not a result of the manuscript under review.

#45
Inconclusive results.

The qPCR results highlight a problem and points to one of the major points of this manuscript which lacks significant discussion: the inconsistency of some RNAseq results and the qPCR results both in terms of trend and more often in terms of differing fold changes as compared to the control.

Here are three possible scenarios:
In scenario one, the RNAseq data is limited and incorrect for some fold changes maybe due to current limitations in the alignment. If this is true, analysis using this type of data and generating heat maps and clustering based on the quantitative data is little informative and might even be misleading.

In scenario two, correctly conducting a qPCR experiment is limited e.g. due to missing information preventing an accurate primer design avoiding off-targets of the same or of different size. Another option might be that the reference gene is not suited for the conditions and tissue under investigation. qPCR experiments measure the steady state levels of a specific transcript (therefore “transcript level” is correct in contrast to “expression”, which is widely used). Depending on the choice of primers, splicing might also have an effect on the detected transcript levels. More reasons for the deviations might be found and should be discussed.

In a third scenario, the experimental conditions are not exactly the same and a factor not considered so far differed between the previous RNAseq experiment and the qPCR experiment like the timepoint within a day when the samples were taken, differences in RNA extraction or cDNA synthesis or differences in the growth conditions of the parental plants as this might affect seedling traits.

However, I would like to point out that the error bars indicate that with the respective available material the experiments were convincingly executed and there is no doubt that these results were obtained.

As this journal supports to report negative and inconclusive results, I suggest to extend the discussion on this aspect. The deviations underline that experiments like RNAseq - and meta-analysis based thereon - require careful verification ideally by different types of experiments but provide a rich and relevant resource for screening for interesting candidates and targets of breeding approaches as done by the authors in this study.
Additional comments: With their manuscript “MYB transcription factors in alfalfa (Medicago sativa): genome-wide identification and expression analysis under abiotic stresses” the authors Qiang Zhou, Wenxue Ma, Yue Cui, Chenglin Jia, Xiaoyu Jin, Dong Luo, Xueyang Min, Yanrong Wang and Zhipeng Liu will provide a resource for potential applications of these genes for the improvement of alfalfa tolerance against abiotic stresses. Moreover the discussion of conclusive but also inconclusive results from the intended verification qPCR experiment of a previous RNAseq experiment will be beneficial for other researchers.

I very much appreciated the efforts of the authors to provide such an extensive compilation of data analysis using previous RNAseq results on alfalfa and data from various other resources. I would like to highlight five points for improving the manuscript from various other points I recorded while reviewing the above mentioned manuscript.

All points are provided in the suggested scheme of the journal divided by three topics above.
1)	The improvement of the citation of web resources and web tools.
2)	Ensuring the availability of the used data.
3)	Improve the unambiguously differentiation of in silico analysis and experimental data as well as results from previous studies and those obtained within the manuscript under review.
4)	Improve the presentation and discussion of the qPCR experiment.
5)	Extend the discussion and emphasize on the inconclusive results obtained in the qPCR experiments as compared to previous RNAseq results.


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

Input:
Title: MYB TRANSCRIPTION FACTORS IN ALFALFA (MEDICAGO SATIVA): GENOME-WIDE IDENTIFICATION AND EXPRESSION ANALYSIS UNDER ABIOTIC STRESSES
Review round: 1
Reviewer: 2

Output:
Basic reporting: I would suggest that a further edit of the manuscript is made to improve the English standard. The language edits would greatly improve the flow of the manuscript.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The manuscript entitled "MYB transcription factors in alfalfa (Medicago sativa): genome-wide identification and expression analysis under abiotic stresses" by Zhou et al. presents a comprehensive analysis on the MsMYB family genes. The in-silico studies have been carried out systematically The expression profiles indicated that several MsMYB genes might play a crucial role in
development and abiotic stresses.

