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: PREDICTION OF ACTIVE INGREDIENTS IN SALVIA MILTIORRHIZA BUNGE. BASED ON SOIL ELEMENTS AND ARTIFICIAL NEURAL NETWORK
Review round: 1
Reviewer: 2

Output:
Basic reporting: This paper is written in proper English, suitable for international audience. Paper background and the overall problem are described in clear and comprehensible way. Previous research referred by the authors are relevant and referenced correctly and in consistent form. Paper structure match the PeerJ standards. Figures and tables are adequate, properly described and supplement well the description of obtained results. Authors supplied raw data as well as additional figures. Nonetheless, minor drawbacks appear in several lines - detailed comments are given below.

Lines 45-49.
Please check the completeness of the first part of the sentence ‘Currently, 19 provinces in China, including Shandong, Henan, Sichuan, Hebei, Hubei, Jiangsu, Shanxi, and Shaanxi (Lu 2019), have been introduced and cultivated, but there are significant differences in the content of active ingredients in the same species of S. miltiorrhiza planted in different regions (Huang et al. 2019; Yang et al. 2011b).’ - the object is probably in wrong position.

Please consider such solution: ‘Currently, 19 provinces in China, including Shandong, Henan, Sichuan, Hebei, Hubei, Jiangsu, Shanxi, and Shaanxi (Lu 2019), have been introduced and cultivated S. miltiorrhiza, but there are significant differences in the content of active ingredients in the same species planted in different regions (Huang et al. 2019; Yang et al. 2011b).’

Lines 129-132.
I suggest to revise an order of the sentence ‘Therefore, in the BP training, select the input Xi of the input layer neuron i, the weight Wij from the input layer neuron i to the hidden layer neuron j, the threshold value θj of the hidden layer neuron j, and the output yj of the hidden layer neuron j is calculated by Equation 1:’ since it’s a little bit incomprehensible. The better solution would be to start the sentence with ‘The output yj of the hidden layer neuron j is calculated by Equation 1:’ or something similar, then give the formula followed by description of its all components (a description similar to given in lines 144-145 should be suitable).

Lines 135-137.
The same comment as above.

Lines 212-213.
There is probably lack of object in sentence ‘The water-soluble components (D1-D4, D11, and D12) showed a higher degree with Mn, Cu, Zn, N, P, and K.’ Degree of what? Please review this sentence.
Experimental design: Line 140.
Please check if the formula given in line 140 is correct. Usually the error function is the sum of squares (or sum of squared error known as SSE), which is minimized during training process. The formula given by Authors is rather square of sum, which could give the same results but only if in parenthesis the minuend is higher than the subtrahend. Moreover, Wang et al. (2019) after Rumelhart et al. (1986) claims that ‘The algorithm is a multilayer supervised learning network trained using the methods of mean square error (MSE) and gradient descent, which involves adopting a learning rule with the steepest gradient descent method to achieve the minimum sum of squared error.’, which suggests that formula given by Authors may be just written incorrectly. However, if formula is correct, please include some reference to justify this way of error computing.

Please provide information about transfer (activation) functions between input and hidden layer as well as between hidden and output layer of ANN. What were the functions? identity, sigmoid (S-shape), hiperbolic tangent, sinusoid, negatively exponential, Gaussian? Please provide the formulas or references, if possible. Additionally, information about number of training epochs, learning rate and convergence criterion should be given.

I suggest to explain clearly somewhere in the sub-division BP (back propagation) neural network what authors mean using term ‘Test data’ (it appears in Table 1). Generally, in ANN-related issues 3 kinds of data sets are reported: training set (other words input data or input vectors) - data presented constantly to the ANN in order to realise training process according to appropriate algorithm, validation set (the subset of input data) - data used for supervision of ANN training process, most of all to avoid network overfitting, which is very common in case of BP learning algorithm, and test set - data never used for training but necessary for final evaluation of ANN and calculation of fit-measures. I suppose that term 'Test data' concerns the latter, but very often term ‘validation set’ is used interchangeably with the term ‘test set’ and refers to assessment the performance of a fully-trained model, which might be confusing, especially for readers unfamiliar with ANN terminology. To avoid confusion please define clearly the role of test set or refer to an appropriate source (e.g. Ripley, 1996).

