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: TRANSCRIPTOME ANALYSIS OF FLAVONOID BIOSYNTHESIS IN SAFFLOWER FLOWERS GROWN UNDER DIFFERENT LIGHT INTENSITIES
Review round: 1
Reviewer: 1

Output:
Basic reporting: The paper “Transcriptome analysis of flavonoid accumulation in safflower flowers grown under different light intensities” is well written, but still needs some work to make it clearer. For instance, in the introduction a phrase linking the first and the second paragraphs is missing.
Some paragraphs later in the results part can be cut out.

The background is well researched.

The raw data is shared, the link to figshare is missing.

It is self-contained, but the hypothesis needs to be stated more clearly.
Experimental design: As mentioned above, the hypothesis and research question needs to be stated more clearly.

The main hypothesis, i.e. the relation of the light intensity UV protection and flavonoid content is unfortunately not clearly highlighted and explained in the paper. For instance, in the “Background”, following phrase reads: “Flavonoids are the main active ingredients in safflower and have many pharmacological effects.” It might be worth adding something like: “and play a role in UV light protection”. Multiple papers have been published on the subject. The missing hypothesis is more clear in p6. Lines 41-45, were the experimental setup to study relationship between light and flavonoid production is not clearly described.

Here I state some of the missing parts, that can provide more detail:

p.6, l. 51. “plant materials used in this study were safflower … obtained” something is missing here: seeds? Leafs? Complete plants?

p.6, l. 59. “Safflower plants with the same growth status” Please indicate the age/time here, for instance 5 weeks old… also, how long were the plants grown in the different light treatments? Please indicate!

p.6, l.63. Materials and Methods: please cite (or describe) here which RNA extraction protocol you used.
Validity of the findings: If the authors address most of my concerns then the paper will be a novel and robust paper, with an original research question and well supporting results.
Additional comments: The paper “Transcriptome analysis of flavonoid accumulation in safflower flowers grown under different light intensities” is well written. It is an exploration of the flavonoid biosynthesis genes under different light regimes, which is a complement to previous more descriptive transcript analyses. The role of flavonoids in plant UV protection and a probable link and/or tradeoff with secondary metabolite production is a question that is still not been extensively explored.
The main hypothesis, i.e. the relation of the light intensity UV protection and flavonoid content is unfortunately not clearly highlighted and explained in the paper. For instance, in the “Background”, following phrase reads: “Flavonoids are the main active ingredients in safflower and have many pharmacological effects.” It might be worth adding something like: “and play a role in UV light protection”. Multiple papers have been published on the subject. The missing hypothesis is more clear in p6. Lines 41-45, were the experimental setup to study relationship between light and flavonoid production is not clearly described.

Detailed important comments and possible corrections:
p.5, l.3. “Safflower is a strongly domesticated species” what does strongly mean here? i.e. underwent a strong bottleneck? From this phrase it’s not clear what the authors mean.

p.5, l.16-17. A transition phrase between paragraph 1 (l.16) and paragraph 2 is not existing. Please spell out a logical transition.

p.6, l. 51. “plant materials used in this study were safflower … obtained” something is missing here: seeds? Leafs? Complete plants?

p.6, l. 59. “Safflower plants with the same growth status” Please indicate the age/time here, for instance 5 weeks old… also, how long were the plants grown in the different light treatments? Please indicate!

p.6, l.63. Materials and Methods: please cite (or describe) here which RNA extraction protocol you used.

p.7, l.72. complete raw reads were eliminated? In most pipelines, only ends - both containing adaptors and/or low quality are trimmed from the sequences.

p.7, l.88. TGICL is quite an old tool therefore I suggest that they use a newer one, like BUSCO. Also, did they re-map the annotated data to the denovo transcriptome? this should be done to have an estimation of transcript representation of the transcriptome.

p.8, l.118. please indicate what type of spectrophotometer.

p.8, l.135. are the 78.8 Gb total? per sample? I guess total from Table 1. Please spell out.

p.9, paragraph functional annotation and classification. I would only mention the best annotation and swissprot and GO terms and include this in a phrase in the former paragraph. Not in a complete paragraph, IMO.

p.10, line 208-209. …two of them were significantly regulated by MeJA…. Please indicate which of the HMT genes were regulated in the text.

To include in the discussion:
How were the light treatments decided upon? The authors say: “The HL (high light) group
was grown under 40,000 Lux. The second group, ML (middle light), was grown under 20,000 Lux. The third group, LL (low light), was grown under 10,000 Lux.” Are this light conditions reflecting conditions at natural settings (which can vary between 2,000 Lux in the shadow and 120,000 Lux https://en.m.wikipedia.org/wiki/Daylight) ? Or rather reflecting illumination in greenhouses?

A discussion on the possibility of subfunctionalization of the HCT genes is missing, as HCT1 and HCT2 are upregulated in the HL condition (protective function), whereas HCT3 seems to be upregulated in the low light condition, a classical example of subfunctionalization.
It would be very informative to see the phylogenetic relationship among the three HTC genes, that seem to have undergone a subfunctionalization, as HCT1 and HCT2 are upregulated in the HL condition (protective function), whereas HCT3 seems to be upregulated in the low light condition, which might indicate a functional switch.

Suggestions for Figures and Tables:
Table 1: quality data should go into a supplementary table, so please move this Figure to the supplementals. Instead a Figure including the analyzed genes (HCT, FLS, ANS...) from the flavonin pathway in safflower would be useful.
Figure 3 and Figure 4 should be put together into one Figure.


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: TRANSCRIPTOME ANALYSIS OF FLAVONOID BIOSYNTHESIS IN SAFFLOWER FLOWERS GROWN UNDER DIFFERENT LIGHT INTENSITIES
Review round: 1
Reviewer: 2

Output:
Basic reporting: -Title it is not very appropriate. With a transcriptome analysis it is impossible to “measure” flavonoid accumulation, so “Transcriptome analysis of flavonoid accumulation” it is not correct.

-For a better understanding of a broad audience, introduction needs more background about the structure of the phenylpropanoid pathway and specially of the anthocyanin biosynthetic pathway (hereafter ABP) and how flavonoids compounds are synthesized. Maybe a figure it would be interesting.

-Information in Lines 11-16 are not very interesting for this study.

-Lines 17-24 describe extensively a previous work of the authors, however there are too much details of that paper that are unnecessary in the context of the present study. It can be resumed in this sentence:  “light factors played an important role in controlling the distribution boundary of safflower (Wu, 2017).”

-Lines 25-35 focus on the pharmacological effects of flavonoids of safflower however this is not the  aim of this study and it can be also resumed. In addition to the pharmacological effects it would be more interesting previous knowledge of the ABP in Carthamus: which enzymes are involved, which compounds are known, how are they synthesized, etc.

-It would be also important to include previous knowledge of the effect of light intensity and other environmental factors in flavonoid production or accumulation. Maybe there is no information for Carthamus, but there is in many other species.

-On the other hand, why do the authors use transcriptomics to answer their questions? Why is this methodology appropriate for their study? Maybe some phrases about this topic are needed.

-Figures and tables are of high quality and well labelled in general. However the description of some of them is not very accurate.

-Table S2 should be included in the main text (with some formatting), this information is one of the most important results of the paper. Or maybe a bars graphic with the expression values of those genes with differential expression.

-In Table 2, the full explanation of the N50 parameter it should be in M&M, in the table description only a short reminder.
- In Figures 3 & 4 it would be nice a larger font size for the y axis if possible. It would be helpful if the comparison (e.g. HLvsML) could be added above or below the figures.
- Figure 5: Since the actual fill of the bars does not give any information, it would be more informative to colour the bars by treatment (HL,ML and LL). Explain what the asterisks mean and which test was performed in the figure legend. In the figure legend, results should not be described.
- Figure 6: Since B and C are results from HPLC I suggest to put them together instead of A and B. On the other hand, instead of describing the results in the figure legend, describe which method was used for each quantification. As for Figure 5, explain what asterisks mean.

-Line 88: do you mean redundant?

-Lines 139-140: From which assembly come this results? From the new reference created? (line 86). Clarify this.

-Lines 168-171: This phrases should be in M&M. Describe what HCT, FLS and ANS means.

-Lines 180-181: I suppose that some tests were performed to test for differences in total flavonoid content but differences were not significant. Add this information in text.

-Lines 193-200: It would be interesting to know which enzymes were identified. There were DEG analyses in those papers? If so, it would be very interesting to know the results to compare with your results.

-Lines 207-210: Is this related with light intensity somehow? Explain

-In the discussion: How this new results fit with previous knowledge? How light intensity affects the synthesis of flavonoid in other species? Are these findings similar to previous results found?
Experimental design: -Lines 42-43: “To explore the relationship between light intensity and flavonoid accumulation in safflower flowers, transcriptome analysis was performed in this study”. Like a previous comment on the title, with a transcriptome analysis, flavonoid accumulation cannot be measured. The transcriptome experiment is useful to obtain the genes sequences and allows the digital expression analyses, but not the quantification of the flavonoids. For the quantification of flavonoids HPLC, the spectrophotometer or even photographs (in the case of anthocyanins) make it possible.

-Lines 46-47: “The correlation between gene expression and the flavonoid content was analyzed”. How was this analyzed? To correlate the expression analysis with the flavonoid content, those genes used for the expression analysis should be the responsible of the synthesis of those flavonoids quantified. This is true for the FLS and kaempferol but not for the HSYA.

-Line 60 specifies that 3 biological replicates were used for each treatment (I assume it is for the transcriptome experiment). However it is unclear how many libraries were finally constructed. The reader has to wait to results to see in table 1 that there was nine samples, three per treatment. In addition I can’t find how many replicates were used for the expression analysis with the RT-PCR or for the flavonoid quantification.