However, I would suggest  some changes in the current structure of the manuscript. Below please find my specific comments:
In the third para of introduction section, authors mentioned “So far, studies on the MYB gene family have been reported in many plants. A total of 104, 166, 155, 197, 524, and 475 MYB genes were identified and analyzed in Lotus japonicas, M.truncatula, Oryza sativa, Arabidopsis thaliana, Gossypium hirsutum, and Brassica rapa ssp. respectively” From the text it is not clear to which category these MYBs belong. Are these numbers represent all the types of MYB or only R2R3 MYB? Please mention in the revised text clearly.
As Medicago sativa is a close relative of Medicago truncatula. In Medicago truncatula, the genome-wide analysis of R2R3MYB gene family has already been carried out (Zheng et al 2017, J. Int. Agri, 16: 1576-91). Therefore, it would be better to do the comparative analysis of the MsMYB genes with MtMYB genes.
Add one figure showing distribution of MsMYB genes over the chromosome with graphical arrangement of segmental and WGD of these MsMYB.
For phylogenetic analysis why only 37 MtMYB  have been selected? For better classification of all the sub groups it would be much worth to add few functionally characterized MYB genes from Medicago, Lotus and Arabidopsis to develop phylogenetic tree. The description of legends of figure 1 is not clear. Please elaborate it and clearly mention according to the biological process of each sub groups from A to N.
Figure quality of S2 and S3 is not clear so improve it in the revised manuscript.
In the supplementary Table S4 and S5, also add the orthologous MtMYB gene along with locus ID.  After addition of this authors move it into main text. However authors have flexibility to decide it.  Additionally, it would be much worth if the authors can validate transiently Protein-DNA/ Protein-Protein interaction of the few MsMYB. This will further strengthen the quality of the manuscript.

I would suggest that a further edit of the manuscript is made to improve the English standard. The language edits would greatly improve the flow 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: URINE PROTEOME CHANGES IN RATS SUBCUTANEOUSLY INOCULATED WITH APPROXIMATELY TEN TUMOR CELLS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Although the hypothesis for the present study is well defined in the Introduction section, authors fail in giving sufficient background. Are there previous reports (excluding those from the group) that have successfully employed urine to detect changes in the proteome of tumor-bearing rodents? In this context, previous studies of the group related to other pathological states do not provide any significant background for this section. The English style is ok. Raw data has been shared.
Experimental design: Authors have employed the rat-derived Walker-256 carcinosarcoma cell line. Remarkably, authors have injected a low number of cells (~10 cells) to interrogate whether it is sufficient to cause any changes in the urine protein content. Changes in the body weight as well as in the behavior were followed for 41 days (according to figure 2). However, there is no mention of tumor growth. Do the animals develop any detectable tumor mass upon injection of 10 cells? Results clearly demonstrate that inoculation of a low number of tumor cells is sufficient to alter the protein composition of tumor cell-inoculated rats at days 13 and 21 post-inoculum. The proteomic analysis performed here is fine. However, in order to attribute the proteomic changes to tumor development, it would be essential to repeat the assays employing non-viable tumor cells.
Validity of the findings: As state above, the results presented in this study should be clearly correlated with the presence of viable, proliferating tumor cells. Remarkably, the discussion section largely fails in comparing the proteomic profiles herein found to previous studies that employed urine form tumor-bearing animal models or patients. Rather, the authors discussed the possible roles of proteins/pathways identified. Consequently, most of the cited articles are review 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: URINE PROTEOME CHANGES IN RATS SUBCUTANEOUSLY INOCULATED WITH APPROXIMATELY TEN TUMOR CELLS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Paper has tons of english language issues.  When the english is ignored the idea of the paper mentioned is to analyze the effect of small amount of cancerous cells on the proteomic/biomarker profile of the urine and using this proteomic profile to detect cancer.

Abstract:

The abstract in not well written, the abstract background is not clear and needs to be rewritten. The abstract results has grammatical errors "were almost clustered together".

Introduction:

line 64-66: rephrase the sentence and grammar
line 68: rephrase "therapeutic measures for patients are inevitable"
line 72: please mention what makes up external influence factors
line 77: rephrase and check grammar of "appearance of pathology changes"
line 79-81: rephrase these two lines as they are not clear
line 83: check grammar
line 86: Please dont use questions, rephrase with passive voice

Materials and Methods:
line 121: rephrase "three volumes of ..."

Results:
line 190: rephrase and check grammar of "All identified urinary proteins were performed with unsupervised clustering analysis
line 190: "almost clustered together" makes very little scientific sense, mention why these are close or similar with distances between centroids or something

Discussion:
line 236: incorrect usage: "stars"
line 238: incorrect usage of "protein changed"
line 239: use "respectively"

line 240-276: needs complete rewrite, discussion feels rushed. Each paragraph is written as one complete sentence with numbers. I would suggest the authors to create separate paragraphs for each important result obtained and elaborate on each of these results. There are sufficient references, but how the results obtained here match or corroborate with previously reported studies needs connection.

line 277: incorrect usage "be avoided"
line 289: incorrect usage "helped do"
Experimental design: The materials and methods are descriptive enough for replication and has sufficient references for various sources.