Ripley B.D. 1996. Pattern Recognition and Neural Networks. Cambridge University Press, UK., pp. 354.
Validity of the findings: Lines 146-147 and 223-264
I have a serious objection to the size of training data set, especially taking into account ANN architecture. It is not about the percentage (this is ok) but the quantity of training vectors (18 cases).  Figure 6 presents ANN structure with 8 inputs, 16 hidden nodes and 7 outputs which give 8×16×7 = 896 network connections (other words parameters (weights) to select in each iteration). Although selection of weights is realised on the training set, the final task of ANN is reproduction, which means that the real goal of network training is to minimize so-called generalization error, not training error. Generalization error is a function of data density and network complexity measure, called VCdim (Vapnik-Chervonenkis) measure (Vapnik, 1992). A small number of training samples (low data density), with a fixed measure of VCdim, leads to model overfitting and bad generalization, as the approximation task is reduced to interpolation and the model becomes susceptible to reproduction of disturbances as a basic features. According to Hush and Horne (1993) good generalization of ANN may be obtained if number of training vectors exceeds VCdim by at least 10 times. Since there is no simple relationship between VCdim measure and network architecture, it is only possible to estimate its lower and upper limit, which approximately correspond to number of weights connecting input with hidden layer and twice the number of all network weights, respectively. In practice, VCdim measure is assumed as the number of all network weights (number of all network connections). Having ANN architecture proposed by the Authors, minimum 8960 training vectors are required to ensure good generalization abilities of such model. Dataset contains only 26 observations, one for each specific location and plant type (cultivated or wild). Since there are no biological replications for each location, no variability of studied natural phenomenon was captured which also provides to uncertainty of other results presented in this paper. I strongly suggest to include additional training vectors (much more than 18) by making biological replications. With additional data Authors may use also some analytical methods based on known algorithms, e.g. bootstrap, to increase the size of training set – nonetheless these methods need data dispersion (variance). Then I suggest to (a) revise the ANN architecture towards reducing the number of hidden neurons or (b) try different solution, e.g. the set of networks (one for each dependent variable to reduce network connections) or (c) try different kind of network which realizes multidimensional regression and is much more resistant to a limited number of training vectors, e.g. Support Vector Machine for regression (Drucker et al., 1997).

Vapnik V.N. 1992. Principle of risk minimization for learning theory. In: Moody J., Hanson S., Lippmann R., (eds). Advances in Neural Information Processing Systems 4 (NIPS 1991), Morgan Kaufmann, San Mateo, pp. 831 – 838.

Hush D., Horne B. 1993. Progress in Supervised Neural Networks. IEEE Signal Processing Magazine, 10: 8 – 39.

Drucker H., Burges C.C, Kaufman L., Smola A.J., Vapnik  V.N. 1997. Support Vector Regression Machines. In: Advances in Neural Information Processing Systems 9 (NIPS 1996), MIT Press, pp. 155 –161.

Line 193.
Since cluster analysis is not a statistical analysis, no statistical tests (like multiple comparisons) are performed to assess the significance of differences between data vectors. In addition there is impossible to perform such comparisons using statistical approach, since there is no data replication in dataset, hence no dispersion measure may be calculated. Therefore, it may be written that Shandong and other producing areas differ in terms of analysed features, but nothing confirms that these differences are significant (this term is reserved for specific, statistical uses). Moreover, please justify the need for conducting cluster analysis. Did its results have any impact on further work on the neural model?

Lines 197-198.
It is about a part of the sentence which starts in line 194.  Authors write: ‘…while other active ingredients were correlated with major and trace elements, but not significantly’ - if correlation is not significant (null hypothesis about r = 0 should not be rejected), it means that there is no correlation (linear one of course), thus further consideration of such dependency is not justified.

Line 202-204.
Collinearity is not the same as correlation. Authors did not check collinearity statistics, e.g. VIF, condition index or proportion of variation. Where conclusion about multicollinearity came from? Finally collinearity is not an error and does not exclude possibility of building a valid regression model.

Figures 4 - 5 and corresponding description in lines 209-212.
This comment concern mainly description of B, Fe and less Cu trends in figures 4 and 5. Scatter plots between these soil elements and components of S. miltiorrhiza shows that some points may be ‘suspected’ of being outliers. It is also possible that dataset consists of more than one subpopulation but it is impossible to find out if it is so since dataset contains too few observations. It is risky to discuss a trend when it is formed by only one or two observations, without checking their influence statistics. For the future, please check influence statistics and identify potential outliers or consider data which form different clusters separately to avoid so called Simpson paradox (Simpson, 1951).

Simpson E. H. 1951. The Interpretation of Interaction in Contingency Tables. Journal of the Royal Statistical Society, Series B, 13: 238-241.

Conclusion

In my opinion research issue described here is very interesting, which is one of the strengths of the paper. Another strength is clear, concise and generally reliable description of research tasks (with minor failings).