-Lines 85-86: I am not familiar with knowledge in Carthamus, however a quick search in genbank showed me that there is a genome available. Why did the authors performed a de novo assembly instead of using the genome as reference? I’m sure there are good reasons, however I think it should be justified. Another question arises from this lines, how was this new reference built? With all the libraries mixed? Or each sample was assembled independently and then a step of clusterization was performed? Clarify this.

-In general a better description of the methods should be made: which parameters were used when using the different programs (the default?, or customized ones?), which version of the software ( e.g line 87, line 97), how many replicates, etc. Or if authors are following a protocol from another paper it should be cited.

-Line 86: which program was used for read cleaning and filtering? Which criteria was used?

-Lines 86-91: I’m not sure to understand this paragraph. If TGICL was used to remove clusters of redundant transcripts and obtain unigenes, why unigenes were divided into two types: clusters and singletons? Rephrase or clarify this.

-Line 98: Specify RSEM version, and cite the authors of the software. DESeq2 is a package implemented in R? If so, this should be mentioned.

-Line 107: How were this genes selected and why? This should be described here, not in Results. How many samples and replicates? Is CHS the closest known enzyme that leads to the synthesis of HSYA? If so, why it hasn’t been included in the expression analyses?

-Line 114: “Data was analysed using SPSS” not “data were generated using SPSS”. Which tests were performed to find differences between treatments?

-Line 118: absorbance was measured at 255nm for total flavonoid content. Is this absorbance commonly used in Carthamus? Flavonols and flavons use to be measured at 300 or 350nm, and anthocyanins at 520nm. I find 255nm a bit low, however if it is correct please cite some literature.

-Lines 164-166: A figure with the digital expression values of these genes is essential (in adittion to statistical tests) to compare the digital expression values with the RT-PCR results.
Validity of the findings: - Please clarify if there are replicates in the expression and flavonoid quantification experiments
-How are the digital expression values within treatments? Are they regular?
-Lines 116-132: How authors test for differences between treatments? Which tests were used? There were replicates? How many?
-Lines 224-226: To say that CHS increases its substrate catalysis, CHS should be included in the expression analysis as well as the product (any chalcone) of its action in the flavonoid quatification. Why it wasn’t included?
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: TRANSCRIPTOME ANALYSIS OF FLAVONOID BIOSYNTHESIS IN SAFFLOWER FLOWERS GROWN UNDER DIFFERENT LIGHT INTENSITIES
Review round: 2
Reviewer: 1

Output:
Basic reporting: Many things are not clear and ambigous. See review.

References are missing to be able to better asses versions of software used, etc.

Article structure not very professional, Figure and Table captions are too brief to decipher the information, Quality of Figures is not good. Labeling in Figures too small to read.

Data are shared on NCBI and figshare. Figshare link is missing.

Yes, although the minimum was made and the importance of the transcriptome is not very clear.
Experimental design: As mentioned in the review, for qPCR the 2-ΔΔCT method (Livak & Schmittgen, 2001) might be inappropiate.
I think it’s a problem to use of 2 as the efficiency base, this assumes perfect binding of the primers to a base 2 amplification per cycle. Instead the formula is E-ΔΔCT, where the primer efficiency needs to be calculated as described in Medrano & Wong 2018.

See above.

Since light appears to be the essential factor in the experiment, information on the chamber type- lamps might be needed to replicate the experiments.
Validity of the findings: Corrections need to be made to increase the validity.
Additional comments: Dear authors,

Please find enclosed an extensive review of your re-submitted paper: Transcriptome analysis of flavonoid biosynthesis in safflower flowers grown under different light intensities. Most of these are formulated as questions, hopefully this helps.

Review:
Main text :

L. 18- 19. In this study, we aimed to explore the relationship between different greenhouse light intensities used for growth and

L. 13 : Please remove the strongly and replace by “ancient” or a more precise meaning, what do they mean ? « widely used » ? Strongly implies that there’s low genetic variability within the cultivar, which I think is not really the case.

L. 142 : Why did they not use the safflower genome at least to remap the transcriptome data to. Is there a logical explanation?

L. 156 : The information on the version of the different databases KEGG, SwissProt is missing !

L. 176 : The 2-ΔΔCT method (Livak & Schmittgen, 2001) was employed to analyze relative gene expressions.
I think it’s a problem to use of 2 as the efficiency base, this assumes perfect binding of the primers to a base 2 amplification per cycle. Instead the formula is E-ΔΔCT, where the efficiency needs to be calculated as described in Medrano & Wong 2018 (https://doi.org/10.2144/05391RV01)

L. 245, 287, 289 : Total flavonol contents are said to be higher, then to be lower and then to “peak”, this is not true, as total flavonoid contents do not display significant differences among the 3 conditions.

L. 291- 292 : « Therefore, we believe that the downregulation of HCT may have a positive effect on flavonoid accumulation.” Do they mean CtHCT3? Else the statement is not clear.

L. 302- 305. “Through the above experiments, we suggested that a decrease in light intensity led to the downregulation of HCT genes in safflower, then the downregulation of HCT genes resulted in the repression of lignin synthesis and increased substrate catalysis by CHS, promoting flavonoid accumulation.” But this is not shown in the experiments! ML promotes flavonoids and this is not what this statement implies.

Comments on Figures and Tables :
The captions are too brief. Many times more information is needed for readers that will only look at the Figures. For instance :

Table 2 » Unigene annotation and assembly statistics.” Can be completed with:  “for safflower de novo transcriptome assembly using Trinity v. 2.2.3”
The same is valid for all other Figures.

Fig. 1: “Proposed a part of flavonoid biosynthesis pathway in safflower”, showing enzymes involved in kaempferol and HCYA biosynthesis analyzed in this work. HCT= Hydroxi….; CHS= chalcone synthase; CHI=…; FLS=…..
At least the enzymes discussed in the main text and leading to the compounds of interest should be highlighted.

Fig. 2 is uninformative

Fig. 3” Statistic of differently expressed genes” obtained by DESeq2 (version X ) in safflower transcriptome subject to different light regimes HL= High light, ML=… LL=.

Fig. 4  Descriptions in Fig. are impossible to read as it stands. Maybe representation as Pie Charts, or other type of graphics allows for larger descriptions.

Fig. 5 Selected 7 DEGs related to flavonoid biosynthesis and their FPKM.
Change to “seven selected differential expressed genes from the transcriptome. Average FKPM values for biological replicates are shown…”.

Fig. 7: Spell out Hydroxysafflor yellow A then display abbreviation.

Table 1 can be moved to Supplementals

Table 2 Please cite the information on the version of the different databases KEGG, SwissProt,  and GO as a Figure foot.


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: TRANSCRIPTOME ANALYSIS OF FLAVONOID BIOSYNTHESIS IN SAFFLOWER FLOWERS GROWN UNDER DIFFERENT LIGHT INTENSITIES
Review round: 2
Reviewer: 2

Output:
Basic reporting: English must be improved.

L29: The previous version said that 13 DEGs were found to be related with flavonoid biosynthesis. However now are only 7. I think authors have focused their attention in 7, but 13 is the original number. Am I right? In any case, the result is 13 DEGs found, and this should be said not only in the abstract, also in the results section.

Introduction has been substantially improved in terms of background information. However I have some comments. I suggest some restructuring of phrases and paragraphs. Since first paragraph it is an introduction to safflower crop, I suggest to maybe finish it with the explanation of its distribution with something like this: “Previous studies on the effect of environmental factors on safflower distribution, showed that light was the most important factor explaining safflower distribution boundaries (Wu, 2017)”. And then I would start the second paragraph with the effects of light on plants (If this comment is considered, then phrases 53-59 should be deleted).

Phrases 59-66 should be better connected. The same words are repeated too frequently.

Phrases 64-65 “Numerous studies have shown that light intensity can influence the biosynthesis of flavonoid in other species (Deng et al., 2012; Pan et al., 2017;Wang et al., 2018)”. Describe how is this influence. Does light increase or decrease (or both) flavonoid biosynthesis? Maybe a review about this topic should be cited.

L80: “only a few genes have been identified in flavonoid biosynthesis pathway of safflower”. Describe somehow which genes have been identified.

L167: “Relative DEGs expression analysis” is not a very descriptive and clear title. Maybe something like “RT-PCR expression analysis of DEGs” is more clear for the reader.

L252-253: There is a relative correlation between kaempferol concentration and CtHCT3 expression, ok, but is there any other significant relationship? What about FLS expression and kaempferol concentration? If there are no other significant relationships this should be mentioned.

L259-263: Related with the number of genes identified with flavonoid biosynthesis, in the discussion authors said that other transcriptomes found 156, 22, and 44 unigenes or isoforms related with flavonoid biosynthesis. In this study 13 DEGs were found, however this comparison is not very useful because in this case only the DEGs have been reported and analysed. Do you have an estimation of how many genes related with flavonoid biosynthesis there are in this experiment not only the DEGs? If this information is available it would be nice to put it.