The experiments are well planned and the inference of the obtained data is valid. The authors should elaborate on each findings of the differential proteomic biomarker identified in this paper. It is great that the results match the hypothesis, but a little more explanation on why these proteomic profile differ needs expanded on.
Validity of the findings: I understand that the mice are expensive to carry out these experiments, but the number of mice that yielded these results are low and the authors have mentioned this limitation in line 278-279.
Additional comments: Please use a english language editor to check for grammar and spelling mistakes.


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

Input:
Title: EFFECTS OF SHOKYO (ZINGIBERIS RHIZOMA) AND KANKYO (ZINGIBERIS PROCESSUM RHIZOMA) ON PROSTAGLANDIN E2 PRODUCTION IN LIPOPOLYSACCHARIDE-TREATED MOUSE MACROPHAGE RAW264.7 CELLS
Review round: 1
Reviewer: 1

Output:
Basic reporting: This manuscript dose an excellent job demonstrating significant effect of Shokyo and Kankyo on prostaglandin E2 production. The article is well written, treats an actual problem. Also, it has focus on importance of traditional Asian medicine.
Experimental design: The experiment has designed very well, with appropriate statistical analysis.
Validity of the findings: The findings are valid and interesting and it has focused on role of traditional Japanese medicine and also spread organic and easier way for more usage of traditional anti-inflammatory drugs.
Japanese traditional medicine uses most of the Chinese therapies including acupuncture and moxibustion, but Kampō in its present-day sense is primarily concerned with the study of herbs. Kambo formulas were introduced from China and it is fully integrated into the modern health care system in Japan. Kampo can be an ideal choice of therapy.
Additional comments: I think this article excellent and is an important link between Shokyo and Kankyo and their water-extracted fractions of ginger with mechanisms of action via the inhibition of cPLA2 activity. This is an interesting topic of research. I personally enjoyed the role of gingerols and shogaols in the reduction of LPS-induced PGE2 production. And a new significant role of these extracts for inflammatory diseases.


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

Input:
Title: EFFECTS OF SHOKYO (ZINGIBERIS RHIZOMA) AND KANKYO (ZINGIBERIS PROCESSUM RHIZOMA) ON PROSTAGLANDIN E2 PRODUCTION IN LIPOPOLYSACCHARIDE-TREATED MOUSE MACROPHAGE RAW264.7 CELLS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Some specific questions are as follows:
1.  Lines 96 and 97: The method of measuring cyclooxygenase (COX)-2 activity is inconsistent with Figure 3A.
2．Line 160: Authors claimed that Shokyo and kankyo (both 100 µg/ml) strongly reduced LPS-induced PGE2 production (Figure 2B), and the effects of kankyo were stronger than those of kankyo. The expression is ambiguous and inconsistent with it in the section “ABSTRACT”.
3.  The dosage of 6-shogaol (Lines 215-223) is inconsistent with Figure 4A. There are some confusion and mistakes of its units.
4.  Some experimental results are delineated vaguely. For example, the descriptions of cell viability on line 151, Western Blot analysis of ERK phosphorylation in “RESULTS” (lines 215 to 223) and in “DISCUSSION” (line 239) are all confusing.
Experimental design: 5.  It was absent of direct evidences to prove that shokyo and kankyo inhibited cPLA 2 activity. The conclusion about COX-2 activity (lines 174 to 184), one of the main point of this article, was obtained through an indirect experiment. It is better to have more direct experimental evidence here, for example, enzyme activity testing.
 6.  In addition, the Western blot results (Figures 3 and 4) showed that neither the herbs extraction nor 6-shogaol altered LPS-induced expression of COX-2, cPLA2 or annexin 1, and there was also no significant inhibitory activity on LPS-induced phosphorylation of NF-κB p65 and ERK. The experiment lacked corresponding positive control. Besides, the relative gray value should be calculated.
7.  It is not enough just to evaluate p65 phosphorylation level for researching the role of shokyo and kankyo on NF-κB signal pathway, and some complementary experiments will make the results more convincing, for example, some detection about the phosphorylation of I-κB or p65 transportation to the nucleus.
Validity of the findings: no comment
Additional comments: The literature and background introduction are quite sufficient.
There are many obvious errors, confusing statements in this manuscript and the experimental design needs to be improved.