Nonetheless, this work has serious methodological drawback and that is why I suggest a major revision of this paper. I'm not convinced to the results obtained by the Authors. This is due to the lack of proper representation of analysed phenomenon. At the same time, I strongly emphasize that I have no objection to the method of obtaining data from the collected soil and plant samples, but to the lack of fundamental feature of experimental data - replication, which provide the knowledge about variability/diversity of natural phenomena. Consequently, the authors obtained high value of correlation coefficients which turned out to be insignificant (this is an indication that something is wrong with the data). In addition, it is not possible to check the basic properties of variables (distributions, collinearity, the influence of observations) and to use classical statistical methods to learn them more deeply (the lack of repetition excludes most analyses based on variance). Unfortunately, it is highly likely that the results obtained for the neural network are the effect of model overfitting, what may be seen in Figures 7-10 (too much adjustment to some data and the inability to reproduce the general relationship).
Additional comments: I would like to encouraged the Authors to improve the dataset, revise ANN architecture as I recommended above and repeat procedure of ANN model creating. Please treat this experimental work as a preliminary study of your problem. I will be glad to read the paper again, after improvements.


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: PREDICTION OF ACTIVE INGREDIENTS IN SALVIA MILTIORRHIZA BUNGE. BASED ON SOIL ELEMENTS AND ARTIFICIAL NEURAL NETWORK
Review round: 1
Reviewer: 3

Output:
Basic reporting: No comment.
Experimental design: This study is required because the roots of Salvia miltiorrhiza Bge. are used in the treatment of cardiovascular diseases. The article will not be accepted in this format. Some major revisions needed.
•	The author described that content of active ingredients of S. miltiorrhiza planted in different regions of the soil environment are different. In my opinion the content of active ingredients not only varies due to some soil factors but also due to environmental factors, altitude, pH of the soil etc. The developed model is based upon soil factors only. Without the environmental factors it is impossible to select the suitable cultivations sites. So if possible develop the model with both soil and environmental factors.
•	Give voucher no for each collected areas.
•	Add the article:   Abdul Akbar, Ananya Kuanar, Raj K Joshi, I S Sandeep, Sujata Mohanty, Pradeep K Naik, Antaryami Mishra, Sanghamitra Nayak (2016) Development of Prediction Model and Experimental Validation in Predicting the Curcumin Content of Turmeric (Curcuma longa L.). Frontiers in Plant Science 7: 1507.
Validity of the findings: no comment
Additional comments: no comment


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

Input:
Title: PREDICTION OF ACTIVE INGREDIENTS IN SALVIA MILTIORRHIZA BUNGE. BASED ON SOIL ELEMENTS AND ARTIFICIAL NEURAL NETWORK
Review round: 1
Reviewer: 4

Output:
Basic reporting: The experiment was inappropriately designed for the stated goals and research topic.
The authors did not define research hypotheses, but briefly mentioned the purpose in the introduction. The introduction is a loose discussion of the problems that were described in the paper. The data obtained from the experiment do not allow the use of most of the used statistical methods and artificial neural networks.
Experimental design: In designed experiment 26 data records were obtained from 25 locations.
ANN assume learning based on a very large number of responsive data records.
A network model with 8 neurons in input layer, 16 neurons in hidden layer and 7 in output layer was presented. The assumption is correct, but the implementation is not. The number of connections between the input layer and the hidden layer is 128. a minimum of 1280 experimental cases is needed to teach such a network. The authors trained it with 18 samples and verified with 8. This is against the assumptions of the use of ANN.
Correlation values between elements and active ingredients were rightly given in the results. Unfortunately, the obtained data do not meet the assumptions of a normal distribution, which the authors ignore in the study. Figures 4 and 5 show that only a few of the studied variables had a distribution that did not differ from the normal distribution for which the correlation method can be used.
Validity of the findings: On the basis of such a small experiment, it is not possible to generalize the relationships and dependencies between the elements in the tested soil and the content of active substances in the plant.
The authors do not present the source data, but it can be concluded from the description that the only method used correctly was hierarchical cluster analysis. Unfortunately, the methodology does not describe it in detail.
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: PREDICTION OF ACTIVE INGREDIENTS IN SALVIA MILTIORRHIZA BUNGE. BASED ON SOIL ELEMENTS AND ARTIFICIAL NEURAL NETWORK
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The manuscript has been revised accordingly.