Figure 5: Title of the figure should be something like: “Expression levels of the DEGs related to flavonoid biosynthesis”. Although for the RT-PCR you have selected 7 DEGs, I think that this graphic should include the complete information, that is, the ecpression levels of the 13 DEGs.
Figure 6: Title should also be more descriptive: “Expression analysis by RT-PCR of seven flavonoid related genes  under different light intensities”. Be consistent with the fill of the bars across the manuscript. Figure 5 and 6 have different colors for conditions HL and LL. As previously suggested, since B and C are results from HPLC and specific flavonoids, I suggest to put them together instead of A and B. This way the reader can easily appreciate the difference in flavonoid concentration. In the description of the figure, instead of “ANOVA- Tukey test was used for this analysis . Some of the differences were marked in the figure (*P<0.05,**P<0.01)” something like “Results of ANOVA-Tukey tests are shown with asterisks (*P<0.05,**P<0.01)” sounds better.
Experimental design: No comment
Validity of the findings: No comment
Additional comments: MINOR COMMENTS

L17: ingredients → compounds
L17: There is a rare symbol in the word “effects”.
L23: by the DESeq2 method → With the DESeq2 package
L25-26: “Flavonoids accumulation…. by HPLC” → “Flavonoids accumulation…. by HPLC and spectrophotometer”
L27: Light intensity → light intensities
L35: confirmed →  suggest
L61: responds →respond
L67: Flavonoid is ubiquitous → Flavonoids are ubiquitous
L69: very clear → well known
L70: p-coumaroyl → P-coumaroyl
L71: Cite Figure 1 after “… phenylpropanoid compounds”.
L78: At the downstream → Downstream
L83: that the flavonoid in safflower → that flavonoids in safflower
L86: Hydroxysafflor yellow A → Specifically,  Hydroxysafflor yellow A
L92: ingredient → compound.
L92: Does exist a reference for Chinese Pharmacopoeia? If so, cite.
L143: Delete “After sequencing and read filtering”. Start the sentence with “Clean reads in each sample….”
L147: redundant→ redundant transcripts
L153-154: Move this paragraph to the end of this section
L221: were → are
L276: safflower growth better → safflower growth was better
L276: reached high level → reached the highest level
L296: might → might be
L314: Seven → Thirteen
L316: and their expressions under.. → and the expression of seven of them under..


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: TRANSCRIPTOME ANALYSIS OF FLAVONOID BIOSYNTHESIS IN SAFFLOWER FLOWERS GROWN UNDER DIFFERENT LIGHT INTENSITIES
Review round: 3
Reviewer: 1

Output:
Basic reporting: Professional english is used. Some minor grammatical errors should be corrected.
For instance, in the last phrase of the introduction: “ In this study, we aimed to explore the relationship between different light intensities and ﬂavonoid biosynthesis in safflower ﬂowers which cultivated in greenhouse.”  should be:  " we aimed to explore the relationship between  different light intensities and ﬂavonoid biosynthesis in safflower ﬂowers cultivated in the greenhouse."

I still miss an important reference of an article published this year on flavonoid biosynthesis in safflower: D. Guo et al. BMC Plant Biol. 2019 Aug 27;19(1):376. doi: 10.1186/s12870-019-1962-0. They discuss the role of CHI an enzyme upstream of the studied enzymes in this paper. It would be nice to see it referred to and possibly show the expression of at least CtCHI1, a nice control to their data.

Figures have captions labeled X- and Y- axis, this is not really standard. Normally the depicted relationships should be described.

The results partially support their hypotheses.
Experimental design: The research is original.

Research question was well defined.

Technical standard needs to be improved, it is difficult to see which is the control in the experiment- as you cannot grow a plant in absence of light. But maybe a "standard" growth from the literature?

They also didn't mention the sample size in each experimental group. Please take care of this.

Also, they didn't show the calculation of primer efficiencies and their answer to the critique point of the reviewer is unsatisfactory. They should show this "95%-105% efficiency" they claim to have in the supplementals.
Their response: "...The method you said should be more accurate, but when we did this part experiment, we generally used the easy-to-use 2 - ΔΔ CT method. Because the amplification efficiency of the target gene and the reference gene was preliminarily determined. The amplification efficiency of target gene and reference gene was close to 100%, and the efficiency deviation between them was within 5%. So we could achieve our goal by using 2 – ΔΔ CT method."

The statistical test is called “ANOVA” with a post-hoc “Tukey- Test” and not  ANOVA-Tukey as they call it in the paper. Please check that all assumptions are met, the Tukey test assumes that:
- Observations are independent within and among groups.
- The groups for each mean in the test are normally distributed.
- There is equal within-group variance across the groups associated with each mean in the test (homogeneity of variance).

The other methods seem accurate.
Validity of the findings: Correcting and regarding the technical details mentiones above will improve the paper. The underlying missing data should be provided (sample sizes, primer efficiencies, discussion over controls)

In the discussion (L.272-273) they state: "However, 104 Unigenes were related with flavonoid biosynthesis in this experiment and 13 DEGs related with flavonoids biosynthesis were not found in all above studies." The other studies were not on light intensities, so maybe the statements should relate to the novel results due to the experimental conditions.

Conclusions are well stated.
Additional comments: Dear authors,

The submitted paper “Transcriptome analysis of flavonoid accumulation in safflower flowers grown under different light intensities” is an transcriptome investigation of the flavonoid biosynthesis genes grown under the mentioned conditions. It is a novel aspect for Carthamus tinctorius and is therefore of interest to explore.

Most of the review points I stated above.

In addition to the comments above, please correct the conclusions (CORRECTIONS IN CAPITAL LETTERS):
13 NOVEL genes related to flavonoid biosynthesis were identified based on the functional annotation of differentially expressed genes, and the DIFFERENTIAL expressions of 7 of them under different light intensities were analySed by QRT-PCR. The concentrations of total flavonoid and two active compounds were measured in safflower flowers grown under 3 different LIGHT conditions.

These corrections will lead to a paper in publishing quality.


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 EFFECTS OF PHYLOGENY, BODY SIZE, AND LOCOMOTOR BEHAVIOR ON THE THREE-DIMENSIONAL SHAPE OF THE PELVIS IN EXTANT CARNIVORANS
Review round: 1
Reviewer: 1

Output:
Basic reporting: One issue I noticed was is in Table 1, where the list of taxa studied is accompanied by a listing of behavioral references.  That list really includes few primary sources of behavioral information, and instead cites quite a few other ecomorphology studies.  I think it is important to list more primary or secondary sources for those ecological attributes (i.e. behavioral studies or even mammalian species accounts).  For example, Nowak (1999, or even better 7th edition from 2005) is an appropriate source for most taxa without more detailed primary info, and Sunquist and Sunquist 2002 is a very appropriate source to list for ecology of the Felidae.  As a useful reference, Samuels et al. (2013) had a sample that included all species studied here, and the locomotion references listed in Appendix 1 of that study could easily be incorporated into Table 1 here.

Some of the reporting in the results would be clearer if rephrased and expanded.  In particular, in lines 162, 163, 164, 173, and 174 all describe principal components as separating taxa.  However, PCA does not really "separate" things, it creates multivariate axes that summarize primary sources of variation in the data set.  I think description of dispersal in morphospace or variation along PC axes would be best here, which is really what the authors have done in line 167.  For example, I would recommend the following for lines 162-163: "PC1 reflects variation in ischiopubic shape, and describes 31% of sample variation. PC2 relates to ilium width and orientation, and describes 23% of sample variation". For each PC axis discussed, I would recommend the authors be certain both the distribution of taxa in morphospace and morphological variation along the axis are clearly described.  For example, negative PC1 scores are seen in ursids, Ailurus, and Procyon, in addition to the only skunk in the sample Mephitis. For PC3, most canids have positive scores and most felids and herpestids have negative scores, and negative scores reflect a longer ischium, while positive scores reflect a longer ilium. For PC4, while Paradoxurus is most distant from other taxa in morphospace, there are other patterns worth mentioning; specifically, most canids and felids have positive PC4 scores, while Mephitis and most herpestids have negaive scores.

Similarly, in the PGLS results (lines 182-186), I would avoid saying the analysis "demonstrated that neither locomotor mode nor size had a significant effect on pelvis shape".  As written, I think that could be misinterpreted by readers, I would recommend rephrasing those sections (lines 183, 186) as "The PGLS model demonstrated that neither locomotor mode nor size had a significant effect on PC1..." and "The PGLS models demonstrated that locomotor mode did not have a significant impact on PC2 or PC3..., but size did have a significant effect on PC2 and PC3...".
Experimental design: The abstract (line 41) indicates "37 species of carnivorans" were studied, but both the methods and Table 1 list only 33 species.

In the methods (line 117) the authors indicate that data were collected on the "right os coxae of adult specimens", but later (lines 134-135) it indicates that "For specimens for which data were collected on a left os coxae...".  I would recommend simply indicated up from (lines 117-118) that data were preferentially collected for right os coxae, but some specimens required collection for left os coxae and subsequent mirroring of data.

Another important point in Table 1 is that Nandinia binotata is listed among the Viverridae, but that taxon has been placed in its own family (Nandiniidae) by various recent studies.  That arrangement is what was used by Nyakatura and Bininda-Emonds 2012, which the phylogeny used in this study is based upon.

The authors include 33 species (from 11 families) and analyze 6 locomotor groups.  However, among the sample studied there are only two groups (terrestrial, scansorial) with a relatively diverse set of taxa, and those two groups are the least specialized. The natatorial group has one species, the cursorial group includes 3 canids (which have been hypothesized to be ancestrally cursorial in Caninae) and one felid, and the semi-fossorial group has one mephitid and one herpestid.  I understand gathering the sort of data used here is challenging, but most of the phylogenetic groups used in this study are not that ecologically diverse (Table 1).  As such, I would wonder if the authors would really expect to see locomotion as a primary source of variation in the data set (principal components analysis) or to have much impact of locomotion in the PGLS analyses.
Validity of the findings: No Comment
Additional comments: The manuscript was well-written and interesting, the figures were of high quality, the tables were appropriate (but see comments above), and the text generally included a thorough inclusion of relevant literature.  A few other points to consider follow:

The results are compared to the findings of a number of other recent studies using 3D geometric morphometric methods (Martin-Serra et al. 2014).  While this study used a more detailed documentation of pelvis morphology (as indicated in the text) and sampled some additional families, I think it is important to point out that the Martin-Serra et al. 2014 study actually had a larger sample of species.  Not only were there more taxa, but the sample also had more locomotor specialists, including more cursorial canids and hyaenids, and also a more diverse sample of mustelids.

In the discussion (lines 235 - 246), the authors make comparison to some studies of primates and discuss orthograde postures.  I do think it is important to note that while ursids do not typically use orthograde postures (as stated in line 245), bears seem much more capable of those postures than do most other carnivorans.  While it may be speculation, pointing out that the family may have a pelvis well-suited for facultative use of orthograde postures.