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

Input:
Title: EFFECTS OF SHOKYO (ZINGIBERIS RHIZOMA) AND KANKYO (ZINGIBERIS PROCESSUM RHIZOMA) ON PROSTAGLANDIN E2 PRODUCTION IN LIPOPOLYSACCHARIDE-TREATED MOUSE MACROPHAGE RAW264.7 CELLS
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript "Effects of shokyo (Zingiberis Rhizoma) and kankyo (Zingiberis Processum Rhizoma) on prostaglandin E2 production in lipopolysaccharide-treated mouse macrophage RAW264.7 cells” was professionally represented in unambiguous clear English language throughout the manuscript with structure aligned with PerrJ standard.
Additionally, raw data have been supplied with high quality relevant figures.  The introduction elaborated the context with a need for further consideration of certain studies and reviews of aqueous ginger and its effect of PGE2 levels in-vitro and in-vivo which will be thoroughly mentioned later on.
Experimental design: Toshiaki Ara et al represented a manuscript aligned with the PeerJ scope with consistently defined research questions. All the methodologies are well described and the data is analysed in a satisfactory modern manner.
Validity of the findings: Robust data with well discussed findings. Nevertheless, The metabolomic profiling of shokyo and kankyo materials is to be provided if not declared in the certificated of analysis provided by Tsumura & Co and the COA provided by the commercial vendor is to be added to the supplementary section.
Additional comments: A) The anti-inflammatory mechanisms of the ginger armoury of phytochemicals and either aqueous or alcoholic extracts were comprehensively vetted in the recent review (Ameliorative and protective effects of ginger and its main constituents against natural, chemical and radiation-induced toxicities: A comprehensive review, Food and Chemical Toxicology, 2018 https://doi.org/10.1016/j.fct.2018.10.048) for both the in-vitro and the in-vivo studies. This review should have been considered and utilised in the introductory section to draw the need for more studies on the aqueous ginger extract to be discussed briefly. Furthermore, some studies elaborated in the aforementioned review are to be considered in the discussion and/or the introduction sections for examples:
- The inhibition of induced cytokines in HaCaT cells and Mice by aqueous ginger extract (Zingiber officinale protects HaCaT cells and C57BL/6 mice from ultraviolet B-induced inflammation J. Med. Food, 13 (3) (2010), pp. 673-680).
- The declined expression of IFN-γ, IL-6 and iNOS with inhibition of the NF-k B activation in LPS challenged mice by aqueous ginger extract (Y.Y. Choi, M.H. Kim, J. Hong, S.-H. Kim, W.M. YangDried ginger (zingiber officinalis) inhibits inflammation in a lipopolysaccharide-induced mouse model Evid. Based Complement Alternat. Med., 2013 (2013), p. 914563).
- The orally administered Cuttlebone complex (CBC) including fresh ginger roots demonstrated a protective potentiality against indomethathin-induced gastric ulcer in rats via reducing the gastric ulcerous lesions and significant increment of the indomethacin-declined PGE2 levels in the stomach in a dose dependent manner (M.-Y. Chien, Y.-T. Lin, F.-C. Peng, H.-J. Lee, J.-M. Chang, C.-M.Yang, C.-H. Chen, Gastroprotective potential against indomethacin and safety assessment of the homology of medicine and food formula cuttlebone complex Food Funct., 6 (8) (2015), pp. 2803-2812).

B) Discuss the ginger up-regulatory effect on PGE2 in the stomach and its down-regulatory effect in macrophages and inflammation site.

C) Global metabolomic profiling of shokyo and kankyo used materials for robustness and repeatability and QC purposes in any further studies.
D) A section on the limitations of the study and ideas for future research could be included.


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

Input:
Title: EFFECTS OF SHOKYO (ZINGIBERIS RHIZOMA) AND KANKYO (ZINGIBERIS PROCESSUM RHIZOMA) ON PROSTAGLANDIN E2 PRODUCTION IN LIPOPOLYSACCHARIDE-TREATED MOUSE MACROPHAGE RAW264.7 CELLS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors of this manuscript have carefully answered the questions raised in the previous review. Some experiments were repeated or supplemented, and several obvious errors in the writing and data were corrected. However, there are still a few minor issues in this article that need to be noted and modified.
Experimental design: no comment
Validity of the findings: 1.	The author does not have a good understanding of the Questions 1. They tried to convert the amount of PGE2 to percent (%), and indicate as COX-2 activities (Figures 2B and 2C). Obviously, this is not appropriate. The problem with this part is the inconsistency between figures and texts. The author claimed in MATERIALS AND METHODS that the cells were treated with LPS and each herb for 6 h (simultaneous treatment) or LPS for 6 h and thereafter with each herb for 1 h (sequential treatment). Then, exogenous arachidonic acid (10 µM) for 30 min (Lines 118-121). This is consistent with the experimental method described in the annotation(Lines 507-511), But it is completely inconsistent with the “Time schedule of treatment” shown in Figure 3A.
2.	I still don't see any image results of Western blots were quantified by densitometry using image software. Of course, this is just a suggestion.
3.	A large number of WB tests were repeated and positive controls were added. But the stripes of WB should not be spliced( in Figure 6C).
Additional comments: no comment