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

Input:
Title: PREDICTION OF ACTIVE INGREDIENTS IN SALVIA MILTIORRHIZA BUNGE. BASED ON SOIL ELEMENTS AND ARTIFICIAL NEURAL NETWORK
Review round: 2
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: In general I'm satisfied with all improvements made by the Authors and I recomend this paper for publication in PeerJ.
My only comment concerns the caption of Figure 6. Please check the captions (A) and (B) for proper singular and plural forms of words 'plots' and 'datasets'. I suppose that each scatterplot represents outputs varsus targets of one dataset where n = 26 or n=41 means the number of observations. Thus, shouldn't these captions be:
'(A) Scatter plot of outputs versus targets values of dataset (n=26); (B) Scatter plot of outputs versus targets values of new dataset (n=41).' ?


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: PREDICTION OF ACTIVE INGREDIENTS IN SALVIA MILTIORRHIZA BUNGE. BASED ON SOIL ELEMENTS AND ARTIFICIAL NEURAL NETWORK
Review round: 2
Reviewer: 3

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


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

Input:
Title: A REVIEW OF THE COMMON CRAB GENUS MACROMEDAEUS WARD, 1942 (BRACHYURA, XANTHIDAE) FROM CHINA SEAS WITH DESCRIPTION OF A NEW SPECIES USING INTEGRATIVE TAXONOMY METHODS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Before all, the text requires much elaboration, which should be made before formal submission.
Experimental design: See above.
Validity of the findings: The recognition of the new species seems to be warranted by genetic and morphological evidence.
Additional comments: No additional comments


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

Input:
Title: A REVIEW OF THE COMMON CRAB GENUS MACROMEDAEUS WARD, 1942 (BRACHYURA, XANTHIDAE) FROM CHINA SEAS WITH DESCRIPTION OF A NEW SPECIES USING INTEGRATIVE TAXONOMY METHODS
Review round: 1
Reviewer: 2

Output:
Basic reporting: TThe MS deals with a taxonomically very complicated genus of brachyuran crabs. Its well-structured and clear written. It describes a new species, which I think is a good distinct specie. The drawings and photos are in high quality condition.

But there are some comments, which I propose to be applied before get it published

1.	Abstract is very short and vague. Please provide some details of your result and conclusion. Your abstract has actually no data, just some vague citation. You can give name of your new records, amount of recorded species of the genus some phylogenetic and biogeographical information.

2.	The English of the MS must be checked and improved. For instance, in the first line of Introduction "The genus Macromedaeus Ward, 1942, currently including six described species found from Indo-West Pacific"

"including" must be "includes"
"Indo-West Pacific" must be "the Indo-West Pacific"

3.	There is no mention about the state of propodus dactylus articulation in M. orientalis.

4.	The subterminal spine of ambulatory legs, usually, is present in the species with propodus-dactylus articulation. What is the state of this key (subterminal spine) character in other species? and what the implications might be?

5.	The syntypes of M. demani are juvenile and the detailed morphology of G1 are not distinguished. In addition, one of the characteristics of M. demani is lack of anterolateral teeth and presence of anterolateral lobes–based on serene (1984). How can we be sure that the characters stated in the manuscript belong to M. demani and not from a new species? Given the characteristics of the syntype M. demani slightly differ from those of the specimen in the manuscript.

6.	There is no taxonomical account on M. crassimanus.

7.	The taxonomical account of M. demani is the iteration of what Serene (1984) and Guinot (1968) stated. Additionally, the authors mention that some authors like serene (1984) list M. demani as a synonym of L. subacutus. However, Serene (1984) listed M. demani a separated species.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: no comment


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

Input:
Title: EXPLORING THE POTENTIAL BIOMARKERS FOR PROGNOSIS OF GLIOBLASTOMA VIA WEIGHTED GENE CO-EXPRESSION NETWORK ANALYSIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: In the present study, the authors are intended to identify biomarkers that can be used for predicting the prognosis of glioblastoma. Overall, the study was well conducted, and the manuscript was clearly written. However, there are still some major/minor concerns.

1. The study is intended to identify biomarkers for GBM prognosis but not therapeutic targets, so I think the title of the manuscript is accurate and should be revised.

