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

Input:
Title: TARGETED METABOLOMICS STUDY OF SERUM BILE ACID PROFILE IN PATIENTS WITH END-STAGE RENAL DISEASE UNDERGOING HEMODIALYSIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Li et al., present a manuscript describing the serum bile acid profile in patients with end-stage renal disease. While these patients are known to have elevated total bile acids, this is the first report describing the specific bile acid species that are altered in disease. Moreover, the authors propose that these alterations may be due to the known disturbances in the gut microbiota. The manuscript is generally well written with the exception of some typographical errors and confusing phrases throughout.

Typographical errors throughout: For example:
line 28 “the” should be “The”,
line 59 “secret” should be “secrete”,
line 94, “were” should be “was”.

Line 68 “G protein-coupled receptor” should be “G protein-coupled bile acid receptor (GPBAR)” and GPBAR subsequently.
Line 195: it is unclear what “change rules” means.
Line 208: “variables” should be “variance”
Experimental design: The introduction and background are sufficient to orient the reader to the question being asked, and clearly states the purpose of the work.  However, further introduction of current diagnostic methods for ESRD, and how this work may fill a diagnostic need and gap in knowledge should be addressed.

The authors present subgroup analysis data prior to telling the reader how these subgroups were obtained.  With respect to this, the authors should not only present age differential between “death group” and “survival group”, but should provide further information regarding disease duration in patients for each group.
Validity of the findings: Several conclusions are not supported by the data. For example, the authors state that FXR activity is inhibited due to decreased concentration of agonists (line 315-318). However, FXR signaling was never assessed, for example by serum fgf19. The authors could also use 7aC4 test to assess Cyp7a activity more directly. Moreover, the authors should more clearly define other possible mechanisms for elevated serum bile acids including alterations in hepatic function, which is alluded to in the discussion. The following reference assesses bile acid transporters in rodent models of disease and may of use: Gai Z. 2014. Am J. Physiol. Renal Physiol.

The authors state as a conclusion that the increased level of taurine-conjugated bile acids… causing impaired barrier function and increased accumulation of urotoxin in ESRD patients (lines 328-331). The data do not support this conclusion and this should be stated as a speculation.

The authors should better explain how a bile acid profile improves upon current diagnostic methods (e.g., GFR).

The work presented provides rationale for hypothesis driven research, for example, the elevation of conjugated bile acids in ESRD patients may be the result of altered microbiota, but could also result from altered enterohepatic circulation and/or hepatic dysfunction, both testable hypothesis, this should be discussed with greater clarity.
Additional comments: The utility of the findings should be better explained. Does this work have the potential to change diagnostics in ESRD?
Speculations should be stated as such, not as conclusions from the work presented.
More detailed explanations as to why it is believed that these findings are not directly related to hepatic dysfunction as a consequence of ESLD.
Fix typographical errors/spelling mistakes throughout.


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

Input:
Title: TARGETED METABOLOMICS STUDY OF SERUM BILE ACID PROFILE IN PATIENTS WITH END-STAGE RENAL DISEASE UNDERGOING HEMODIALYSIS
Review round: 2
Reviewer: 1

Output:
Basic reporting: -In order to provide a more credible and professional contact email, can Professor Yuan Yu provide his/her professional email address? (i.e. ...@hospital.cqmu.edu.cn).
-Rather than having "P<" and "p<", please pick one and be consistent in the reporting.
-Line 297: Change "They are recently found" to "They were recently found".
-See title of Figure 1.  My request to change "Metabonomic" to "Metabolomic" has not been addressed.
-Please remove the sentence "Bile acid synthesis is primarily involved in hepatic cholesterol metabolism, which causes an enhanced demand of cholesterol in the liver", as I don't believe that hepatic cholesterol metabolism is not the sole reason for an enhanced demand of cholesterol in the liver.
-See Table 1.  Please keep the presentation of the superscripts consistent.  You have "kg/m2", "10^9", and superscript a.  I'd suggest changing everything to a true superscript.
-Please relabel the axes of the PCA plots in Fig. 1A.  Simply replace t[1] and t[2] as PC1 and PC2, respectively (this is standard reporting for PCA plots).  Same for Fig. 1B, as the general audience will not know what to[1] and t[1] will mean without any explanations in the legends. Please correctly label the x and y-axes.  "200 permutations 1 components" is NOT the correct description of the measurement in the x-axis.  Why are you just providing a screenshot? (I see the "SIMCA 14.1 - 2019/4/23 21:57:43...").
-See legend for Figure 2.  "The curve is closet to the top left"...this does not make any sense. Please revise. There is enough standard text in the literature to use as a model for providing a basic description of an ROC curve.
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: TARGETED METABOLOMICS STUDY OF SERUM BILE ACID PROFILE IN PATIENTS WITH END-STAGE RENAL DISEASE UNDERGOING HEMODIALYSIS
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors have extensively modified the text and greatly improved the language. However, a few minor typographical errors remain which can be easily addressed (e.g., metabolism is misspelled in line 59).
Experimental design: My concerns have been addressed.
Validity of the findings: My concerns have been adequately addressed.
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: TARGETED METABOLOMICS STUDY OF SERUM BILE ACID PROFILE IN PATIENTS WITH END-STAGE RENAL DISEASE UNDERGOING HEMODIALYSIS
Review round: 3
Reviewer: 1

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: Thank you for taking the time to address all my concerns.


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: IMPORTANCE OF ACTIVE LAYER FREEZE-THAW CYCLES ON THE RIVERINE DISSOLVED CARBON EXPORT ON THE QINGHAI-TIBET PLATEAU PERMAFROST REGION
Review round: 1
Reviewer: 1

Output:
Basic reporting: See general comment
Experimental design: See general comment
Validity of the findings: No comment
Additional comments: Comment to“Importance of active layer freeze-thaw cycles on the riverine dissolved carbon export on the Qinghai-Tibet Plateau permafrost region”

In the context of global warming, the active layer and permafrost in the Qinghai-Tibet Plateau (QTP) are experiencing quick change, the severe permafrost degradation shows great effects on hydrological and biogeochemical processes, however, riverine dissolved carbon transport changes and their responses to freeze-thaw cycles remains poor. Authors used data in thaw and freeze seasons of 2014 and 2016, DIC and DOC concentrations and fluxes and their relationships with freeze-thaw cycles were consequently unveiled following a hypothesis. Generally, the Ms is interesting and provides good data of riverine C in the permafrost region. The structure, figure and Tables look good. While English should be improved (I have given some specific suggestions as follows). Methods were well described with sufficient detail while hypothesis should be well stated and should be more meaningful.

The authors get main findings of fluxes, concentrations and relative importance of DIC and DOC relating to thawing and freezing process. i.e., As the freezing process, DIC and DOC concentrations increased, while their fluxes decreased. As the thawing process, fluxes and concentrations of dissolved C increased. Authors discussed their patterns and controls, and also compared to other studies, while I think authors also should related to contrary patterns of other studies (i.e., L81-82 Thawing of active layer could result in the decrease of DOC and increase of DIC due to DOC mineralization and respiration (Drake et al., 2015; Striegl et al., 2005; 2007; L102-103 A previous study at northern QTP found that DOC export decreased as thaw depth increased (Mu et al., 2017). What resulted in the different results?.

Methods
Authors must clarify the measurements of thawed depth, frozen depth, land use composition etc.

Some specific comments including languages.
L 33 delete “while”
L43 change “was highly depended on” to “was highly dependent on”
L44 Please rewrite
L56 Please check
L67 Maybe change “Riverine exported carbon” “riverine carbon” or “riverine carbon export”?
L68 “according to studies conducted in a variety of scales and locations”, these words do not make sense, please delete
L79 “deepen”?
L85-87 Please re-organise
L106-107 Hypothesis testing is good, please be more clear
L127 What is the highest temperature? Please show the number
L136 Please Check the citation
L149 Your findings of major ions?
L155-157 I can not catch the sentence
L174 Please need to re-organize
L186 maybe “after sampling”
There are many typos such as the format of citations: L306, L313, L321, L327 any problem of citation? while I can not point out all issues

Tables
Table 1 Please indicate that how to get the catchment characteristics (i.e., land use composition)
Table 3 Please indicate the significant difference between DOC and DIC?

Figures
Fig S5 There are no plots in the ternary diagram?


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: IMPORTANCE OF ACTIVE LAYER FREEZE-THAW CYCLES ON THE RIVERINE DISSOLVED CARBON EXPORT ON THE QINGHAI-TIBET PLATEAU PERMAFROST REGION
Review round: 1
Reviewer: 2

Output:
Basic reporting: When I am reviewing manuscripts, I usually raise several major comments and then give 2 or 3 pages of minor comments. But I read this manuscript, I found the field work was solid，and the manuscript was well organized, the data have been properly analyzed, and the conclusions were interesting. It is a pleasure to review this manuscript.
Experimental design: The experiments were designed rigorously.
Validity of the findings: It is interesting, and the results were in agreement with our expectations.
Additional comments: There are only minor suggestions for the authors, which I think they can do this work during the proof stage, so I need not see the revised version.