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

Input:
Title: LIVER SLICE CULTURE AS A MODEL FOR LIPID METABOLISM IN FISH
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1.	Basic Reporting
The quality of writing  is quite good, only a few minor editorial suggestions (e.g. L17 “…interacting cell types essential for liver function.”)

L26:  not sure what “…exceedingly more similar to whole liver..”   means?  This is not a typical comparator comment.  Suggest an alternate wording to indicate that PCLS profiles, as measure in this study, are statistically, more similar to whole liver than that of hepatocytes.  I think this is what is meant here.

L100 & 106:  .”..(empty BCD)..”  suggest change to “(BCD only)” for clarity
Figure legends do not indicate n values (see comments under Experimental design section).

L107. Assume this refers to ALA concentration gradient?  “..70, 100 and 140 on day three ..”  units missing.

Figure Legends in general, require some additional detail for clarity.
Figure 1: no indication what the individual symbols represent in 1B…a single liver slice?? In panel C, no labels to indicate important features.  L191-192, no mention of features to support this statement.
Figure 4: it would be helpful to indicate n values on which the means are based..yes, the individual data are shown as separate circles, but difficult to assess, especially if some overlap.  Also, spell out abbreviations.

L253 Figure 4C is not mentioned prior to Fig 4D..rather the former is not mentioned until L279. Suggest changing figure order or mention order to correct this.
L268: “…(FDR <0.01; Fig 5A)” This is not the correct reference…I think it should be Supplemental Fig 2A.
L327: there is no Fig 6C.
Experimental design: 2.	Experimental Design
The research in question is well defined and will assist in the fields of piscine toxicology and aquaculture moving forward, as it relates to enhanced throughput for feed and toxicity assessment.  The rationale is well described and convincing.

The experiments performed are  appropriate, as is the analysis.  The text describing  the methods is somewhat confusing and lacks some crucial detail, as outlined below.

The Methods section is rather confusing as written and some details are missing.
L 77..descrioption of culture method lacks information about ambient gas content…were the cultures in air? Or were they incubated in conditions typical for mammalian culture (5%CO2:95%O2)?  This is important, as it may impact the acid-base status of the culture medium.  The latter was not mentioned...what was the pH of the media? Representative of Atlantic salmon ECF at 15°C?

It was also not clear why salmon of different sizes (50g or 500g) were used, and why freshwater and seawater fish were used.  It wasn’t clear if these differences may have confounded the data, especially those reported in Fig 5.

Determining the n value for each experiment was most difficult…it was not clear whether the liver was taken from a single fish, and multiple slices were obtained and thus, n refers to slice number, not number of fish.  (if so, then, arguably, then data represent technical,  not biological replicates.). The figure legends do not report n values.  This needs to be addressed.

L113-114:  How were livers injected? Via a blood vessel?
L114: type of collagenase?  There are many different kinds.
L117: pellet likely resuspended, rather than dissolved.
L117:  Why different source of insulin  (Merk vs. Sigma).  Human insulin?
L125-126: how were sections of liver slices chosen for imaging and assessment?
L128: How many liver slices were examined?
L172: what minor changes?
Validity of the findings: The authors are to be congratulated on collecting and analyzing a comprehensive data set.  The conclusions drawn are well supported by the data.  It is convincingly argued that PCLS is far superior to 2D hepatocyte culture for studying hepatic function.  Interestingly, the insulin and ALA interactions were not as predicted...which opens an new window for investigation.

One disconnect (at least for me) are in the steroid biosynthesis data.  In Fig 5A,  the PCLS are shown to have similar expression as whole liver, and that appears to contradict what is shown in Fig 3, where steroid biosynthesis pathway expression appears to be significantly reduced compared to intact liver.

L388:   “…all of the insulin receptors are bound at 10 nM.” Suggest softening here…to “are likely bound”.
Additional comments: No additional comments