2. Please add the reference for the following sentences:
Introduction
(1) “Glioblastoma (GBM) is the most common primary neurogenic tumor, and the prognosis of most subtypes is poor.”
(2) “The main reasons for the poor prognosis of GBM are due to tumor metastasis and postoperative recurrence.”
(3) “Given that tumors invade the brain aggressively, GBM tumors can rarely be completely removed by surgery.”
(4) “In the past few decades, gene sequencing and bioinformatics analysis have been widely used for genetic variation screening at the gene level…”
Results
(1) “Among them, CDC73 and CDC123 are cyclins of cell division, B4GALT1 is one of …”
(2) “These four genes have been shown to have a close relationship with many kinds of cancers, such as thyroid carcinoma (Sarquis et al. 2019), breast cancer, etc”
Discussion
(1) “…molecular heterogeneity affects the effectiveness of single-molecule markers in predicting …”
(2) “At the same time, some studies have found that the high recurrence rate of GBM is …”
(3) “And these processes usually involve multiple genes.”
(4) “Among them, transition metals are critical for many metabolic processes, and their steady state is vital to life.”
(5) “…DARS gene encodes the aspartyl-tRNA synthetase，which pairs aspartate with its corresponding …”
(6) “… never been reported to be associated with the development and progression of GBM.”
(7) “… advantages, such as high prediction accuracy and personalized detection results.”

3. Please indicate why some samples are highlighted (green color) in figure 1a.

4. The resolution of some figures is low and the legends in those figures cannot be seen clearly.

5. Some abbreviations do not have full names (e.g. SCNAs, CNV). Please provide their full names.

6. As the authors described at the beginning of the manuscript, single gene analysis may not best predict the pathogenesis and prognosis of glioblastoma. Why not the authors establish a prediction model by combining all the hub genes and/or other factors together?
Experimental design: 1. I am confused about the research goals of this study since the authors described that they want to identify biomarkers for glioblastoma while they compared the co-expressed gene modules between untreated glioblastoma patients and the recurrent cancer patients. I think a better experiment for knowing the biomarkers is to compare glioblastoma tissue with normal brain glia cells. If the authors want to know what genes are related to the recurrence of glioblastoma, a better comparison would be the recurrent cancer patients vs. non-recurrent cancer patients. Please explain why comparing untreated cancer patients with recurrent patients.

2. Please list all specimen IDs acquired from the TCGA database in a supplementary table.

3. Please clearly describe the criteria for choosing the four hub genes (DARS/GDI2/P4HA2/TRUB1) in the methods.

4. Please define “mRNA high” and “mRNA low” in figure 7.
Validity of the findings: 1. The findings in figure 6a is not fully described in the context (e.g. P4HA2 and CD8+ T cells are also highly correlated). Please clearly describe all findings in the results.

2. Figure 6B shows that different mutations in the 4 hub genes can significantly affect immune infiltration, but not many genetic alterations were found in the GBM samples that are used in the study. Please explain this difference.
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: EXPLORING THE POTENTIAL BIOMARKERS FOR PROGNOSIS OF GLIOBLASTOMA VIA WEIGHTED GENE CO-EXPRESSION NETWORK ANALYSIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Zhang et al. used co-expression to identify several genes that are associated with the relapse/survival of glioblastoma. They subsequently investigated how these candidate genes were associated with known cancer-related pathways, drug response, immune response etc. While this study is well motivated, there are a few issues in how the authors designed the analyses and validated their findings.
Experimental design: See comments to authors.
Validity of the findings: See comments to authors.
Additional comments: 1. The authors used GSE145128 to identify candidate genes. Since this is not a particularly big dataset, the generality of the authors’ findings is a concern. In addition, the samples of glioblastoma relapse in this dataset were all treated by temozolomide, raising the possibility that the markers identified by the authors can be related to this treatment. Unless the authors have a valid reason to use this single dataset (which they did not explain in the manuscript), I am not convinced that their findings generally apply to glioblastoma cases.
2. How the four hub genes were identified is not clearly described. Table 1 shows that there are 14 genes that are significantly associated with patient survival – how did the authors filter these 14 genes down to four candidates? Did the authors analyze GSE145128 to find the 14 genes, and then validate them with TCGA to find the four candidates? Since this part is unclear, I am not sure if the authors have tested whether their markers can predict patient survival, which undermines the validity of their central findings.
4. While the authors claimed that they were searching for “hub” genes in the darkturquoise module, they did not use the connectivity of a gene as the searching criteria. Instead, they used the correlation between genes and patient survival. To me, what they found should not be called hub genes, and the authors should rephrase the related texts.
5. I am not sure why the authors searched TCGA for genes that co-expressed with the four candidate genes. The rationale of WGCNA is clustering genes based on how well they co-express with each other, therefore the additional genes found in TCGA should already present in the darkturquoise module. On the other hand, if these genes were not found by WGCNA already, then the authors need to explain why they ignored the lack of the co-expression in GSE145128 and focused on TCGA.
6. The authors need to be careful with the analysis on immune cell filtration. Despite recent studies have suggested that certain T cells can populate brain, there may not be enough evidences that support the presence and functions of other immune cells in brain (I can very well be wrong on this point since I am not an expert in immunology). Can authors provide literature to support the presence and functions of B cells and neutrophils in brain?
7. I found the following figure legends unclear:
Fig2C: is each node a gene? What does the size of the node mean? The texts “Enrich the term network” are unclear.
Fig2B: the Y axis label “Gene significance for luminal” needs more explanation.
Fig3: what is the cutoff for low versus high expression?
Fig5A: what does the size of an area in the pie chart mean?