L76-77. Delete this sentence.
L77, The deepening AL could. You need not to repeat the seasonal thawed active layer.
L86, I would prefer passive voice.
L89, Seasonal change in soil thaw depth. The active layer is almost the same as the deepest thaw depth in the autumn.
L94, the largest. Not the only. Even in Hawaii, there is permafrost.
L94 wrong citation. It is Zou et al. 2017.
L97. There are several other reports, such as Zhao et al., 2018, Mu et al., 2015. Although Ding reported the SOC pools using SVM, there are also large uncertainties. For example, they used the kriging method to retrieve the spatial soil particle distribution, which is unacceptable.
L103, while it is unknown about the DIC exports changes during the freeze-thaw cycles in AL.
L107, delete potential.
L113-115. Remove these sentences.
L479, delete “since…freeze”.
L486, Climate warming-induced permafrost degradation may result in higher disturbance of permafrost carbon
L490 Connections-----relationships
L491. Delete this sentence.


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: IMPORTANCE OF ACTIVE LAYER FREEZE-THAW CYCLES ON THE RIVERINE DISSOLVED CARBON EXPORT ON THE QINGHAI-TIBET PLATEAU PERMAFROST REGION
Review round: 1
Reviewer: 3

Output:
Basic reporting: This is an interesting, solidly executed pieces of work. The topic is of relevance and the findings should be of interest to the international readership. The methodological approach is appropriate and is sufficiently described. The data are well presented and discussed, and the conclusions drawn are supported by the findings. The paper can be accepted after a thorough language editing.
Experimental design: The study design is appropriate.
Validity of the findings: The findings are justifiable.
Additional comments: This is an interesting, solidly executed pieces of work and I enjoyed reading it. The topic is of relevance and the findings should be of interest to the international readership. The methodological approach is appropriate and is sufficiently described. The data are well presented and discussed, and the conclusions drawn are supported by the findings. The paper needs, however, a thorough language editing before it can be published. Below are just a couple of examples of grammar/usage errors:

Line 30: Change “riverine exported carbon” to “riverine carbon export”

Line 32: Add “the” before “central QTP”

Lines 37-39: The sentence reads awkward. You may rephrase it to something like: “Spatially, DIC and DOC concentrations were positively correlated with vegetation coverage and were lowest in bare land.”

Line 45: Change “in QTP” to “in the QTP”

Line 192: Runkel et al 2004 is missing in Refs.

Figure 3 caption: Do you mean monthly average fluxes in kg per day? Please be clear.

Figure 3 caption: Is monthly mean discharge or actually total monthly runoff and precipitation?


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: GENOME-SCALE METABOLIC RECONSTRUCTION AND METABOLIC VERSATILITY OF AN OBLIGATE METHANOTROPH METHYLOCOCCUS CAPSULATUS STR. BATH
Review round: 1
Reviewer: 1

Output:
Basic reporting: The quality of language is satisfactory. The text of the manuscript is easy to read and understand. The paper is well organized, the literature references look sufficient.
Nonetheless, legends for all the figures and supplementary materials are absent. Please, add the legends or modify the figures so that they became more clear.

Also, there are some minor comments to the text.

1. Lines 72: It is better to write "the ribulose monophosphate (RuMP)" than  "the ribulose monophosphate RuMP".

2. Lines 130-131: "relative percentages of ribonucleotides, and ash were assumed to similar to that in M. buryatense 5GB1". What is an "ash"?
Experimental design: Unfortunately, experimental design is so limited and biased, so this paper cannot be accepted without major revisions.

1. Lines 193-195. It is absolutely unacceptable to use such a reaction for the ATP production. This reaction can be used for the mitochondria but not for any bacterium. At first, Bacteria can use various electron acceptor, not only an oxygen (see e.g. Welte & Deppenmeier 2014, PMID: 24333786). Moreover, in lines 381-384 a nitrite reductase is mentioned. This points to oxygen is not an only electron acceptor that can be used by Methylococcus capsulatus str. Bath. Second, various reactions are possible even for oxygen-dependent respiration, e.g. compare https://www.vmh.life/#reaction/CYTBD2 and https://www.vmh.life/#reaction/CYOmq. Third, NADH is not an only possible electron donor. Thus multiple intermediates of the central carbon catabolism as succinate (https://www.vmh.life/#reaction/SUCDimq) or glycerol-3-phosphate (https://www.vmh.life/#reaction/G3PD6) are used as electron donors. Thus, usage of only one reaction for NADH oxidation, oxygen reduction, and ATP synthesis is absolutely unacceptable. Please, use separate reactions for an oxidation of electron donors, reduction of electron acceptors, and APT synthesis (e.g. https://www.vmh.life/#reaction/ATPS3).

2. Lines 344-345: "The different carbon sources such as methanol, formaldehyde, formate, acetate, and CO2, were tested on iMC535." - please, explain a selection of these carbon sources.
Validity of the findings: Because of ill-conditioned experimental design, it is not possible to estimate the validity of the findings.
Additional comments: No comments.


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

Input:
Title: GENOME-SCALE METABOLIC RECONSTRUCTION AND METABOLIC VERSATILITY OF AN OBLIGATE METHANOTROPH METHYLOCOCCUS CAPSULATUS STR. BATH
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: everything is fine
Validity of the findings: The MS has one serious issue preventing it to be published in its current state: the lack of relevant experimental data. Authors collected the experimental data (among highly important are ATP maintenance, methane consumption an other) for their model evaluation from i) published Methylococcus capsulatus str. Bath, ii) data, published for another methanotroph M. buryatense 5GB1 and iii) even from E. coli. All of these can add serious bias to current calculations making the model of low value. I understand that it is most probably impossible to obtain this information ASAP, however, authors can possibly obtain more available experimental data to evaluate it with the model.
Additional comments: There also several specific comments:

L 39. Why Methylococcus capsulatus str. Bath is unique?
L 53-54 It is still questionable
L 76 In microbiology 45C cannot be regarded as high temperature – it is closer to mesophily than thermophily
L 77 Again, uniqueness of M. capsulatus. Could you please list the unique features of this microorganism in comparison with other Methylococcaceae and other methanotrophs?
L 79 Not speaking of other methylotrophs and methanotrops in particular, the only Methylococcaceae family has already around 60 sequenced genomes in IMG or NCBI databases
L129-130 amino acid composition in proteins. Why not calculated in silico?
L177 what does it mean? 10-6 of what?
L 196 how this equation was calculated? from the calculations down the text?
L 236 what are these categories? look like COGs, but COGs weren`t mentioned in the methods
L 242-244 what does it mean? Reconstructed de novo? I assume authors manually verified the KEGG maps-based (or MetaCyc) automatic reconstructions.
L 440-441 It is strange since according to the original publication of Foster and Davis, 1966, NO3- was much better than ammonium or amino acids


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: GENOME-SCALE METABOLIC RECONSTRUCTION AND METABOLIC VERSATILITY OF AN OBLIGATE METHANOTROPH METHYLOCOCCUS CAPSULATUS STR. BATH
Review round: 2
Reviewer: 1

Output:
Basic reporting: everything is fine
Experimental design: everything is fine
Validity of the findings: I am not satisfied with the response on my main concern on relationship of the model and experimental data.
Additional comments: The answers on the particular questions are fine. Overall, the MS is well-written, designed etc. However, besides the main concern on the validity of the model, authors still did not convince me their study is of particular scientific novelty or impact.


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: GENOME-WIDE CHARACTERIZATION AND EXPRESSION ANALYSIS OF PP2CA FAMILY MEMBERS IN RESPONSE TO ABA AND OSMOTIC STRESS IN GOSSYPIUM
Review round: 1
Reviewer: 1

Output:
Basic reporting: Abstract
It is not clear what GhPYL2-2D, etc. are?  Are they Genebank sequences of PP2CA sequences or another gene, localized to subgenomes D08, A06, and A11?  The introduction says that PP2CAs are co-receptors, interacting with PYL and controlling ABA signaling.  I would somehow mention this in a sentence in the abstract so the audience understands the relationship and further understands the reason for the yeast two-way hybrid experiments.