The authors discuss the lack of evidence for morphological adaptations for locomotor habits in the pelvis of carnivorans, and how this may relate to constraint, I would like to see more discussion of other aspects of the postcranial skeleton as well.  Among mammals there seems to be a general pattern of more pronounced modification of more distal appendicular elements. That may be a consequence of developmental, structural, or some other evolutionary constraint, but it also may be related to how organisms actually move their bodies.  The distal elements of the body are the ones that interact with substrates and modification of distal elements can have profound impacts on mechanical advantage or velocity of limb movements, while not necessarily being so strongly linked to direct support of the body..


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 EFFECTS OF PHYLOGENY, BODY SIZE, AND LOCOMOTOR BEHAVIOR ON THE THREE-DIMENSIONAL SHAPE OF THE PELVIS IN EXTANT CARNIVORANS
Review round: 1
Reviewer: 2

Output:
Basic reporting: I think it is a rather good idea to use 3D surface reconstruction to study the overall shape of the pelvis in carnivorans. I think authors made quite a good job with their paper that could meet PeerJ journal standards pending several corrections that are suggested below.

Authors use clear and unambiguous, professional English and conform to professional standards

Relevant prior literature is appropriately referenced.

The structure of the article conforms to standard sections format.

Figures are relevant to the content of the article, of sufficient resolution, and appropriately described and labeled.

Appropriate raw data have been made available in accordance with PeerJ Data Sharing policy. Some should be added such as the origin of the specimens if known (captive or wild caught).

All results are relevant to the hypothesis.
Experimental design: This paper brings relevant results in the field of morphofunctional study of the post-cranial system in carnivorans. Ecological and behavioral stressors impact on the pelvis studies are starting to bring new understandings of the role of this poorly described girdle.

Nevertheless, methods could be described with more details (see specific comments). Data are properly shared to replicate.
Validity of the findings: The manuscript brings novel results on pelvis 3D shape in relationship with phylogeny, size, and locomotor behavior. The sample size is relevant for covering a large number of taxa but, unfortunately, there are quite a few numbers of specimens for each taxon. That brings a poor statistical reliability to assess the impact of locomotor behavior on the pelvis shape, especially because intraspecific variability is not considered. Furthermore, intra-individual variability is forgotten when authors built their study on the right side of the pelvis and switch to the left side for some specimens. The impact of the specimen sex on the overall shape of the pelvis, which could be important, was not studied either on the sample. See specific comments below.

Some topics are discussed by the authors,(e.g. captive origin of the specimens) without being stated in methods and results.

However, conclusions are well driven and linked to the original research question.
Additional comments: Title: I would suggest changing “locomotion” for “locomotor behavior”.

L 33, L 35, L 37 : Try to find a synonym for “previous” to avoid the repetition (4 times).

L 88-89 This has already been explained in the first paragraph.

L 100: change “Martín-Serra and colleagues (2014a)” to “Martin-Serra et al. (2014a)”

L 104: change “Martín-Serra and colleagues (2014a)” to “ Martin-Serra et al. (2014a)”

L 111: The sample is rather interesting with a lot of taxa but shows a rather poor number of specimens per taxon (half of the species are represented by one specimen). This might be a problem to evaluate the variation of the overall pelvis shape in relationship with phylogeny, body mass and locomotor behavior since the intraspecific variation is not taken in account. Furthermore, you do not mention the sex of the specimens. There might be a risk of intraspecific variation in the pelvis shape in relationship with the sex of the individual, especially in a group of mammals. How did you deal with this question?

L 117 : you mention that data were collected on the right os coxae and then, line 135, you mention that some data were collected on the left os coxea ? How many specimens are you talking about? Which specimens are they? I found these data neither in Table 1 nor in the raw data table. As far as I know, there are quite a few intra individual variations in the articular surfaces of the pelvis and we can assume that there might be an intra-individual variation between the right and left coxal bones. Did you test this difference? This should be explained in Methods section.
Refer to:

Pallandre, J. P., Cornette, R., Placide, M. A., Pelle, E., Lavenne, F., Abad, V., ... & Bels, V. L. (2019). Iliac auricular surface morphofunctional study in Felidae. Zoology, 125714.

L 216: change “Martín-Serra and colleagues’ “ to  “Martin-Serra et al. (2014a)”

L 220 : change  “forelimb by (Martín-Serra et al., 2014b)” to “forelimb by Martín-Serra et al. (2014b)”

L 242 : change “(e.g., Davis, 1964; (Martín-Serra et al., 2015)” to  “(Davis, 1964; Martín-Serra et al., 2015)”

L 243 : change “(Davis, 1964, p. 110)”.to “(Davis, 1964)”.
L 245-246: I would not be so directive with this conclusion. First, because ursids are known to practice bipedalism in a facultative way. Among the heaviest carnivorans species they are able to stand up and to step on hind limbs in several situations such as fighting, feeding, and threat posture…
Refer to :

Tuttle, R. H., Webb, D. M., Tuttle, N. I., & Baksh, M. (1992). Footprints and gaits of bipedal apes, bears, and barefoot people: perspectives on Pliocene tracks. Topics in primatology, 3, 221-242.

Zimmerman, A. M. (2013). Comparative Digital Examination of the Talocrural (ankle) Joint Provides Insight into Human bipedal locomotion (Doctoral dissertation, Case Western Reserve University).

Harcourt-Smith, W. E. (2015). Origin of bipedal locomotion. Handbook of paleoanthropology, 1919-1959.

Second, because ursids share, in different ways, common features with orthograde mammals (e.g. lumbar morphology).
Refer to :

Russo, G. A., & Williams, S. A. (2015). Giant pandas (Carnivora: Ailuropoda melanoleuca) and living hominoids converge on lumbar vertebral adaptations to orthograde trunk posture. Journal of human evolution, 88, 160-179.

Shapiro, L. J., & Russo, G. A. (2019). The Lumbar Spine. Spinal Evolution: Morphology, Function, and Pathology of the Spine in Hominoid Evolution, 51

L 249-250: “In addition, we used both captive and wild-reared animals in an effort to increase sample sizes“. I agree with this willingness to increase sample size but this was not mentioned in methods. This might be a bias in your study. Due to bone plasticity, because locomotor behavior and foraging strategy are dramatically reduced, captivity might impact the general pelvis shape in captive specimens. I think it would be worth it to mention how many specimens of the study are from wild or captive origin and to give the percentage of captive/wild caught in methods. You should mention this origin, when you know it, in your raw table too. You could then discuss the influence of this ratio on your results.
Refer to:

Hollister, N. (1917) Some effects of environment and habit on captive lions. Proceedings of the United States National Museum, 53, 177–193

O'REGAN, H. J., & Kitchener, A. C. (2005). The effects of captivity on the morphology of captive, domesticated and feral mammals. Mammal Review, 35(3‐4), 215-230.

Table 2
May be this is due to PDF building but landmarks (Ln) are not always in front of the definition. Can you check this please?

L2: “the opint in the center” should be “the point in the center”

C3: “to point #8” should be “to point 8”

Supplementary data:
The phylogenic tree word file is not really readable for me. Could you provide a more readable cladogram?


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 EFFECTS OF PHYLOGENY, BODY SIZE, AND LOCOMOTOR BEHAVIOR ON THE THREE-DIMENSIONAL SHAPE OF THE PELVIS IN EXTANT CARNIVORANS
Review round: 1
Reviewer: 3

Output:
Basic reporting: Raw data: The assumption here is that the data provided are the raw and unadjusted landmark configurations. However, that is not absolutely clear and could be made more explicit. If the landmarks provided are not the raw landmarks then the authors should also include a column for centroid size.
Experimental design: This study expands on multiple prior studies. However, most specifically it expands on the work of Martín-Serra et al. (2014) who sampled 11 families and ~ 53 carnivore genera. In this study Lawton et al. sample 10 families and 33 carnivore genera and the authors indicate in lines 104-108, that their methodology expands on this prior work by first capturing anatomical regions of the os coxae not previously sampled, and via the inclusion of three carnivore families not sampled by Martín-Serra et al. (2014) (herpestids, mephitids and viverrids). The inclusion of these additional anatomical regions as well as increased sampling across the Carnivora may make sense to those who explicitly study comparative carnivore skeletal morphology, but even in those cases that would require speculation by a reader familiar with the topic as to why these differences are important. The rationale for this study would be significantly strengthened by providing explanations as to why the additional anatomical data might be advantageous (i.e. capturing relevant regions of muscle attachment or joint articulation that are critical during locomotion) and why the inclusion of these families (improved sampling at the smaller body size range?) benefits the dataset and analyses.

In the methods, the authors state that they formulated species means of Procrustes coordinates. Can the authors clarify if they created species means when: 1. Multiple specimens for that species existed, 2. If by means, they meant that they produced mean landmark configurations for each species and 3. If they generated the means from the raw landmark data and then Procrustes transformed or if they Procrustes transformed and then generated the means (and explain why).

The authors state that they used centroid size as an estimate of overall body size, which makes logical sense since the os coxae is a major weight bearing skeletal component and larger taxa should have larger ox coxae (and centroid sizes for that structure) and vice a versa for smaller taxa. However, providing some/any evidence for the correlation between body size and pelvic size in mammals seems appropriate. Either from the literature or if for at least a small subset of these museum specimens for which body size data are available and correlations between cerroid size and a body size metric could be calculated.
Validity of the findings: no comment
Additional comments: This study os well thought out and orchestrated while being well placed in the context of existing research  on carnivore pelvic morphology and thus represents both meaningful replication of prior work as well as an expansion of that work. I enjoyed reading this well written article very much and think it will make a great addition to this body of work. Most of my comments are minor and strive primarily for explansion of both rationale and context by the authors with some clarification of the methodology.

Below is a final minor suggestions that I believe might make the manuscript more broadly accesible.