Minor:
1. The sections “Gene functional analysis” and “Cancer-related pathways and drug sensitivity analysis” are under the same topic. Perhaps the authors should reorder the sections?
2. The authors used “darkturquoise” to refer the WGCNA module of interest, but the term itself does not have a biological meaning, making it especially confusing when placed in the abstract. The authors should find a better term.
3. I think Fig1 should be moved to the supplement; it shows only technical details of the WGCNA analysis. Same for the most panels of Fig4, as they are showing redundant information.
4. The section “Genomic alterations of DARS/GDI2/…” is a bit thin. Can the authors say more on this topic, like are these mutations common enough to be used as therapeutic markers in GBM?


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: EXPLORING THE POTENTIAL BIOMARKERS FOR PROGNOSIS OF GLIOBLASTOMA VIA WEIGHTED GENE CO-EXPRESSION NETWORK ANALYSIS
Review round: 2
Reviewer: 1

Output:
Basic reporting: All my questions were resolved satisfactorily by the authors. I suggest accepting the manuscript for publication.
Experimental design: N/A
Validity of the findings: N/A
Additional comments: N/A


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

Input:
Title: EXPLORING THE POTENTIAL BIOMARKERS FOR PROGNOSIS OF GLIOBLASTOMA VIA WEIGHTED GENE CO-EXPRESSION NETWORK ANALYSIS
Review round: 2
Reviewer: 2

Output:
Basic reporting: I thank the authors for revising the manuscript, but some of my concerns were not adequately addressed. I am willing to consider the manuscript again, if the authors can address my questions seriously.
Experimental design: Please see comments to the authors.
Validity of the findings: Please see comments to the authors.
Additional comments: 1.  How the four hub genes were identified is still unclear, despite the authors’ explanation in the response. The authors wrote that “our primary criterion for identifying core genes was whether they had an impact on tumour prognosis, followed by KM survival analysis of all genes using the DARK module, in which DARS/GDI2/P4HA2/TRUB1 had a strong impact on survival and ranked higher.” Table 1 shows that genes like FKBP1B and DREU1 have lower P-value than DARS and TRUB1, then why aren’t these genes considered candidate genes? How was the strength of the impact on survival measured and ranked?
In addition, I had difficulty identifying the relevant changes in the manuscript, because there are a lot changes in the manuscript. Can the authors include a copy of the revised texts in their response?

2. To identify potentially important genes from a WGCNA module, both the connectivity of the genes and the associations between the genes and patient survival are valid criteria. But when the authors wrote that “hub genes have the highest connectivity in the module” and did nothing related to connectivity, it is really misleading. In their response to my comment, the authors claimed that the way they determined hub genes was identical to Lin et al. (2019). However, Lin et al. (2019) clearly wrote that “The hub genes of each module were selected based on the number of edges (degree of connectivity) associated with the corresponding nodes present within the network.”
I think “key gene” is a better term. Whether genes that are associated with survival necessarily have high connectivity is irrelevant to the goal of this study.

3. I asked authors to explain why they included additional genes that co-express with the four hub genes in the TCGA dataset. In their response, the authors cited Wang et al (2021) and Zheng et al (2021) to support their use of TCGA. However, these two studies did not use co-expression to identify the candidate genes from the non-TCGA data, so looking for additional genes that co-expressed with the candidate genes in TCGA does not produce contradiction. The current study identified candidate genes based on co-expression. If the expression of the candidate genes and that of the additional genes correlate positively in TCGA but negatively in the non-TCGA data, there will be a contradiction. While I understand it can be impractical to replace this analysis with a new one, the authors did not even try to address this problem in a meaningful way. With minimum effort, the authors could have verified if the expression of the additional genes and the four candidate genes are positively correlated in the non-TCGA data, or better verified if the additional genes are in the same or nearby WGCNA modules.

4. The authors’ response to my question on Fig2B is unfortunately not helping. Please define how “Gene significance for luminal” and “Module membership in darkturqouise module” in Fig2B were calculated.