Line 38 were identified the genomes of these plants, respectively. Analysis results showed that most
CHANGE ‘identified the genomes’ to were identified in the genomic sequences

Line 42 PP2CAs were under purifying selection in Gossypium during evolution. Moreover, majority of the PP2CAs were expressed specifically in diverse tissues, and highly expressed in flowers in G.
CHANGE ‘Morever, majority’ to The majority

Line 45 osmotic stress. Besides, yeast-two hybrid assays revealed that many GhPP2CAs could interact
CHANGE ‘Besides’ yeast-two hybrid assays revealed that many GhPP2CAs could interact’ to Yeast-two hybrid assays revealed that many GhPP2CAs are capable of interaction with

Line 79
2013; Singh et al., 2015). PP2CAs are functional redundant, and their expression is upregulated
CHANGE ‘functional’ to functionally

Line 119 default setting (E-value<e-10). The putative Gossypium PP2Cs were then characterized using the
DEFINE putative.  What criteria lead to the selection of sequences and how many sequences for the next step?  The output of BLAST can be extensive so what is the cutoff?

Line 123 The online tool ExPaSy (http://web.expasy.org/protparam/) was applied to analyze the
Line 124 properties of PP2CAs in Gossypium. The subcellular localizations of Gossypium PP2CAs were predicted using the program in the website (http://www.csbio.sjtu.edu.cn/bioinf/Cell-PLoc/). The
Line 126 structures of the PP2CAs were characterized by the GSDS (http://gsds.cbi.pku.edu.cn/). The
COMMENT That is a hefty lineup of methods.  Add some more information to each method as to the purpose, any other settings used, and cutoff values or quality control measure to continue to the next method.

Line 143 We downloaded the PP2CA databases for Arabidopsis thaliana (http://www.arabidopsis.org/),
CHANGE ‘We downloaded the PP2CA databases for’ to The PP2CA databases were downloaded for

Line 155
collected from TM-1 cotton plants grown in soil for 21 d. Flowers were got 1 d post anthesis, and
CHANGE ‘Flowers were got’ to Flowers were collected
DESCRIBE source of TM-1 because specific clones give different results.  I know the US Cotton Germplasm Collection offers one as SA-2269, PI 607172, but individual labs may have kept their own clones.

Line 158 MS medium in a growth chamber (day/night temperature cycle of 28°C/26°C, 14 h light/10 h
ADD citation for MS medium

Line 208 3A’, GbHAI3-3D, GbHAI3-3A, GbABI1-3D) had not orthologs in G. arboretum and G.
CHANGE ‘not’ to no

233 GhHAI2D, GhABI2-1D, GbAHG3-2A and GbAHG3-1D were located on scaffolds rather than
CLARIFY the meaning of ‘scaffold’.  Is it unassigned to a chromosome?

263 under purifying selection during evolution. The Ka/Ks ratios for the two gene pairs GrAHG3-
KEEP the italics consistent, because Ka/Ks was italicized in line 259, but not in lines 260 and 263.

Line 271 members. This suggests that these PP2CA formed before the divergence of eudicots and monocots.
COMMENT. Figure 5 should be highlighted for the fact that, as expected, Gossypium sequences clustered together before another genus comes close.  Were the Arabidopsis sequences in this figure also in S1- S4?  Those that clustered with a Theobroma sequence, are they on the same locus in Gossypium, because it suggests stability across genera and should be within the genus too.  If not, then they duplicate by rearrangements or transposable elements, etc.  The whole block of just G. barbadense sequences that first cluster with Oryza before another Gossypium species is very curious to me as it seems specific to that species.

Line 312 GhPP2CAs exhibit diverse expression patterns in responses to ABA and osmotic stress.
QUESTION, you mentioned that there is interaction between PP2CAs, so is there a cascade of interactions of those active at 3h, followed by 6h, followed by 12h and 24h?  It is logical that after 3h, those activated or upregulated will trigger those you see active or upregulated at 6h, and so on.

Line 338 Compared to the number of PP2CAs in Arabidopsis
Line 339 (10), rice (10), maize (16) and Brachypodium distachyon (8), that in G. hirsutum and G.
Line 340 barbadense was great (Xue et al., 2008; Wei and Pan, 2014; Cao et al., 2016). This suggests that more complex and elaborate ABA signaling mechanisms modulated by PP2CAs may exist in the upland and island cotton species.
SUGGESTION, emphasize the polyploid nature of cotton and how this could have allowed more duplication in sequences, while still conserving one copy of the original. Also it was adapted to the needs of humans, growing in many regions of the Americas.  Cotton is often grown in hot dry regions because it benefits boll ripening and harvesting of dry, high quality fiber so evolution and selection among these sequences could help fine tune cotton to the farmers needs.  Remember G. raimondii is wild and is photoperiodic in its flowering so I would expect differences here.  Having grown G. raimondii in greenhouses I noticed that it easily drops its flowers so I wonder if ABA is functioning here?  Flowering and development of bolls is a resource intensive process in cotton, even in hot and dry weather conditions so maybe they were selected by man for such areas, when it runs contrary to native Gossypium plants that normally flower in short daylength and cooler winter periods of the tropics, not hot, long days of semi-tropical regions.

352 and G. barbadense are directly descended from those PP2CAs in the two diploid species.
SUGGESTION reword  ‘directly descended from those’ to ancestrally related to those

Line 355 Additionally, no orthologous genes of
Line 356 GaAHG3-3, GrABI1-2 and GrHAI2 were observed in G. barbadense (Fig. 1). This hints that these genes are possibly lost during evolution.
COMMENT Another possibility is that these genes arose after the tetraploid species appeared and are the result of separate evolutionary processes in these diploid species.

361 evolutionary processes even after the divergence of monocotyledon and dicotyledon.
CHANGE ‘monocotyledon and dicotyledon’ to monocotyledons and dicotyledons

371 Transcript abundance analysis indicated that majority of the GhPP2CAs predominantly
CHANGE ‘that majority of the GHPP2CAs predominantly’ to that the majority of the GhPP2CAs were predominantly

Lin 384 Totally, 14, 13, 27 and 29 PP2CA genes were characterized from G. arboretum, G. raimondii, G.
CHANGE ‘totally’ to In total


Literature references, sufficient field background/context provided.

I am impressed with the mastery of the English language.  The structure is logical and it flows well.  The introduction was easy to follow.  I would consider finding some citations that show that ABA or protein phosphatases were implicated in cotton yield, physiology, disease resistance, etc. so it appeals to a wide audience.  By itself the research is fascinating and a great contribution to basic science but some are reading it with an idea of application to cotton improvement. What follows is a listing of grammatical changes and comments that I accumulated while reading the article.

Line 101 The yield and quality of cotton are adversely affected by many abiotic
Line 102 stresses such as drought and high salinity, which are governed by ABA signaling. Therefore, it is
ADD references to this statement that show the current state of knowledge of ABA in cotton.

Line 118 (http://database.chgc.sh.cn/cotton/index.html), respectively, using the BLAST program with
ADD the reference for BLAST

Ohta et al., 2003 should go after Park et al., 2009 in your citation section because they are all arranged alphabetically by main author’s last name.  Otherwise it appears that the citations in the text are all spelled out in the citation section.


Professional article structure, figs, tables. Raw data shared.

The figures, and tables were essential to the article.

COMMENTS, in Figure 6. You display resolution in the regulation of different PP2CAs but what is the relevance or what could be happening?  The differences in flower and fiber should be relevant for cotton breeders so they can separate fiber quality genes (not shutting down fiber genes will allow fibers to mature and elongate fully) from those just for yield (flowers that remain would increase total yield)? G. raimondii does not produce relevant fiber so those sequences could be further subtracted so breeders just narrow it down to those affecting fiber quality?  Are they separated by chromosomes and easily selected by a breeder or scientist or are they linked?  I am going to speculate here, osmotic stress normally shuts down flowering but in nature a dry period is sometimes best for flowering and perhaps Gossypium shares this feature.  The pollen is sensitive to moisture and is heavy, requiring insect pollination, further indications that even though moisture is limiting and that normally restricts vegetative growth (possible releases of ABA), it must counter it with PP2CAs in order to continue flowering?  Short daylengths in dry winter months are common in the tropics and normally when native Gossypium species flower so there might be a connection or one that can be investigate with the new data and tools you developed?

Table 1.
Size(aa		and Mass(kDa
)			)
Should be reformatted to
Size		Mass
(aa)		(kDa)

COMMENT, what is the meaning of ‘?  For example what is the difference between GaAHG3-2 and GaAHG3-2’?

Figures 6, 7, and 8
QUESTION, how did you calculate the Standard error?  I saw mention of experiments replicated three times, but how were they replicated?  What are the actual data and where is mention of an ANOVA of the data?

All appropriate raw data has been made available in accordance with our Data Sharing policy.

I am not as familiar with this journal or how data is presented.  Is there access to the sequences you found, because it could be difficult for a research to get the exact same sequences, even if they duplicated every thing exactly because of the size of the genomes, how one selects output and what order they use it, etc.