Starting at line 79 and elsewhere, there is an introduction of the concept of “high locomotor loads".  A brief explanation of what is meant by this term would be helpful.


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: MULTIDIMENSIONAL EVALUATION OF THE TRMM 3B43V7 SATELLITE-BASED PRECIPITATION PRODUCT IN MAINLAND CHINA FROM 1998–2016
Review round: 1
Reviewer: 1

Output:
Basic reporting: The paper with NO. peerj-40927 focused on the evaluation of TRMM 3B43V7 with ground observations over mainland China. This work was conducted based on three traditional metrics (R values, RMSE and RB). The paper is generally well written and the results are useful for the improvement of algorithms and applications of 3B43V7. However, the study is regular without enough novelty. I suggest a major modification. Here are some comments may be useful for the improvement of this paper.
Experimental design: The evaluation metrics were widely used and traditional. It is some kind too simple to provide enough evaluation information by using just R, RMSE and RB. The evaluation work was conducted at multiple scales which could provide useful reference for the readers of this journal.
Validity of the findings: The results are validated. However, the independence between rain gauges from CMA and rain gauges used for generating TRMM 3B43V7 product. In addition, some redundant sentences in the findings should be reduced to make the text be brief.
Additional comments: 1.	In the study, the grids including at least one rain gauge from TRMM 3B43V7 were extracted and compared with the ground-based rain gauges from CMA. However, part of these observed gauges from CMA have been incorporated in the generation of TRMM 3B43V7 as mentioned: “Some of the data from certain stations utilized in this present study could also be used for bias adjustment of the TRMM 3B43V7 product”. Therefore, the independence between rain gauges from CMA and rain gauges used in generating 3B43V7 should be analyzed in the dataset section. This is very important for the accuracy of the evaluation results. Otherwise, the evaluation results can hardly be to convince.
2.	The caption of figures and tables cannot be found in the text which make the review inconvinient.
3.	Some result sentences are repeated. For example, the authors analyzed results by repeating the statistical values (e.g. R values, RMSE values). For saving space, please remove the repeated sentences which can easily be obtained from the figures. For example: “The R values at annual, seasonal, and monthly timescales were 0.96, 0.95, and 0.93, respectively”; “The values from RMSE at annual, seasonal, and monthly timescales were 54.56 mm, 41.79 mm, and 34.86 mm, respectively”; “It is obvious that 3B43V7 had higher R values of 0.96, 0.92, 0.92, and 0.95 for spring, summer, autumn, and winter, respectively.”….
4.	Datasets: I suggest the authors improve the dataset section with great modifications by considering the following aspects:
(1)	Give a clear and detailed description for 3B43V7 datasets especially the generation processes and the correction process by using rain gauges.
(2)	More information on the benchmarked dataset (rain gauges from CMA) should be clearly described such as the background, the generation processes, the quality check processes, and the potential errors.
(3)	Independence analysis between rain gauge from CMA and rain gauges used in generating 3B43V7 should be added.
5.	The words should be well re-considered, like precipitation and rainfall. For example, in the text of the study area, the authors used “annual precipitation”, however, used “rainfall” in the legend of Fig. 1.
6.	English should be improved by native speaking people in this area.
7.	Lines 47-59: The sentence is not clear. Reword it.
8.	English problems: The English should be improved greatly with polishing by local speaking experts.
9.	Lines 60-61: what’s your evidence to conclude that “the TMPA precipitation estimate is considered to be one of the most reliable and widely-utilized products”?
10.	Line 63: why you evaluate the product again even though the TRMM had retired since several years ago?
11.	Lines 73-99: The authors left a large space for the previous studies on evaluation of TRMM products by listing the main findings. However, it is not clear and easy comparable enough for the readers. The following aspects should be improved for this part: (1) the historical studies should focus on the 3B43 product studied in this study rather than all products from TMPA algorithm (e.g. 3B42V7, 3B42V5, 3B42RTV7) as the performance for different versions of TRMM TMPA algorithm vary greatly due to its different inherent algorithms. In other words, the performances between different versions cannot be compared with the same standard. (2) Actually, the product TRMM 3B43V7 has been evaluated by using different methods over different regions (including mainland China, for example (Chen and Li, 2016)) compared with other satellite-based precipitation estimates or even improved by using different methods. The authors should pay more attention to these studies. To provide a better background knowledge, I suggest the authors compare these previous studies in one table with time, area, standard data, period, main findings, references. (3) after adding one previous study table, the novelty or at least the difference between the current study and the listed previous studies should be highlighted.
12.	The significance of this study is too general or not specific by just reading as “The results of the study can provide a basis for hydrological research and water resource management”. In addition, daily datasets may be more frequently used in hydrological research. Therefore, the significance of this evaluation work should be re-considered and highlighted with more specific applications like drought monitoring.
13.	Study area: Why did the authors divide the mainland China into 9 basins? The original purpose should be mentioned in the study area section. In addition, if the authors intend to use these 9 basins as evaluation sub-regions, the introduction of these basins should be added. For saving space, a table could be used here.
14.	Lines 132-133: I cannot agree with “This satellite mission obtained the longest data series of almost the total amount of global precipitation”. The data records of TRMM is not the longest one.
15.	The sentences are not accurate, the latest version of the TRMM TMPA algorithm is Version 7, while the version 7 products include different estimates like 3B42V7, 3B42RTV7, 3B43V7.
16.	Line 160: Based on the “relatively complete measurements were selected”, I can guess that part of the 620 gauges selected is not complete. The accurate standard for selecting gauges from 756 gauges should be given here. In addition, the quality check should also be mentioned like what is the type of rain gauges for measuring rainfall. Could the snowfall be also measured by the rain gauges?
17.	Line 194: remove the dot.
18.	Line 201: replace “higher” with “high”.
19.	Line 205: replace “(with RB of 14.0%)” by “(RB=14.0%)”
20.	Line 207: “This may be the result of snowfall, which is a factor that seriously affects the precipitation estimates made by satellites in North China (Chen & Li 2016).” Could the rain gauges from CMA capture snowfall? What kind of rain gauges used for capturing snowfall? The answer to these questions should be mentioned in the dataset section.
21.	Line 297: modify the title as “Evaluation of elevation-dependent error”
22.	Line 301-303: why only four classes used in the analysis? Is there any standard to split the elevation range into “<500 m, 500-1500 m, 1500-3000 m, and >3000 m”? I suggest the authors split elevation range into more number classes, which will be more useful for application users of 3B42V7.

References
Chen, F.R., Li, X., 2016. Evaluation of IMERG and TRMM 3B43 Monthly Precipitation Products over Mainland China. Remote Sensing, 8(6): 472. DOI:10.3390/rs8060472


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: MULTIDIMENSIONAL EVALUATION OF THE TRMM 3B43V7 SATELLITE-BASED PRECIPITATION PRODUCT IN MAINLAND CHINA FROM 1998–2016
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript needs some improvement in English. Generally, the manuscript organization is good. The figures and table are well made.
Experimental design: I suggest the authors to add a strong statistical metric, for example, Nash-Sutcliife Efficiency. The metrics the authors used in the manuscript are weak in terms measuring the performance of the TRMM data as well as pointing out the causes of the bias in the data.
Validity of the findings: The statistical tests needs to be supplemented by significance test results.
Additional comments: I have provided detail comments/changes and suggestions annotated with the pdf of the manuscript attached.


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: MULTIDIMENSIONAL EVALUATION OF THE TRMM 3B43V7 SATELLITE-BASED PRECIPITATION PRODUCT IN MAINLAND CHINA FROM 1998–2016
Review round: 2
Reviewer: 1

Output:
Basic reporting: The paper has been well improved.
Experimental design: The independence problem should be explained further.
Validity of the findings: None
Additional comments: The paper with NO. peerj-40927-v1 focused on the evaluation of TRMM 3B43V7 with ground observations over mainland China. The paper is improved after the first round modification. Most comments have been responded well except one. Therefore, I suggest a minor modification. Here are some comments may be useful for the improvement of this paper. The response for independence problem is not acceptable by listing some references or “discussed with several famous experts in this fields”.
The independence problem should be mentioned in the manuscript. I can understand that “the station name and station number cannot be obtained”, but you can obtain the grids contains “the number of sites in each box”. As we all know that the TRMM 3B43V7 is corrected based on GPCC at a monthly scale. In China mainland area, there is a possibility that the dependence could be high to make the validation results unstable. According to CMA, at least 160 international stations are distributed over mainland China. In my opinion, the authors could assume that all repeated grids containing gauges in GPCC are repeated with gauges you used as reference. In this case, if the 3B43V7 still have a consistent performance, then the authors could conclude that “the results are credibility”. Otherwise, the authors should explain the independence problem in the manuscript for the readers.


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: MULTIDIMENSIONAL EVALUATION OF THE TRMM 3B43V7 SATELLITE-BASED PRECIPITATION PRODUCT IN MAINLAND CHINA FROM 1998–2016
Review round: 2
Reviewer: 2

Output:
Basic reporting: The writing is much improved. Figures and Tables are organized and clear.
Experimental design: No further comments.
Validity of the findings: No further comments.
Additional comments: No additional comments


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

Input:
Title: UPREGULATED EXPRESSION OF PYRUVATE KINASE M2 MRNA PREDICTS POOR PROGNOSIS IN LUNG ADENOCARCINOMA
Review round: 1
Reviewer: 1

Output:
Basic reporting: The present manuscript provides useful information how PKM2 expression is important in lung cancer as a biomarker of prognosis and pathogenesis. Their skillful analysis of data from all subsets reveal its role in different clinical cohorts. These results are important for clinical oncologists who have been treating the patients with lung cancer.