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: EXPLORING THE POTENTIAL BIOMARKERS FOR PROGNOSIS OF GLIOBLASTOMA VIA WEIGHTED GENE CO-EXPRESSION NETWORK ANALYSIS
Review round: 3
Reviewer: 1

Output:
Basic reporting: I thank the authors for clarifying my confusions! I can recommend the paper for acceptation, pending on a few minor revisions:

1. The authors have added texts to explain how the four candidate genes were chosen. Some references are needed after these sentences: Line 174 (PDF) "previous research experience in our lab" and Line 176 "confirmed to be related to tumorigenesis".

2. I am glad the authors found most of the co-expressed genes are also present in the darkturquoise module. To prevent other readers from being confused like I was, I suggest the authors notify readers about the membership of these genes in the co-expression analysis. Line 184, after "TRUB1 and CUL2 have the highest correlation", seems a good place for the notice and a reference to supplement 2.

3. Regarding Fig2B, the authors should explain the the x- and y-axis labels in the legend as they did in the response to my comment. Not all readers are familiar with WGCNA. I think explaining "module membership" as "correlation in expression between the given gene with the eigengene of the module" is sufficient. "Significance to luminal" seems to be a correlation between the expression of the gene and the trait luminal. The key question is how did the authors quantify this trait? Did they calculate it from certain information of the patients? Please clarifying this in the Materials and Methods section.

I trust the authors will fix these minor points, and I do not need to see a formal response again.
Experimental design: NA
Validity of the findings: NA
Additional comments: NA


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: FUNCTIONAL GENETIC VARIANTS IN COMPLEMENT COMPONENT 7 CONFER SUSCEPTIBILITY TO GASTRIC CANCER
Review round: 1
Reviewer: 1

Output:
Basic reporting: Please see my additional comments.
Experimental design: Please see my additional comments.
Validity of the findings: Please see my additional comments.
Additional comments: This manuscript by Siyue Wang et al. entitled “Functional genetic variants in complement component 7 confer susceptibility to gastric cancer” investigated the relationship between complement component 7 polymorphisms and the risk of gastric cancer (GC). This study is interesting and of potential clinical significance if the following issues are revised.
1.	In text line 143, “After adjusted by gender, age, drinking and smoking status, non-conditional logistic regression analysis showed that the distribution of C7 rs1376178 AA and CA genotypes was statistically different between cases and controls (P<0.001)”. Why were these variables, such as gender, age, drinking and smoking status selected?
2.	In text line 163 “individuals with rs1061429 AA had an increased risk of gastric cancer among youngers (OR=2.84, 95%CI=1.39-5.80, P=0.004) and non-smokers (OR=2.79, 95%CI=1.63-4.77, P<0.001), but not among elders (OR=1.81, 95%CI=1.00-3.28, P=0.050) and smokers (OR=1.03, 95%CI=0.43-2.5, P=0.947)”. What are the possible explanations?
3.	In text line 166, “the stratification analysis by gender or drinking status showed that rs1061429 AA genotype was contributed to the risk of gastric cancer regardless of gender and drinking status (OR = 1.94, 95%CI = 1.13-3.33, P = 0.017 for males; OR=2.87, 95%CI=1.26-6.54, P=0.012 for females; OR=1.92, 95%CI=1.18-3.13, P=0.008 for nondrinkers; OR=5.23, 95%CI=1.34-20.40, P=0.017 for drinkers)”. Isn’t OR=5.23 significantly different from OR=1.92?
4.	In text line 176, “we explore the potential function of gene C7 in gastric cancer, based on GEPIA data, our results showed that the level of C7 mRNA in gastric cancer tissues (n=408) was significantly depressed compared with that in adjacent normal tissues (n=211)”. How does the expression of C7 reflect the potential function? Is SNP correlated with C7 expression level? Is C7 level associated to survival in gastric cancer?
5.	In Discussion, authors discussed the role of C7 in other types of cancers. Are C7 rs1376178 and rs1061429 SNPs also associated to other cancers?
6.	In text line 203, “we speculated that the change of C to A in C7 rs1376178 site enhanced the binding capability of transcription factors STAT1”. In C7 rs1376178 C>A samples, is STAT1 upregulated?
7.	In text line 242, “functional experiments are still needed to be done in the future”. What functional experiments? Although the title is “Functional genetic variants in complement component 7 confer susceptibility to gastric cancer”, authors did not provide any functional data in 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: FUNCTIONAL GENETIC VARIANTS IN COMPLEMENT COMPONENT 7 CONFER SUSCEPTIBILITY TO GASTRIC CANCER
Review round: 1
Reviewer: 2