COMMENT, figures S1-S4, I would expect every sequence of Gossypium to cluster with one another Gossypium sequence before even coming close to a sequence in Arabidopsis.  So how did some Gossypium sequences cluster with an Arabidopsis sequence before clustering with another Gossypium sequence?  Maybe I am noticing the obvious but it would be good to have an explanation accompany the nice figures.  It would suggest to me that certain enzymes in certain places on certain chromosomes may predate the evolutionary separation of these two families and therefore there is a grandfather sequence that ended up in both families.  Evolution protected them to the extent that they resemble that sequence in other families more strongly than duplicated and descendant sequences within the same species.  When you have a close association of a putative sequence in two families, but with one that is highly annotated as in Arabidopsis, it makes a compelling case for assigning genetic function.

Self-contained with relevant results to hypotheses.

I appreciate the whole package of the research as it started with genomic sequences and ended up with putative genes.  It should be made clearer to the reader the purposes and details of the extensive methodology.  The reader can benefit from an explanation of why each step is necessary and how they feed into each other.  Then the hypothesis structure becomes more relevant.  When each step continues to suggest the function as PP2CAs then your conclusions are stronger and keeps narrowing down the role of each sequence in the biochemistry of the plant.  It allowed you to do the PCR primer reactions, two-way hybrid experiments, etc. all from starting just with 4 genomic sequences.

It would appear that the hypotheses are something like, phosphatases assigned by function and genetic sequences in well studied plants like Arabidopsis will confer gene function to similar genetic sequences in Gossypium.  Genetic sequences in four species with published genomes that show homology to known phosphatase genes in other families can assign these broad functions to these new sequences in Gossypium.  A wide assortment of research tools are used to find the closest homologies of sequences to those known to function in phosphatases and ABA, because they have strong effects in cotton physiology (references specific to cotton are needed, you have one related to potassium, which is interesting as potassium is often consumed the highest during boll filling).  Because there is evolutionary structure implicit in Gossypium with tetraploid species sharing very high homology to two diploid species and no other explanation is possible other than they merged to give rise to the tetraploids, the sequences are compared among these living species to infer similar gene functions among homologous genetic sequences.  Higher order structures of genetic sequences such as amino acid codes, intron/exon sizes and patterns, protein structure and activity and locations on chromosomes and activity in nucleus and expression in specific tissues are further indications of their likely roles as phosphatases in ABA pathways.  As long as the experimental data continue to align, then the hypothesis that these are putative sequences in PP2CAs and ABA pathways becomes more likely.  Also Interactions are expected and are tested in yeast two way hybrids and are further indications of function, homology and likely evolutionary relationships among these species.  The tetraploid nature of G. hirsutum and G. barbadense and even selection pressures by man to produce cotton in agricultural settings are expected to create additional variation in sequences and expression, measured further with tissue specific assays.

Some statement(s) like these or some rationale before each description of each method will greatly assist the readers in following the purpose and hypotheses specific to each study.  There is a lot of information and evidence gathering but the reader still needs to know the significance of each.  Perhaps citations to another article that used the same method would suffice for brevity.
Experimental design: I appreciate the thorough collection of the data, but want more information on details of each, and how the output is generated, analyzed and fed into the next method/step.  It was assumed that the readers all know the steps, but if that is the case, your audience becomes very small.  Some sentences with each method can help understand the expected results or the hypotheses and help the reader to try to repeat your results.  The variety of methods and data output is acceptable to me and to the investigative goals of the article.  I have confidence that the findings could lead to more research on PP2CAs and ABA in cotton.


Rigorous investigation performed to a high technical & ethical standard.

Give more detail on how you review the results of each method to assure quality for the next method.
The methods in this article are all common to this type of research and the data was well applied to each step and to the conclusions of the research.


Methods described with sufficient detail & information to replicate.

More detail is needed because so many methods, programs, sequences are utilized in this study.  There are hundreds, or thousands of other lines of the four species that you used that could also be analyzed.  There are 40 some other species of Gossypium that could also be used in this type of study.  Ask yourself, how would another scientist repeat every step of your study in order to contribute the same useful data and make similar conclusions about PP2CAs and ABA pathways in Gossypium?  Imagine if I stated my research as, planted Gossypium trilobum, harvested leaves, extracted DNA, ran AFLP and created a linkage map.  Obviously, there is a lot of detail to each step.  It seems to be an easy fix as you have plenty of methods but the readers need more details and then can be inserted or clarified.

One example where more detail is needed is:
Line 153 Measurements of PP2CAs expression in tissues and in response to ABA or osmotic stress
It indicates TM-1 plants, but how many plants were grown?  It also indicates flowers and ovules were harvested, but how many flowers were harvested?  At least tell the readers if there is a quantitative product needed like so many ug/ml of cDNA and it would indicate that say 50 flowers were harvested to generate at least 2 mL of a 20 ug/ml concentration of cDNA, something to help the readers understand and replicate this study.  It ends with saying it was replicated three times, but this is vague.  Was it done with different plants to avoid injury effects of resampling?  Was it done a week apart, a month apart?  Plant expression, especially with ABA can be very sensitive and even forgetting to water the plants one day could alter the results so the details are important.  How was the PEG6000 applied, into the MS media or in a spray?  I am assuming that this replication was how you got the standard errors, but what were the actual values put in and the results of ANOVA?  Even though you cite some previous articles so they can read up on the methodology the readers still need some detail to visualize your work and relate it to their methods.  Many readers will be learning this for the first time, or at least the first time with respect to cotton, so the more detail the more helpful.  Results from one are funneled into another method so how is the quality control assured in each step, or the cutoff values to selecting sequences for the next step?  For example, the results from a BLAST search can be longer than an encyclopedia set so how was it trimmed down to a best set of sequences to continue?
Validity of the findings: The novelty of the research makes it difficult to determine validity.  Be aware that more proofs are needed before one is 100% sure that a given sequence is a specific enzyme.  The wealth of methods and data give strength to your findings and this should be emphasized.

How were the standard errors generated?  The consistency of putative PP2CA sequences throughout all of your methods gives weight to the data.

The conclusions are conservative but appropriate.  Meaningful data was generated and presented.  I would explain more about figure 3, because I see it in many places but not much is concluded.  It is pretty but I am not sure what conclusions to draw from it.

Emphasize the collinearity among A chromosomes 1 – 13 (G. arboreum) and D (G. raimondii) and At and Dt because it is central to all this research, and it ties decades of classical genetics, breeding and cytology into evolution of the Gossypium genus.  It is a contributing reason you went so quickly from just genomic sequences to finding a class of genetic sequences for function as PP2CAs.  It also helps to show where is the genetic diversity, how it was formed, and how it can be dissected and better understood and utilized.  I am curious about the brief mention of ‘seed germination’ in your introduction because maybe these pathways have an effect in my work with exotic cotton germplasm.  You have a very good introduction that provided a information on these pathways so how much more do we know from your research, because it is not always obvious to the readers.  The conclusion should list the ways it can spawn new research especially if you think it is too early to speculate too much on the results.

It seems to be an article that opens many doors to future research.  If more speculation is desired than an example would be that the separation of PP2CAs according to time and tissue and stress responses indicates separate loci amenable to selection by cotton breeders for trait improvement or by cotton scientists for research into complex pathways in Gossypium.  Extension of these experiments to wild species or other cotton pedigrees will further dissect the regulation and genetics of PP2CAs and the role of PP2CAs and ABA in cotton growth and productivity.
Additional comments: It is good research because it starts with mere genomic sequences and then ends up with putative genes and means to dissect the evolution and physiology of Gossypium.  Make it easier for another researcher to duplicate the research so that more knowledge can be added.


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: GENOME-WIDE CHARACTERIZATION AND EXPRESSION ANALYSIS OF PP2CA FAMILY MEMBERS IN RESPONSE TO ABA AND OSMOTIC STRESS IN GOSSYPIUM
Review round: 1
Reviewer: 2

Output:
Basic reporting: As a pivotal phytohormone under water-limited conditions, abscisic acid (ABA) has been intensively studied in model plants such as Arabidopsis thaliana, and its signaling has been well characterized at molecular levels. In this manuscript, the authors examined one of the important players of ABA signaling, type 2C protein phosphatases (PP2Cs) classified in clade A, in four cotton (Gossypium) species. This study might provide interesting insights into ABA signaling in fiber crops, and the results were presented in a standard manner. However, there were several concerns.

1-1. The authors should reconsider designation of the names of PP2Cs and the protein subfamily. First, I disagree with the designation of Gossypium clade A PP2Cs named after the Arabidopsis PP2Cs (Table 1; line 183-186). Because the nine Arabidopsis PP2Cs were not one-to-one orthologs of the Gossypium PP2Cs according to the phylogenetic relationships (Fig. S1-S4), it should not be objective. In addition, all of the nine Arabidopsis PP2Cs have been named based on the characteristic of genetics and transcripts, such as ABA insensitive 1 (ABI1). If the authors used the same terms for Gossypium, readers should be confused. I would suggest simply numbering the PP2Cs as the previous report (Xue et al., 2008) cited in this manuscript.
Secondly, to avoid confusion, the abbreviation of clade A PP2Cs, PP2CAs, should not be used because one well-characterized clade A PP2C in Arabidopsis has been named as PP2CA (Tähtiharju and Palva, 2001), the alias of which is AHG3 (Yoshida et al., 2006).