The manuscript is well designed and written to convey the main message to the scientific community. Introduction, study design and data analysis sections systematically outline the study undertaken. They have discussed their results by performing a comparative assessment adequately. They have also investigated the data whether PKM2 mRNA expression was related to its methylation using MEXPRESS, which is a web-based and user-friendly tool for the visualization of TCGA gene expression to strengthen their conclusion.
Experimental design: In this manuscript, the authors have examined overexpression of PKM2 in lung adenocarcinoma (LUAD) using an online microarray database (ONCOMINE database) for mining PKM2 gene information, which is significantly associated with increasing tumor stage, lymph node metastasis, and the copy number variations, survival time. This study includes seven datasets such as Beer Lung, Selamat lung, Landi lung, Stearman lung, Hou lung, Okayama lung and Su lung to analyze PKM2 expression pattern in
LUAD. Further, the study demonstrates that DNA methylation and copy number variations regulate the expression of PKM2 in LUAD, which suggest that PKM2 can serve as a promising prognostic biomarker and potential therapeutic target against LUAD or other tumors.
Validity of the findings: The manuscript is well designed and written to convey the main message to the scientific community. Introduction, study design and data analysis sections systematically outline the study undertaken. They have discussed their results by performing a comparative assessment adequately. They have also investigated the data whether PKM2 mRNA expression was related to its methylation using MEXPRESS, which is a web-based and user-friendly tool for the visualization of TCGA gene expression to strengthen their conclusion.
Additional comments: The present study outlines the role of key cancer metabolism enzyme PKM2 in the disease pathogenesis and prognosis of lung adenocarcinoma to elucidate the prognostic value of PKM2 in LUAD based on integrated analysis of LUAD samples in different subsets. The study focuses on examining the association between PKM2 expression and clinical parameters, as well as prognostic values. whether PKM2 expression is related with the changes of PKM2 methylation and copy number variations (CNVs).

The authors should address following comments and revise the manuscript.
1.	How survival was defined in these subsets? Whether it was the time interval from the date of surgery to the date of death? In order to elucidate a possible correlation between PKM2 gene expression and the clinical outcome, please be selective to include the selected patients with survival>certain days (X number of days), indicating that the patient survived from the initial therapeutic intervention (surgery and radiation treatments).
2.	The Materials and Methods section should have a separate detailed description for Statistical Analysis including various statistical tools used in the data analysis.  To further delineate the role of PKM2 expression, the authors should provide more details to describe plots of the Kaplan–Meier analysis with appropriate sample size, which may provide the information on the length of survival, median survival time of the distinct sample populations, and significance of the difference between the survival curves.
3.	What were the common determinants among patients among seven subsets? The authors should include a paragraph in the Result section to describe Patient Characteristics and analyze the data for conveying more meaningful message.
4.	Introduction section (lines 86-90) and Discussion section (lines 213-215) summarize the tumor biology of PKM2, its role as diagnostic and prognostic biomarker of the pathogenesis. But, it lacks recently published information by other investigators for its role as either diagnostic or prognostic marker at subcellular levels in a variety of pre-clinical animal models of human cancers including small cell and non-small cell lung cancer. The authors should run a comparative analysis of their results with these published data for a clearer interpretation.

5.	The authors should increase the font size of figure title, X and y-axes legends of Figure 2 A-F, Figure 3 A-C, Figure 4 for better readability.


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: UPREGULATED EXPRESSION OF PYRUVATE KINASE M2 MRNA PREDICTS POOR PROGNOSIS IN LUNG ADENOCARCINOMA
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Title:
Upregulated expression of pyruvate kinase M2 predicts poor prognosis in lung adenocarcinoma

Comments:

1.	The term of “mRNA” should be added to the title of figure 2 and 3, and modified as following:
Figure 2 Box plots showing expression of PKM2 mRNA in LUAC based on clinical parameters.
Figure 3 Survive curves evaluating the prognostic value of PKM2 mRNA expression in LUAC.

2.	Figure 2C: Expression of PKM2 in LUAD based on patient’s race.
Comment:
Most of the patients in this study were the Caucasian (N=387). Therefore, the title of this paper should be modified as: Upregulated expression of pyruvate kinase M2 predicts poor prognosis in the Caucasian lung adenocarcinoma
P.S. Asian, N=8; African-American, N= 51.

3.	Figure 3 Survive curves evaluating the prognostic value of PKM2 expression in LUAC.
Question:
3.1 How your determined the mRNA threshold of PKM2 to tell the difference between the high and low expression groups? And what was the rationale?
3.2 We would like to know how many percentage of lung adenocarcinoma patients were with high expression of PKM2 mRNA in this study.

4.	Figure 4B The relationship between PKM2 expression and its methylation levels in promoter
Figure 4C The relationship between PKM2 expression and its methylation levels in genebody.
Suggestion:
4.1 The labelling of Y axis should be modified from “methylation” to “DNA methylation level”. For X-axis labelling, please modified from “expression” to “relative mRNA expression of promotor” or “relative mRNA expression of genebody”
4.2 The correlation Coefficients (r) of figure 4B and 4C were 0.171 and 0.280 respectively. Please show the p value of each figure for us.
4.3 The significance of hypo-methylation of PKM2 in genebody region should be explored more extensively in the section of discussion if p value of figure 4C less than 0.05.
4.4 What is percentage of hypo-methylation of PKM2 in promoter or genebody of lung adenocarcinoma patients in your study?

5.	Figure 4C The Pie char was plotted for showing PKM2 CNVs changs in LUAC samples. The value of diploid normal copy (0) was 27% (231/861).
5.1	There was a wrong spelling, i.e. changs, please correct it as changes.
5.2	The figures shown in line 253~255 were--, CNG of PKM2 was observed in 49 (5.69%) patients, and CNL in 236 (27.41%) patients. It was not match to the labelling of pie chart as shown in figure 4C. Please clarify the mismatch figures in pie char and text.

6.	 Line240~241, --, hypomethylation of PKM2 in genebody region might be strongly correlated with overexpressed.
Line 254~255, Over-expressed PKM2 is strongly correlated with its CNVs (Fig.
255 4A, r=0.341, p<0.0001),
Question
Both methylation and CNVs had effect on PKM2 expression of lung adenocarcinoma patients as stated by the authors. We would like to which one is more important? Would you please do a couple of comments in the section of discussion?

7.	Did the PKM2 can be detected by immuno-histochemical (IHC) method? What is percentage of high expression of PKM2 protein of lung adenocarcinoma patients in this study?

8.	The figure of Inverse correlation between DNA methylation(Y axis) and relative mRNA expression (X axis) of promotor or genebody were shown in Figure 4B and C. Please show another figure with Inverse correlation between DNA methylation(Y axis) and protein expression (X axis), if PKM2 protein could be detected by immune-histochemical method.

9.	P166, 166 PKM2 was aslo identified to be positively correlated with lymph node metastasis (Fig. 2B)
Suggestion: wrong spelling, asloalso

10.	Figure 2 Box plots showing expression of PKM2 in LUAC based on clinical parameters. The mRNA levels of PKM2 in patients with stage II and III were apparently higher than that in patients with stage I (Fig. 2A).
Over-expressed PKM2 was also identified to be positively correlated with lymph node metastasis (Fig. 2B).
Fig. 3, Kaplan–Meier plot revealed that over-expressed PKM2 was significantly associated with worse OS.
Comment:
The TNM staging is still one of powerful survival predictive factors, therefor, the power of PKM2 should be tested in the same TNM stage, or been analyzed with multi-variable Cox regression method. Please show the survival curve of high and low expression of PKM2 of lung adenocarcinoma patients within the same stage.

11.	L82~84, Pyruvate kinase (PKM2), a key metabolic enzyme for the last rate-limiting step of glycolysis, catalyzes the transfer of a phosphoryl group from phosphoenolpyruvate to ADP, generating ATP and pyruvate.
L236~238, Importantly, nine abnormal methylation sites, including cg19687230, cg00635674, cg27032813, 237 cg07365018, cg22234930, g24327132, cg05888487, cg23160336 and cg09651136, were238 identified to be negatively related with PKM2 expression in LUAC.
Comment
The PKM2 was treated as oncogene as authors stated, I just wonder what will be happened if PKM2 were hyper-methylation? Have you ever tried to use Gene Ontology (GO) to clarify what biological process will be altered if PKM2 were hyper-methylation?


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: UPREGULATED EXPRESSION OF PYRUVATE KINASE M2 MRNA PREDICTS POOR PROGNOSIS IN LUNG ADENOCARCINOMA
Review round: 2
Reviewer: 1

Output:
Basic reporting: The present manuscript focuses on a potential role of PKM2 expression as a biomarker of disease severity and prognosis of lung cancer subjects. Their skillful analysis of data from all subsets reveal its role in different clinical cohorts. These results are important for clinical oncologists who have been treating the patients with lung cancer.

The manuscript is well designed and written to convey the main message to the scientific community. The authors have presented their results adequately and incorporated the changes recommended by the reviewers.

I have observed spelling mistakes at certain places, which I have listed in the section "General Comments for Authors" . I have also noticed that there are few key references missing that are pertinent to the study are not covered yet for a comparative assessment of their data.
Experimental design: The authors have improved the experimental design by incorporating the comments and suggestions made by the reviewers.

I have no additional comments.
Validity of the findings: The quality of revised version of the manuscript is improved and conveys the main message to the scientific community adequately. Introduction, study design and data analysis sections systematically outline the study undertaken. They have also investigated the data whether PKM2 mRNA expression was related to its methylation using MEXPRESS, which is a web-based and user-friendly tool for the visualization of TCGA gene expression to strengthen their conclusion.

I noticed that there is a room to introduce some recent references published by other scientists, which they can discuss on a comparative assessment platform for conveying their outcome precisely.
Additional comments: The revised manuscript is improved for its readability and quality. The authors should fix spelling mistakes still uncorrected in lines 193, 195, 265 and 267. It will be better to get the manuscript be reviewed by a English speaking person for narration and grammatical errors.