References: Tähtiharju, Sari, and Tapio Palva. "Antisense inhibition of protein phosphatase 2C accelerates cold acclimation in Arabidopsis thaliana." The Plant Journal 26.4 (2001): 461-470.
Yoshida, Tomo, et al. "ABA-hypersensitive germination3 encodes a protein phosphatase 2C (AtPP2CA) that strongly regulates abscisic acid signaling during germination among Arabidopsis protein phosphatase 2Cs." Plant physiology 140.1 (2006): 115-126.

1-2. Please cite the original studies for the contexts (lines 82-84 and 90-92). The followings should be more appropriate.

References: Fujii, Hiroaki, and Jian-Kang Zhu. "Arabidopsis mutant deficient in 3 abscisic acid-activated protein kinases reveals critical roles in growth, reproduction, and stress." Proceedings of the National Academy of Sciences 106.20 (2009): 8380-8385.
Fujii, Hiroaki, et al. "In vitro reconstitution of an abscisic acid signalling pathway." Nature 462.7273 (2009): 660.
Fujita, Yasunari, et al. "Three SnRK2 protein kinases are the main positive regulators of abscisic acid signaling in response to water stress in Arabidopsis." Plant and Cell Physiology 50.12 (2009): 2123-2132.
Geiger, Dietmar, et al. "Activity of guard cell anion channel SLAC1 is controlled by drought-stress signaling kinase-phosphatase pair." Proceedings of the National Academy of Sciences 106.50 (2009): 21425-21430.
Lee, Sung Chul, et al. "A protein kinase-phosphatase pair interacts with an ion channel to regulate ABA signaling in plant guard cells." Proceedings of the National Academy of Sciences 106.50 (2009): 21419-21424.

1-3. Likewise, the references seemed to be incorrect (line 315). It should be Ma et al., 2009 and Park et al., 2009, as cited in the Introduction (lines 86-87).
Experimental design: The authors sought PP2Cs genes in four cotton species, and performed phylogenetic and synteny analyses especially focusing on clade A PP2Cs. There were major concerns on prediction of subcellular localization (Table 1) and protein-protein interaction assays (Figures 9-11).

2-1. As described in the Introduction (lines 79-82), it has been shown that Arabidopsis clade A PP2Cs interact with a number of cytosolic and nuclear proteins. It seemed to be inconsistent with the prediction that most of Gossypium clade A PP2Cs are localized in nucleus (Table 1; lines 192-195). Have the authors verified the accuracy of prediction (Plant-mPLoc: http://www.csbio.sjtu.edu.cn/bioinf/plant-multi/; lines 124-125)? If Arabidopsis clade A PP2Cs are predicted to be localized in different compartments in contrast to literature knowledge and other prediction database (such as SUBA; http://suba.live/), the accuracy of Plant-mPLoc should be carefully verified. Alternatively, since it is just a prediction and not experimentally validated, the result can be excluded from Table 1.
Moreover, the notion would not be rational (lines 345-347). As described above, Arabidopsis clade A PP2Cs are thought to be present in cytosol and nucleus to dephosphorylate plasma membrane proteins and transcription factors. Given that subcellular localization of rice PP2Cs have not been reported in one cited paper (Xue et al., 2008), the sentence was literally wrong.

2-2. The result (Figures 9-11) should be cautiously interpreted. Given that the homology between GhPYLs and AtPYLs was not supported by any data and references, except for the accession numbers (lines 317-318), it was unclear if these GhPYLs have roles as ABA receptors. To argue that they ‘may play roles via interaction with GhPYLs in ABA-dependent or ABA-independent manner in cotton’ (lines 380-381), further supporting data showing that GhPYLs are orthologs of AtPYLs (for example, alignment of protein sequences) are requested.
Meanwhile, the result did not indicate that they are ‘functional phosphatases’ (line 380). Any biochemical evidence are needed.
Validity of the findings: More rational interpretation and further discussion would be required.

3-1. The authors should interpret the result objectively (lines 188-192, 343-344). I would not think that most of the clade A PP2Cs in Gossypium has ‘similar’ properties, because of the varieties in protein size and pI.

3-2. I would feel that most of the text in the Discussion section were already described in the Result section. For example, the paragraph (lines 367-370) was a repetition of the result (lines 274-276). More references should be requested for discussion and interpretation of the results.
Additional comments: 4-1. Abstract: Please precisely describe the biological role of PYL genes. Otherwise, readers could not tell the importance of the result of yeast two-hybrid assays (lines 45-47).

4-2. Please consider revising the description (lines 69-70). Clade A PP2Cs are the ones of well-studied PP2Cs in Arabidopsis, and they have been shown to have important roles in ABA signaling. But, nobody knows if they are the most important in plants.

4-3. The statement should be incorrect (lines 75-77). Not only ABI1 and ABI2, HAB1 and AHG3/PP2CA have been well studied and these PP2Cs are thought to cooperatively function in many signaling events.

Reference: Rubio, Silvia, et al. "Triple loss of function of protein phosphatases type 2C leads to partial constitutive response to endogenous abscisic acid." Plant physiology 150.3 (2009): 1345-1355.

4-4. A minor technical comment. The authors showed protein-protein interaction by yeast two-hybrid assay. Although no information was given in the legends, I assumed that black triangles indicate gradients of yeast culture. The gradients seemed not to be correlated with the spots in most cases, and thus I was wondering if the experiments were performed in a reproducible manner and if it is necessary to show all of the results as main figures.

4-5. Not supported by any experimental evidence (lines 386-387). Please tone down the description.

4-6. The phylogenetic relationships between Gossypium species and Arabidopsis were shown in Supplemental Figures (S1-S4). I would suggest using the same color code for the four figures. It would be much easier for readers to interpret the result if the PP2Cs of the same clade are shaded by the same color in the four figures.

4-7. Please specify the abbreviation ‘ABI’ (line 73).

4-8. It should be ‘Highly ABA-induced PP2C 1’ (line 74).

4-9. References are needed (lines 75-77, 79-80, and 80-82).

4-10.What does ‘structure’ mean (line 95)? It should be ‘domain structure’.

4-11. There were a couple of typographical errors.


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: GENOME-WIDE CHARACTERIZATION AND EXPRESSION ANALYSIS OF PP2CA FAMILY MEMBERS IN RESPONSE TO ABA AND OSMOTIC STRESS IN GOSSYPIUM
Review round: 2
Reviewer: 1

Output:
Basic reporting: Be more consistent in the use of italics.  I am not sure if annotating the PP2CA with Gh, Gb, Ga, or Gr makes it all italics or should just the Gh be italicizeds.  In appeared that the whole annotated sequence is italicized and I tried to correct it in the word doc.  Doublecheck my changes and make sure you are consistent with the use of italics.
Changes should be in Orange color.
Experimental design: I am still unclear on the use of plants one experiment.  It appeared that you were able to start sampling for all leaves, roots, stem and flowers just after 21 days.  We know that a cotton plant is still seedling at 21 days.  Can you be more clear on the age/size of the plant when you harvested the relevant tissue.  It is not conceivable to me that a 21 day old plant can provide flowers for your experiment.

You did supplement better this time with all the materials and methods and citations.
Validity of the findings: I tend to enjoy the speculation part of the discussion and wrote my comments into the manuscript.  Check that you agree with my speculation because you are the expert with your research and I am just a first time reader of it.  I think you could say more about the Ka/Ks ratios as it could be significant for plants that are selected in nature versus selected by man.  Remember you are assigning gene function on the basis of a number of indirect methods so stress the consistency of the many methods to the validity of assigning PP2CA gene function to these sequences.
Additional comments: It is a much improved manuscript this time around.
The additional edits are in orange in the pdf (still contain tracking from the previous edits).


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: GENOME-WIDE CHARACTERIZATION AND EXPRESSION ANALYSIS OF PP2CA FAMILY MEMBERS IN RESPONSE TO ABA AND OSMOTIC STRESS IN GOSSYPIUM
Review round: 2
Reviewer: 2

Output:
Basic reporting: Abscisic acid (ABA) plays important roles in plant abiotic stress responses, and its signaling event has been characterized in the model plant Arabidopsis thaliana. To gain insights into molecular mechanisms of stress responses in cotton, the authors studied type 2C protein phosphatases (PP2Cs), which are crucial components in ABA perception and signal transduction, in four cotton species. The results were concisely presented, and the concerns raised by previous reviewers have been addressed.

There were several minor concerns.

1. The tissue-specific expression of GhPP2CA was presented in Figure 6A and B. It might be inconsistent with the text (lines 290-291), because GhPP2CA27 (Figure 6B) and other GhPP2CAs were highly expressed in fibers. Please consider to include others in Figure 6B or omit classification ‘A’ and ‘B’.

2. The following sentence was incorrect (lines 390-391): GhPYLs are homologs of AtPYLs and some GhPYLs are functional ABA receptors (Liang et al., 2017; Zhang et al., 2017b).
These studies did not provide any direct evidence that GhPYLs perceive ABA molecules. ‘Some GhPYLs have been suggested to be functional ABA receptors’ by these studies.

3. How many PP2CAs in Arabidopsis? It would be nine in Introduction (line 74), whilst it was ten in Discussion (line 337).

4. I would suggest spelling out ‘PP2CA’ at the first appearance in the text (line 70), except for Abstract.

5. Line 32: negatively?

6. Lines 279 and 282: cacao?

7. Line 299: ‘GhPP2CA2-4, 8, 10, 24’?
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: DECONSTRUCTING THE POLYMERASE CHAIN REACTION II: AN IMPROVED WORKFLOW AND EFFECTS ON ARTIFACT FORMATION AND PRIMER DEGENERACY
Review round: 1
Reviewer: 1

Output:
Basic reporting: The paper is clearly and professionally written, with only a few errors.  (Please complete the sentence on line 56, and check the grammar on line 297.)  References are cited as appropriate, spanning over two decades' worth of method development and refinement.  The authors show a comprehensive knowledge of various sources of PCR bias.
Experimental design: The paper provides important but incremental improvements in previously published methods for two stage PCR (TAS and PEX PCR), which were also developed in the same laboratory.

I'd like to make a note that two stage PCR has been around since Bohlander first published the concept in 1992, which was later modified by Joseph Derisi's lab.  The original papers had fully degenerate primers for whole genome amplification, instead of partially degenerate primers for targeted amplification.  The important point is that 2-stage PCR protocols often have a limited numbers of tagging cycles (2-4) in order to prevent the artifacts that were identified as a "novel source of bias" in the PEX PCR paper.
Validity of the findings: The current method has shown improvements in reducing chimera formation, and the addition of barcodes allows pooling of multiple samples and evaluating the primer utilization profiles of different members of the degenerate primer pool.  The addition of a bead-based cleanup step also improves the workflow compared to the restriction enzyme step.
Additional comments: This paper clearly demonstrates how subtle differences in the method can lead to profound differences in the results, which is one of the major criticisms in commercial application of microbial community analysis (e.g. gut microbiome analysis in human health.)  In a microbial community, there is an underlying “Truth” to the diversity and abundance of different organisms, and it’s not clear that this method is any closer to revealing the Truth.

The problem with modern microbial ecology is that the 16S databases are full of vestiges of past priming events-- artifacts of priming, cloning, and sequencing over the past 30 years.  Have we now “converged” on a universal primer set that is now only 18-fold degenerate?  For example, it appears that primer 341F has dropped the degeneracy that was used in earlier studies.  (https://doi.org/10.1016/j.mib.2004.04.015)

The bigger picture is that the 16S gene has a number of faults that make it a lousy biomarker, but it's been swept up by a tidal wave of new users and new contributions to public sequence databases.  This was a problem that I thought about deeply in my graduate career, because the limitations in the methods obscured the ability to comprehend the underlying biology.  (https://doi.org/10.1016/j.mcp.2006.09.003) Now is a different era, though, where high throughput sequencing is inexpensive and accessible. I would love for the authors to consider a new line of inquiry for future method development, which overcomes the biases of the 16S gene.


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: DECONSTRUCTING THE POLYMERASE CHAIN REACTION II: AN IMPROVED WORKFLOW AND EFFECTS ON ARTIFACT FORMATION AND PRIMER DEGENERACY
Review round: 1
Reviewer: 2

Output:
Basic reporting: The article on a novel deconstructed PCR approach, in which the linear and exponential phase of the PCR reaction are separated, is well structured and well written.
The language and phrasing are clear and correct, the referencing and the contextualization of the research are sufficient and appropriate.
Figures and Tables are clear and well structured, and the conclusions are overall well supported.
Experimental design: The experimental setup is exhaustive and well designed in order to evaluate and validate the DePCR methods.

There is some inconsistencies in the methods text, and throughout the manuscript regarding the first step PCR: at some points 2 cycles, at other points 4 cycles are mentioned. As it must be 4 cycles for the setup to work, please correct the 2-cycle mentions to reflect the true experimental setup.

The evaluation of ´low´ DNA input at 1.25 ng per reaction is still quite a high input for many low biomass samples, to be honest. While I do not necessary think additional experiments are needed, the fact that low input DNA might be a limitation (even if many very low input samples are multiplexed before cleanup) should be discussed better. Also, the fact that a rather variable number of reads is retrieved, not directly correlating with DNA input, should be discussed in more detail.
Validity of the findings: The approach is very interesting, and a great improvement of the method previously published by this research group.
All presented conclusions are statistically well supported.
Additional comments: Considering the annealing temp gradient analysis, I think it would be interesting to present, at least as supplementary information, some compositional data (taxonomic classification of the obtained sequences/ASVs/OTUs) - to enable readers to have a glance at the biological consequences of the highly diverging annealing temperature effects between ´normal´PCR and DePCR PUP.


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: DECONSTRUCTING THE POLYMERASE CHAIN REACTION II: AN IMPROVED WORKFLOW AND EFFECTS ON ARTIFACT FORMATION AND PRIMER DEGENERACY
Review round: 2
Reviewer: 1

Output:
Basic reporting: All fine
Experimental design: The revised methods are now easier to follow, in particular trough he improved Figure 2 and the corresponding legend.
Validity of the findings: All fine
Additional comments: Than you for your responses and revisions in response to my comments. I have no further comments to add.


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: GENOME-WIDE ANALYSES OF THE BHLH GENE FAMILY REVEALS STRUCTURAL AND FUNCTIONAL CHARACTERISTICS IN THE AQUATIC PLANT NELUMBO NUCIFERA
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comments
Experimental design: The reason for the authors to select the stress treatment should be described.
Validity of the findings: Acceptable
Additional comments: It is well known that bHLH gene family is a very large family containing over a hundred of genes in most of the sequenced plant species. Because of this, great attention has been paid on the functions of this gene family. However, it is still far away to fully understand their functions because of the diversification during gene evolution. In this study, the author conducted a comprehensive analysis on the members of this gene family in a newly sequenced plant lotus, which is very helpful. The whole manuscript and its analysis are acceptable for me. But I still have some minor concerns as listed below:

1, there is a mistake on reference citing about the lotus database. It should be Wang et al., 2015
2，The authors conducted cold and salt treatment without telling us the reason for them to choose these two, and why should be 4oC and 50 mM NaCl.
3, Based on figure 1B, it seems the sequences among different members are quite conserved, which seems to be conflict with figure 2C about the prediction of their conserved motifs.
4, The authors conducted gene duplication analysis, which must has also been performed in some other species, such as Arabidopsis and rice. Why did not they compare this results with those from Arabidopsis and rice, or even others?
5, Since the authors just analyzed the expression of these genes in 4 tissues, it is hard to determine the tissue-specific genes.
6, English writing may need to be polished further.


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: GENOME-WIDE ANALYSES OF THE BHLH GENE FAMILY REVEALS STRUCTURAL AND FUNCTIONAL CHARACTERISTICS IN THE AQUATIC PLANT NELUMBO NUCIFERA
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript by Mao and colleagues present a large identification and bioinformatic analysis of the bHLH gene family in sacred lotus. This approach will be useful for subsequent research programs, especially on stress resistance.
I however have some reservations about this manuscript, that to my opinion needs serious improvement before publication.

1- It should be corrected by a native English speaker.

2- In accordance with the international nomenclature, “NNbHLH” should be replaced by “NnbHLH”.

2- Figure and table legends are imprecise. For instance (not restricted to):
- Figure 1: For each panel, a first “title” sentence is missing. In the sentence “The amino acid with identify more than …”, “similarity” should be used instead of “identity”. In the last sentence, “similarity” should be replaced by “identity”.
- references in Tables 1 and S5 are not given
- two distinct tables are given under the name “Table S4”

3- Many mistakes in the author names in the “References” section, including (but not restricted to):
- Line 571: “Gabriela” is a surname, not a name. “Gabriela et al.” should be replaced by “Toledo-Ortiz et al.” throughout the manuscript.
- Line 583 : “Hir RL” should be replaced by “Le Hir R”.
- Lines 676, 682 and 688: many first names are indicated instead of family names.

4- Lines 189-190: sources of the ratios in other species?

5- Line 206-209: Ledent et al. (2002) does not report this.

6- Lines 239-247: the sentences are copy-pasted from Niu et al. (2017), cited in the manuscript. I feel some other sentences of the manuscript have been copy-pasted from other publications.