The manuscript is still missing a comparative assessment of their results with pertinent key references published in the recent past. The authors should run a literature search with appropriate key words and incorporate in the revised manuscript. This is still missing.  A few representative references are listed for their review and placing at appropriate sections in the manuscript.  They may find more upon searching in literature search engines.
1.	Guo et al.  The prognostic value of PKM2 and its correlation with tumour cell PD-L1 in lung adenocarcinoma., BMC Cancer.   2019, 19(1), 289
2.	  Yang et al. Cytosolic PKM2 stabilizes mutant EGFR protein expression through regulating HSP90-EGFR association. Oncogene 2016;35: 3387-98.
3.	  Gines et al. Subcellular Localization Is Involved in Oxaliplatin Resistance Acquisition in HT29 Human Colorectal Cancer Cell Lines. PLoS One 2015;10: e0123830.
4.	  Li et al. Nuclear PKM2 contributes to gefitinib resistance via upregulation of STAT3 activation in colorectal cancer. Sci Rep 2015;5: 16082.
5.	  Suzuki et al. Subcellular compartmentalization of PKM2 identifies anti-PKM2 therapy response in vitro and in vivo mouse model of human non-small-cell lung cancer. PLoS One, 2019, 14(5), e0217131
6.   Stone et al. Loss of pyruvate kinase M2 limits growth and triggers innate immune signaling in endothelial cells Nat Commun. 2018; 9(1): 4077
7.   Goldberg MS, Sharp PA. Pyruvate kinase M2-specific siRNA induces apoptosis and tumor regression. J Exp Med 2012;209: 217-24.
8. Li et al. PKM2 and ACVR 1C are prognostic markers for poor prognosis of gallbladder cancer. Clin Transl Oncol 2014;16: 200-7.
9.  Zhou et al. Role of isoenzyme M2 of pyruvate kinase in urothelial tumorigenesis. Oncotarget 2016;7: 23947-60.


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: UPREGULATED EXPRESSION OF PYRUVATE KINASE M2 MRNA PREDICTS POOR PROGNOSIS IN LUNG ADENOCARCINOMA
Review round: 2
Reviewer: 2

Output:
Basic reporting: see below
Experimental design: see below
Validity of the findings: see below
Additional comments: Title:
Upregulated expression of pyruvate kinase M2 predicts poor prognosis in lung adenocarcinoma.

Comments:
1.	Title: Upregulated expression of pyruvate kinase M2 predicts poor prognosis in lung adenocarcinoma.
Suggestion:
Upregulated expression of pyruvate kinase M2 mRNA predicts poor prognosis in lung adenocarcinoma.
2.	P249-252, Thus, the discovery of DNA methylation biomarkers might be a promising approach to improve the early diagnosis. Currently, some DNA methylation signatures have been identified to help improving early detection and predicting the prognosis in LUAC.
Comment:
From my point of view, for clinical application, immunohistochemical staining of PKM2 is more convenient for early diagnosis. Therefore, methylation signatures were not a convenient tool for the early detection and predicting the prognosis in LUICA. So, lack of immunohistochemical stain of PKM2 should be listed as one of limitation of this study.
3.	P256-257, ---, Importantly, 257 nine abnormal methylationsites, including—
Suggestion
A space is needed between the methylation and site
---, Importantly, 257 nine abnormal methylation sites, including—


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: SINGLE NUCLEOTIDE POLYMORPHISM OF MTHFR RS1801133 ASSOCIATED WITH ELEVATED HCY LEVELS AFFECTS SUSCEPTIBILITY TO CEREBRAL SMALL VESSEL DISEASE
Review round: 1
Reviewer: 1

Output:
Basic reporting: Previous studies have correlated MTHFR SNP causing C677T mutation with cerebral small vascular disease and high plasma homocysteine levels. In this study, authors validate correlations by measuring and comparing blood total homocysteine levels in a case-control study where patients selected for the cohort were screened by CSVD inclusion criteria. Therefore, this study overcomes the limitation of prior studies in direct measurement of tHCY levels as a causal factor for CSVD. The manuscript is written in a clear and scientific manner.However, there are few minor suggestions that would make it even better. They are:

1) Line 60: Shi and Wardlaw, 2016 instead of Shi & Wardlaw.
2) Brief mention of why 2:1 ratio was selected for control people: case people.
3) Name the analyzer used for blood Hcy assay.
4) Please clarify that the values in table 4 are tHCY levels or serum IFN-gamma levels because readout from plasma tHCY enzymatic cycling assay should be tHCY levels. Inflammatory cytokine levels are used as indirect measurement.
5) Present Table 2 in better format. In current format, it is somewhat difficult to read and understand it.
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: SINGLE NUCLEOTIDE POLYMORPHISM OF MTHFR RS1801133 ASSOCIATED WITH ELEVATED HCY LEVELS AFFECTS SUSCEPTIBILITY TO CEREBRAL SMALL VESSEL DISEASE
Review round: 1
Reviewer: 2

Output:
Basic reporting: This is a very interesting manuscript investigating the contribution of several SNPs of MTHFR gene to both Hcy levels and susceptibility to cerebral small vessel disease. The authors observed association of rs1801133 with increased risk for CSVD, and the same polymorphism showed a tendency for increased hcy levels. The article is written in good English and professionally structured, but important references have been missed, so that I have some comments to improve its quality (see the next points).
Experimental design: In the introduction (lines 80-81), the authors state "However, the relationship among the MTHFR gene polymorphisms, Hcy, and CSVD has not been investigated". This is not actually true: see for example (a) Increased total homocysteine level is associated with clinical status and severity of white matter changes in symptomatic patients with subcortical small vessel disease. Clin Neurol Neurosurg. 2011 Nov;113(9):711-5. (b) Homocysteine, small-vessel disease, and atherosclerosis: an MRI study of 825 stroke patients. Neurology. 2014 Aug 19;83(8):695-701. (c) Association of MTHFR C677T Genotype With Ischemic Stroke Is Confined to Cerebral Small Vessel Disease Subtype. Stroke. 2016 Mar;47(3):646-51. and many similar articles in the field. The authors must therefore put their research in the context of the available literature in the field, without claiming as original their research hypothesis. Both introduction and discussion should be rewritten in light of the available literature in the field, and particularly their discussion should include and properly discuss previous evidence in the field.
Validity of the findings: 1) Univariate analysis demonstrated that the genotype frequencies of rs1801133 (χ2=12.852, P=0.002) and rs1801131 (χ2=6.203, P=0.045) were statistically different between the case group and the control group, but the multivariate analysis only confirmed association for rs181133. It is well known from the literature that rs1801133 and rs1801131are in strong linkage, giving rise to six common genotype combinations and 3 rare combinations (See the article by Ulvik et al. Hum Genet (2007) 121:57–64). In the same article it was also suggested that the effects on circulating Hcy levels are the result of the combined genotypes. I therefore invite the authors to investigate the haplotypes and the combined genotypes for MTHFR 677C>T (rs1801133) and 1298A>C (rs1801131), and clarify if they are different between patients and controls, and their joint effect on Hcy levels.
Additional comments: Please consider my suggestions in order to improve your manuscript and add originality to your investigation


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: SINGLE NUCLEOTIDE POLYMORPHISM OF MTHFR RS1801133 ASSOCIATED WITH ELEVATED HCY LEVELS AFFECTS SUSCEPTIBILITY TO CEREBRAL SMALL VESSEL DISEASE
Review round: 1
Reviewer: 3

Output:
Basic reporting: Although the authors report an interesting study, the language used in the manuscript should be revised to ensure better comprehension. The introduction and discussion section lacks cohesion and needs to be revised to avoid possible confusion. Some examples where the language could be improved includes lines 89, 95, 96, 164, 218, 219.

The statement made in lines 64, 65 should be supported by citing appropriate literature.
Experimental design: The authors have mentioned the inclusion and exclusion criteria used for selecting the data they have used for the analysis. These criteria should be discussed in more detail and the authors must explain the reason for the choice of criterion. If available authors must report studies from literature that have used these criteria. This will help validate the results.
Validity of the findings: In the conclusion section, the authors merely, state the result obtained during their analysis. However, no attempt has been made to link the result to potential mechanism of association of the MTHFR rs1801133 SNP with the elevated Hcy levels. Also, the conclusion section is very brief. The authors should address the potential limitation of the study by including the risks/limitations of reaching a conclusion based on analysis of a small data set. How does the size of the data set affect the validity of the findings? Addressing this will make the manuscript more transparent.
Additional comments: In the submitted manuscript titled “Single nucleotide polymorphism of MTHFR rs1801133 associated with elevated Hcy levels affects susceptibility to cerebral small vessel disease” the authors employ standard statistical approaches to find a correlation between SNP of MTHFR rs1801133 and elevated levels of Hcy. Populations with elevated Hcy levels are susceptible to cerebral small vessel disease and the results presented in the manuscript could provide insights into identifying possible markers for the detection of CSVD susceptible populations.
Overall, this is an interesting study, but above mentioned issues/comments need to be addressed before being considered for publication in PeerJ.


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: SINGLE NUCLEOTIDE POLYMORPHISM OF MTHFR RS1801133 ASSOCIATED WITH ELEVATED HCY LEVELS AFFECTS SUSCEPTIBILITY TO CEREBRAL SMALL VESSEL DISEASE
Review round: 2
Reviewer: 1

Output:
Basic reporting: A. The authors haven't addressed comments shared by all reviewers regarding correction of grammar errors in the manuscript. I have tried to make some corrections in the "tracked changes" document that I will forward to the editor to share further.

B. Authors have not provided any table in the revised document for review.

C. The explanation regarding sample size ratio has not been added to the revised manuscript. Kindly add the explanation, including how it statistically improves efficiency of the study.