7- Lines 336-383 are a useless re-writing of Table S5

8- Line 397: “functional analysis” should be replaced by “predictive analysis”

9- Line 412: “the other 7” should be replaced by “the other 6”

10-Figure 5: subfamilies should be indicated. NnbHLH could be listed from 1 to 115, for easier reading.
Experimental design: 11- The publication by Hudson and Hudson (2014), on bHLH family in sacred Lotus, should be mentioned in introduction rather than in discussion. The research question of the manuscript should be well explained, in view of this publication by Hudson and Hudson.

12- The growing conditions and treatments of the cold and salt stresses experiments are not well described.

13- In qPCR experiment, at least 3 housekeeping genes should be used.

14- Lines 387-395 : based on the figure, one cannot say that a gene is expressed specifically in a tissue, because the data are normalized by the average expression of bHLHs in each tissue.

15- Figure 6 (qPCR): what are the statistical tests? qPCR on control (non-treated) plants should be provided, to be sure that the variation in gene expression are not due to circadian regulation.
Validity of the findings: 16- Figure 1: why are some protein sequences truncated at the N-term? In the analysis (Table 1), the authors do not take into account these truncated sequences to calculate the frequency of conserved AAs, which is not correct.

17- Figure 2: Subfamilies 16 and 17 should not be separated in such a way, because the most recent common ancestor of subfamily 16 proteins is also ancestor of subfamily 17 proteins.

18- Figure 2: bootstrap values should be indicated.

19- Lines 201-205: the list of given “conserved amino acids” are actually not the most conserved ones. They correspond to the conserved positions identified by Atchley and colleagues (1999) in animals, but do not correspond to the conserved ones in plants (as shown by Toledo-Ortiz and colleagues (2003) and by other publications). It would have been more accurate and more interesting to present the true list of conserved amino acids and to compare them to what has been published in Arabidopsis, rice, brachypodium,…

20- Lines 301-303: the category “DNA binding transcription factor” (85 proteins) is not listed.

21- Lines 314-316: the cis-element ARE (88 promoters) is not listed.

22- Line 332: the new phylogenic tree should be provided. Rice data are not presented.

23- Line 399, “the expression level of the 15 NNbHLH were all increased”: this is untrue, based on the figure.

24- Line 474, “Then GO annotation revealed that most NNbHLH proteins were involved in…”: this is an over-interpretation, as the GO annotation can only suggest this.
Additional comments: 25- Line 192 : how many megascaffolds in total?


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: GENOME-WIDE ANALYSES OF THE BHLH GENE FAMILY REVEALS STRUCTURAL AND FUNCTIONAL CHARACTERISTICS IN THE AQUATIC PLANT NELUMBO NUCIFERA
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have answerred all my concerns. It is now acceptable for me.
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: GENOME-WIDE ANALYSES OF THE BHLH GENE FAMILY REVEALS STRUCTURAL AND FUNCTIONAL CHARACTERISTICS IN THE AQUATIC PLANT NELUMBO NUCIFERA
Review round: 2
Reviewer: 2

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: I would like to acknowledge the work of the authors, that considerably improved the manuscript. To my opinion, it is now suitable for publication in PeerJ and would be very useful for the scientific community.


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: ASSOCIATION BETWEEN COMPLEMENTARY AND ALTERNATIVE MEDICINE USE AND PROLONGED TIME TO CONVENTIONAL TREATMENT AMONG THAI CANCER PATIENTS IN A TERTIARY-CARE HOSPITAL
Review round: 1
Reviewer: 1

Output:
Basic reporting: Thank you for giving me the opportunity to review the article. The authors conducted a survey to reveal the use of alternative therapy among patients with cancer in eastern Thailand. I thought that the topic is interesting, but additional information mainly on the study methods should be provided. Comments have been listed below.

1. Basic reporting
Major Comments:
Title:
1.	The word “unconventional” should be replaced with “alternative”. It because that the authors used the word “alternative” in their questionnaire.
Abstract:
2.	L30: The number and % of the most common type of alternative medicine (i.e. herbal products) should be added.
3.	L35-36: The most important result of the multivariable regression analysis is better to be added.
Introduction:
4.	L48: References about the factor which contributes to the inferior survival rate should be added.
5.	L53: A reference such as a journal article or statements which provided by the government should be added.
Results:
6.	Table 1: The percentage for each group should be provided. This should not be calculated using the whole number of patients (which include both use and non-use patients). For example, a characteristic sex should be shown such like the below.
Example: For the “Use” group, male 115 (59.9%), female 77 (40.1%)

Minor Comments:
Introduction:
7.	Could the authors check the words “…can benefit select”? The authors mean “…can benefit for selected patients”?
Materials and Methods:
8.	L82-: Not only the original version, translated (English) version of the informed consent form should be provided (as for a supplementary material which will be published with the article).
Experimental design: Materials and Methods:
9.	L80: The ethical approval number and date should be added.
10.	L100: How many researchers review the medical record? If two or more researchers did review, they individually perform the review process?
11.	L121: Regression coefficients also should be provided as standardized regression coefficients. In addition to this, coefficients of determination is better to be presented.
12.	L121-: How to select the variables which include in the analysis? These factors may not be all the factors they collected in this study.
Validity of the findings: 13.	L82-: One of the most important point of the experimental design, the authors should provide the structured interview form of this study. If there is no structured form, reliability of the study results can be limited.
14.	L82-Is the structured interview form of this study validated before using? If not, this should be stated in the Discussion section.
15.	Table 1: Did the authors only collect the variables show in the table1? All collected variables should be shown in the Materials and Methods section to reduce the arbitrariness.
Additional comments: None


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: ASSOCIATION BETWEEN COMPLEMENTARY AND ALTERNATIVE MEDICINE USE AND PROLONGED TIME TO CONVENTIONAL TREATMENT AMONG THAI CANCER PATIENTS IN A TERTIARY-CARE HOSPITAL
Review round: 1
Reviewer: 2

Output:
Basic reporting: Reporting is clear and unambiguous.

One concern I have is to use the word complementary and alternative treatment instead of unconventional as that is not the right word to describe.

Something lacking is there is insufficient information on the gaps in literature. More citation is needed especially use of CAM around the world.
Experimental design: Fit the scope of the journal. Research question is meaningful. But the authors will need to be clear how different is the thai population compared to the rest of the world to justify the need for the study.

Methods are generally ok. Last line of statistical analysis is redundant.
Validity of the findings: The findings needed to be changed.

1. Should use word associations rather than correlations.
2. Need to adjust for the variables with p<0.1 in Table 1 as they are likely confounders.
Additional comments: Generally useful article. However, there are some concerns as above:

1. Need to justify a thailand study is important
2. what has been shown in other studies
3. statistical analysis need to be redone and preferably consult a statistician.


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: ASSOCIATION BETWEEN COMPLEMENTARY AND ALTERNATIVE MEDICINE USE AND PROLONGED TIME TO CONVENTIONAL TREATMENT AMONG THAI CANCER PATIENTS IN A TERTIARY-CARE HOSPITAL
Review round: 1
Reviewer: 3

Output:
Basic reporting: The English language should be significantly improved to ensure clarity and consistency.
Experimental design: The experimental design is fine.
Validity of the findings: The authors need to clarify what the waiting time meaning for cancer patients.
Additional comments: The quality of this manuscript needs significant improvement.  The major flaw of this study is that the multivariate linear regression analysis can only indicate correlation but not causality.

The introduction part needs much more details about how previous studies have done on a similar topic.

I would reject this paper for this Journal. Detailed comments please see my attachment.


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

Input:
Title: ASSOCIATION BETWEEN COMPLEMENTARY AND ALTERNATIVE MEDICINE USE AND PROLONGED TIME TO CONVENTIONAL TREATMENT AMONG THAI CANCER PATIENTS IN A TERTIARY-CARE HOSPITAL
Review round: 1
Reviewer: 4

Output:
Basic reporting: This is a paper describing unconventional therapy in the treatment of cancer in eastern Thailand, and the relationship between the use of unconventional therapy and waiting time for conventional therapy. I would appreciate the authors' dedication to the study design, analysis, and the manuscript.

The English language is professional, structure and figures are understandable. And the raw data is clear.
Experimental design: Please see the comments below.
Validity of the findings: Please see the comments below.
Additional comments: Major comments:
1.STROBE Statement (strengthening the reporting of observational studies in epidemiology) is a reporting standard, it will be useful to add detail information following the STROBE checklist.

2. The target population is cancer patients in eastern Thailand in the title, but the sample is cancer patients in one hospital only although it is a referral center. The representativeness is not well enough because patients refusing transfer or seeing a doctor in other places or countries after diagnoses are not included. Besides, the survey includes patients from other places not belong to eastern Thailand according to words in Table1. It could be discussed in the limitation part if possible.

3. Line 73# “Patients were non-randomly enrolled during or after conventional treatment for cancer”, the sampling method could be described in more details of how the investigators chose who will be surveyed, and what is the specific method such as systematic sampling, stratified sampling which belong to probability sampling or convenience sampling and purposive sampling which are part of nonprobability sampling.