D. Table 2 still has some formatting issues.
Experimental design: No comment.
Validity of the findings: Discussion and conclusion still need to be revised to make it into a stronger manuscript.
Additional comments: The manuscript looks better after the changes that were earlier suggested have been incorporated. Kindly look into some more suggestions that have been added to further improve your 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: SINGLE NUCLEOTIDE POLYMORPHISM OF MTHFR RS1801133 ASSOCIATED WITH ELEVATED HCY LEVELS AFFECTS SUSCEPTIBILITY TO CEREBRAL SMALL VESSEL DISEASE
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors made only cosmetic changes to the manuscript, but failed to address my previous concerns.
Experimental design: The authors included the suggested references.
Validity of the findings: I previously suggested to explore the joint effect of the studied polymorphisms to Hcy levels, the existence of linkage disequilibrium and the distribution of haplotypes. This was not done.

The authors reply was "Thanks for your comments. Your recommendation is very instructive. We will explore their joint effects on CSVD susceptibility and Hcy levels in the next manuscript which we plan to submit to Peer J, too".

They shall do it in the present manuscript.
Additional comments: Please address haplotype distribution and the joint effect contribution of the studied polymorphisms in the present study


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

Input:
Title: ENDOPLASMIC RETICULUM STRESS AND THE PROTEIN DEGRADATION SYSTEM IN OPHTHALMIC DISEASES
Review round: 1
Reviewer: 1

Output:
Basic reporting: Song et al. reviewed the literature about ER stress and protein degradation system associated ocular diseases. It is a quite complicated topic, the authors managed to put related information together and draw a clear conclusion.
Experimental design: Well designed.
Validity of the findings: Cite related literature and structure related information together.
Additional comments: In general, the authors tried to collect all related background information and organise these information and present the data in a clear way. However the whole structure needs some changes, though I understand this is a quite broad and complicated topic. I would suggest the authors present the all related background information first, for example the sections of 'Understanding the function of the degradation system during ERS' and 'ERS and ub-proetasome response'  need to be ahead of other section which describe the involvement of ocular disease. There are a lot of small errors, particularly the citation of references in the main text.


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: ENDOPLASMIC RETICULUM STRESS AND THE PROTEIN DEGRADATION SYSTEM IN OPHTHALMIC DISEASES
Review round: 1
Reviewer: 2

Output:
Basic reporting: In this review, Song et al. describe the relationship and cross talk between two important cellular events, ER stress and protein degradation. They describe in detail how protein misfolding in the ER triggers a series of adaptive signalling cascades which extends beyond the ER and is aimed at restoration of cellular proteostasis and homeostasis.
Whilst some recent reviews have highlighted the roles of the UPR effectors and the protein degradation pathways in eye diseases  (see for example Kroeger H et al., FEBS J, 2019; Chan P et al., Brain Res, 2016; Chai P et al., Int J Biol Sci 2016; Zhu and Du, Int J Ophthalmol, 2018), Song et al., draw attention to the specific roles and interrelationship between ER stress, the UPR and the cellular protein degradation machineries in the pathology of several ocular diseases.
The review therefore addresses an important and rapidly evolving area of research and would be of broad interest.
Experimental design: Song et al., appear to have extensively researched the subject matter, and show a good grasp of existing literature, past and present.
The sources are properly cited, save for what could be an editing error; some references are just inserted in the text and some in superscript. That said, the authors rely too heavily on citing review articles.
In terms of organisation, large parts of the review appear to be disjointed; some subsections and paragraphs are introduced without context. Overall, the structural organisation could be clearer and logical.
Validity of the findings: Song et al., have presented a well-researched review of the literature in a rapidly developing area of research. However, the authors could do more to highlight areas and direction of future research and perhaps suggest how our understanding of the interplay between ERS and protein degradation pathways could impact our understanding of ophthalmology.
Additional comments: Abstract
The first sentence should be rephrased, ‘cause of the occurrence’ in line 32

Introduction
In general, the introduction is well written and succinctly describes the subject matter. It also clearly addresses the set objectives and why the review would be of general interest.

The authors should seek a better word/phrase to replace ‘broken’ in line 54.
The authors should replace ‘chamber’ in line 63, with lumen
‘Two pathways associated with…’ line 66-67, should be a new paragraph
Line 95…’that are generally expressed in cells’ do the authors mean ubiquitous expression? This sentence should be rephrased
Line 98…’Studies have found…’ should read another study have found….
Line 99 should be … levels of spliced XBP1
Line 100-102, is same study referenced in line 100, so sentence should be rephrased to reflect this
Line 110 should read ‘a transgenic rat model…’
Line 115…should read ‘activated ATF6 translocates to the nucleus…’
Line 116 - 117…should read ‘and activate gene transcription of proteins including ER chaperones that increase ER protein folding….’
Lines 126 – 128…please rephrase sentence
Line 135 – 136…should read ‘…cytoplasmic domain of ATF6 translocates’
Line 137…’Our study…’  Not sure what study is referred to here
Lines 145 – 150…rephrase to show logic and avoid repetitions
Line 152…UPS is a kind of compound….?
Line 156…’it is involved in…’ what is?
Line 159… pls use more appropriate references
Line 179…’rhodopsin T17M mutant’ is sufficient
Line 184…axa?
Line 196 – 199…sentence too complex, please rephrase
Line 212…’misalignment of the ER membrane’ do the authors mean retrotranslocation to the cytosol?
Line 217…’mannan’ do the authors mean mannose?
Line 217… authors should include also describe briefly process of retrotranslocation of misfolded proteins to conclude this paragraph. The description of the ubiquitin enzymes should form a new paragraph. Perhaps a brief introduction to the process of ubiquitination would be appropriate to start this new paragraph, with brief mention of the specific roles of the E1, E2 and E3 enzymes, and how process primes misfolded/unfolded proteins for degradation by the 26S proteasome
Line 220 – 226…authors should Make a very clear distinction between the yeast and human E3 uniquitin ligases. The sentences could be clearer and more coherent.
Line 226 – 228…the link between the UPR effectors and the UPS needs to be better presented
Line 231…’Researchers have found’…could be replaced by ‘Studies have shown…’
Line 250…’ubiquitous life phenomenon’… authors should please rephrase
Line 262 – 264…’…and are endogenous mechanisms…’ please clarify
Line 277 – 280…sentence too complex, please check punctuations and simplify
Line 281…’researchers have…’ perhaps could be replaced with ‘Studies have shown…’
Line 296 – 298…Please rephrase sentence and include sources/references
Line 317…’we found that…’ could be replaced with ‘It was reported that/It has been shown that….’
Line 326 - 329…. Please rephrase sentence, use an active voice…. ‘The MAPK pathway activates ERK…’
Line 333…Please rephrase sentence
Line 335…’more and more…’ could be replaced with ‘increasing number…’
Line 340…. avoid repetition.  Autophosphorylation of IRE1 occurs on the cytoplasmic region
Line 358…’Then…’ could be replaced by ‘Thus…’
Line 366 – 368…. Please rephrase the sentence
Line 368…the word death, could be replaced by the phrase cell death
Line 370….’one study…’ could be replaced It was shown
Line 381…please rephrase sentence
Line 386…’The latest discovery…’ could be replaced by ‘ A recent study…”
Line 399 – 404…please rephrase and simplify sentence
Line 405…’…between these 2 systems…’ please specify what systems you refer to
Line 413…’…managing the health of the cell…

Figure legends should be provided - these should succinctly explain the figures.


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: ENDOPLASMIC RETICULUM STRESS AND THE PROTEIN DEGRADATION SYSTEM IN OPHTHALMIC DISEASES
Review round: 1
Reviewer: 3

Output:
Basic reporting: The review outlines the response of cells to stress by exploring endoplasmic reticulum stress and protein degradation systems.  It deals with the complexity of the regulation of the systems involved and the role of other proteins in mediating or suppressing the activity of the protein degradation system.

There are some minor aspects that require attention including, lack of standard referencing with superscript used in a number of places, lack of space between the full-stop and the start of a new sentence, and the explanation of all acronyms when they are first used.

Line 81: should this read endoplasmic reticulum stress, autophagy and/or ubiquitin?

Use of casual language is also evident Line 54 ‘ER is broken’, line 95 ‘genes that are generally expressed’. In response to what stimulus?

Other aspects which are more significant to address include the study design and the conclusions drawn from the review.
Experimental design: The article content is within the Aims and sscope of the Journal.  The authors explain the methodology used as a review of academic articles.  If this has been chosen as the methodology it is more usual to put much greater detail in the text and include keywords used, number of articles identified and number of articles used for the review.  This can then be regarded as a systematic review.  If this is a narrative review then there is little merit in proposing a specific methodological approach as it is not possible for the reader to scrutinize the method and to be confident that the methods were comprehensive and unbiased.
Validity of the findings: The authors state in line 81 and 82 that, we propose a future research direction based on the articles.  In the conclusion the authors state that. We should strive to inhibit diseases by simultaneously regulating autophagy and Ub-proteosome in order to achieve cellular homeostasis.  There is clearly merit in this approach in some specific instances but the authors themselves comment on the varied role that activation of the systems have in various ophthalmic diseases (see lines 178 – 182) so this statement should be qualified with a more specific statement regarding the proposed future direction.  In reality the whole system is driven by the ‘stress’ and it would be worthwhile considering how to reduce the stress rather that modify the ‘stress response’.
Additional comments: This is an interesting area and one that has worthwhile content. The authors have discussed complex cellular regulatory pathways relating to the stress response and have focussed this on the endoplamic reticulum, ubiquitin-proteosome and autophagy pathways. However, in its present form it is quite unwieldy and the authors should consider a more structured approach to the different section as there is currently a lot of overlap.  It may be appropriate to consider a more substantial section on the introduction and regulation of the 3 systems and then deal with some of the ophthalmic diseases in a more deliberate way rather than weave them into the regulatory description of the endoplasmic reticulum stress pathways The complexity is covered well but would benefit from further scrutiny of the language used, the formatting of the sections to reduce the overlap and the consolidation of the ways in which the various ophthalmic conditions are identified to facilitate engagement with the text.

  .