4Line224# It would be better to narrow the range of target population instead of “patients with cancer in eastern Thailand”, because of the representativeness mentioned above.

Minor comments:

1. keywords of “cancer wait times” is not clear enough, try to change the expression as “wait time for cancer treatment” or something else. Study type could be added in the keywords list as well.

2. The introduction part briefly shows the background of the study, but medical policies are quite different all over the world so a more detailed background of the waiting time caused by the referral system is needed in this part.

3.Table 3: the definition of “use before Tx” and “use after Tx” could be explained clearer. Do you mean before or after a whole treatment course or for example one cycle chemotherapy?

4. Line 182#-183# There is no significant difference between the high-income group and the low-income group according to the survey, so it is not suitable to give a conclusion that unconventional therapy was more commonly used in the high-income group simply based on the percentage of using unconventional therapy.

5. Line 210# Not only the prevalence but also the constitution of unconventional therapy patterns should be discussed in the limitation part due to a hospital-based population.

Thanks for the opportunity of reviewing this 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: ASSOCIATION BETWEEN COMPLEMENTARY AND ALTERNATIVE MEDICINE USE AND PROLONGED TIME TO CONVENTIONAL TREATMENT AMONG THAI CANCER PATIENTS IN A TERTIARY-CARE HOSPITAL
Review round: 2
Reviewer: 1

Output:
Basic reporting: Thank you for giving me the opportunity to review the revised version of the article. The authors improved the contents of the manuscript according to the comments provided. Additional comments (AC) have been listed below.

1. Basic reporting
Major Comments:
Title:
1.	The word “unconventional” should be replaced with “alternative”. It because that the authors used the word “alternative” in their questionnaire.
Reply: Agreed, we have replaced “unconventional therapy” with “complementary and alternative medicine (CAM)”
AC: This point has been adequately corrected.
Abstract:
2.	L30: The number and % of the most common type of alternative medicine (i.e. herbal products) should be added.
Reply: We have added “Approximately 34.3% of these therapies involved unlabeled herbal products with unidentifiable components” in the abstract.
AC: This point has been adequately corrected.
3.	L35-36: The most important result of the multivariable regression analysis is better to be added.
Reply: We have added the regression coefficient of CAM use in the abstract.
AC: The value (regression coefficient) was added, but the meaning is still unclear in the abstract. So, the authors clearly state that the value means the additional days of waiting compared with the referenced category.
Introduction:
4.	L48: References about the factor which contributes to the inferior survival rate should be added.
Reply: We have added the following reference: ‘’Elit L. Wait times from diagnosis to treatment in cancer. J Gynecol Oncol. 2015;26(4):246-8.” (reference 3)
AC: This point has been adequately corrected.
5.	L53: A reference such as a journal article or statements which provided by the government should be added.
Reply: We have added the following reference: “Singh T, Pearlman PC, Kostelecky B. Supporting Evidence-Based National Cancer Control Planning: The Asia-Pacific Phase II Leadership Forum. J Cancer Policy. 2017;12:75-78.” (reference 4)
AC: This point has been adequately corrected.
Results:
6.	Table 1: The percentage for each group should be provided. This should not be calculated using the whole number of patients (which include both use and non-use patients). For example, a characteristic sex should be shown such like the below.
Example: For the “Use” group, male 115 (59.9%), female 77 (40.1%)
Reply: Agreed, the table 1 has been revised accordingly.
AC: This point has been adequately corrected.

Minor Comments:
Introduction:
7.	Could the authors check the words “…can benefit select”? The authors mean “…can benefit for selected patients”?
Reply: We have reviewed this with a professional language editor. “Select patients” indicates a treatment or intervention may benefit some patients, but not necessarily all patients.
AC: I understood the explanation provided by the authors.
Materials and Methods:
8.	L82-: Not only the original version, translated (English) version of the informed consent form should be provided (as for a supplementary material which will be published with the article).
Reply:  We have provided the English-translated version of the informed consent in the supplementary content.
AC: This point has been adequately revised.
Experimental design: 2. Experimental design
Materials and Methods:
9.	L80: The ethical approval number and date should be added.
Reply:  We have added the IRB approval no. in the material and method section.
AC: This point has been adequately revised.
10.	L100: How many researchers review the medical record? If two or more researchers did review, they individually perform the review process?
Reply: One investigator was responsible for medical record review. We have provided the medical record data collection form in the supplementary content.
AC: I understood the situation, thanks.
11.	L121: Regression coefficients also should be provided as standardized regression coefficients. In addition to this, coefficients of determination is better to be presented.
Reply: We have added regression coefficients and coefficients of determination in the table 3.
AC: This point has been adequately revised.
12.	L121-: How to select the variables which include in the analysis? These factors may not be all the factors they collected in this study.
Reply: All the factors that we had collected were shown in the manuscript. These factors were chosen because they have been found to be related to prolonged wait times in other studies.
AC: I understood the situation, thanks.
Validity of the findings: 3. Validity of the findings
13.	L82-: One of the most important point of the experimental design, the authors should provide the structured interview form of this study. If there is no structured form, reliability of the study results can be limited.
Reply: We used semi-structured interview method in this study. The English-translated version of interview protocol has been provided in the supplementary content. We have added the following sentence in the material and method section: “The interviews were conducted by five interviewers using a semi-structured interview protocol (Article S1).”
AC: This point has been adequately revised.
14.	L82-Is the structured interview form of this study validated before using? If not, this should be stated in the Discussion section.
Reply: The interview protocol was reviewed by hospital specialists, and all interviewers successfully completed practice interviews with the principle researchers before interviewing the patients. We have added this information in the manuscript.
AC: I understood the situation, thanks.
15.	Table 1: Did the authors only collect the variables show in the table1? All collected variables should be shown in the Materials and Methods section to reduce the arbitrariness.
Reply: Yes, all of variables were shown in the manuscript.
AC: I understood the situation, but the authors should discuss about the limited number of variables corrected in this study in the Limitation section.
Additional comments: None


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: ASSOCIATION BETWEEN COMPLEMENTARY AND ALTERNATIVE MEDICINE USE AND PROLONGED TIME TO CONVENTIONAL TREATMENT AMONG THAI CANCER PATIENTS IN A TERTIARY-CARE HOSPITAL
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The authors provided a satisfactory response.


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: ASSOCIATION BETWEEN COMPLEMENTARY AND ALTERNATIVE MEDICINE USE AND PROLONGED TIME TO CONVENTIONAL TREATMENT AMONG THAI CANCER PATIENTS IN A TERTIARY-CARE HOSPITAL
Review round: 2
Reviewer: 3

Output:
Basic reporting: See my detailed comments on the manuscript.
Experimental design: See my detailed comments on the manuscript.
Validity of the findings: See my detailed comments on the manuscript.
Additional comments: See my detailed comments on the manuscript.


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

Input:
Title: ASSOCIATION BETWEEN COMPLEMENTARY AND ALTERNATIVE MEDICINE USE AND PROLONGED TIME TO CONVENTIONAL TREATMENT AMONG THAI CANCER PATIENTS IN A TERTIARY-CARE HOSPITAL
Review round: 2
Reviewer: 4

Output:
Basic reporting: The language has been improved and and the added literature references are  sufficient for readers to understand.
Experimental design: In the translated information sheet, the purpose of the study did not mention "association between CAM use and its association with time to conventional treatment". Whether participants fully understand with the aim of the study?

The procedure of the study is well explained and ethical compliance.
Validity of the findings: The collection of data is acceptable. Conclusions are revised and well stated.
Additional comments: This is a meaningful study and the revised version is adequate to publish.


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: ASSOCIATION BETWEEN COMPLEMENTARY AND ALTERNATIVE MEDICINE USE AND PROLONGED TIME TO CONVENTIONAL TREATMENT AMONG THAI CANCER PATIENTS IN A TERTIARY-CARE HOSPITAL
Review round: 3
Reviewer: 1

Output:
Basic reporting: The manuscript has been revised appropriately according to the comments.
Experimental design: The manuscript has been revised appropriately according to the comments.
Validity of the findings: The manuscript has been revised appropriately according to the comments.
Additional comments: Thank you for giving me the opportunity to review the revised version of the article. The authors appropriately revised the manuscript according to the comments. I thought the manuscript can be accepted for publication in its current form.


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

Input:
Title: ASSOCIATION BETWEEN COMPLEMENTARY AND ALTERNATIVE MEDICINE USE AND PROLONGED TIME TO CONVENTIONAL TREATMENT AMONG THAI CANCER PATIENTS IN A TERTIARY-CARE HOSPITAL
Review round: 3
Reviewer: 2

Output:
Basic reporting: .
Experimental design: .
Validity of the findings: .
Additional comments: Attached