Output:
Basic reporting: Comment 1: Line 67-68, it seems a bit abrupt to talk about DAF without first introducing to the readers on DAF and how it's related to Complement component family. Furthermore, 2-3 cases should be given to support "Complement gene polymorphism is closely related to the occurance of cancer"

Comment 2: In the method part, please provide the criteria for people that were selected as smokers and drinkers.
Experimental design: Comment: It is inappropriate to call Fig. 1 a "Function analysis of C7" when it's merely a expression profiling analysis. In 1(A), the authors have studied only on the rs1061429 genotype but not rs1376178, please explain why. This reviewer is also concerned about the imbalanced group sizes in 1(B), a reduced analysis with an equal number of samples per group should be performed to confirm the findings. It is confusing to the readers to say " gastric cancer compared to levels in normal stomach tissues" when the comparison was actually done between the cancerous tissues and adjacent normal tissues, which could have a totally different C7 expression than completely cancer-free tissues. Last but not least, the authors should consider repeating the same analysis in (A) and (B) using your own genotype and gene expression data which were collected on peripheral blood DNA from cancer patients and healthy people rather than gastric tissue DNA from cancerous and adjacent tissues and compare if the findings remain the same.
Validity of the findings: Comment: More analyses should be done to support the valuable role of C7 as a prognostic tool for gastric cancer. The following are some of the reviewer's suggestions:

1.  Kaplan-Meier curves according to the C7 expression levels for progression free survival and overall survival of patients with gastric cancer

2. Kaplan-Meier curves according to different variants in each C7 genotye for progression free survival and overall survival of patients with gastric cancer

3. Odd ratios in association of C7 expression level with clinicopathological factors such as histology, grade and clinical stage

4. Odd ratios in association of different C7 variant in each genotype with clinicopathological factors such as histology, grade and clinical stage
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: FUNCTIONAL GENETIC VARIANTS IN COMPLEMENT COMPONENT 7 CONFER SUSCEPTIBILITY TO GASTRIC CANCER
Review round: 2
Reviewer: 1

Output:
Basic reporting: Authors have successfully responded to all my comments.
Experimental design: None.
Validity of the findings: None.
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: SIXTEENTH-CENTURY TOMATOES IN EUROPE: WHO SAW THEM, WHAT THEY LOOKED LIKE, AND WHERE THEY CAME FROM
Review round: 1
Reviewer: 1

Output:
Basic reporting: See Additional Comments
Experimental design: See Additional Comments
Validity of the findings: See Additional Comments
Additional comments: Review of Sixteenth-century tomatoes in Europe: who saw them, what did they look like, and where did they come from?

Van Andel et al. present a very valuable study to the origin of tomatoes in Europe, using an approach that leverages the few surviving 16th-century herbaria. Specifically, the paper aims (l 142 143) to provide a more accurate overview of early sixteenth-century descriptions, illustrations and herbarium specimens of the tomato. This is a much needed effort, because discussions on tomato origins span well over a century, and this literature is rather littered with incomplete, incorrect or contradictory information. Also, this must have been rather challenging given the diverse array of languages of the relevant sources. In addition, the study presents a fraction of an ancient tomato genome (ca. 1.2%), which the authors downplay as being not very important due to it being so fragmented.

Overall, I am quite enthusiastic about this paper.  The topic is highly relevant, the methods are suited to address the questions, the historic research is executed with great care and comes across as rather comprehensive.  I also appreciate the effort the authors took in presenting this study rather well-readable; the structure of the Results here certainly helped.  The overall result, that the early tomatos in europe were already quite diverse in traits, and point to both peruvian and mexican heritage, is convincing.

Nonetheless, I have a few comments, which I trust the authors will be able to address.

My main criticism is the presentation and the interpretation of the ancient tomato genome.
- Foremost, the authors continuously refer to a study Michels (2020) which is not cited in the literature.  So either the ancient genome is already published, in which case it should not be presented as Results of this paper, or it is not yet published, in which case many relevant methodological details are missing (see below).
- Also the rather sweeping interpretation of the ancient genome is a bit surprising. In particular, although the authors state further ancient genomes may generate exciting results (l 601), they also state rather strongly that clues on historic traits of tomatos should not be sought in ancient genomes but in extant "ancient-like" cultivars and landraces. As written, it comes across as dismissing ancient-genomic research altogether (possibly because their result of 1.2% genome is rather modest?) which is probably not what the authors intended.
- Finally, the topic of ancient genomics should be properly separated in Methods, Results and Discussion, which is now all presented in Results.

Some examples of missing or confusing information on the ancient genomics part:
- where is the genome made publicly available?
- Illumina TruSeq is a library preparation method, not a sequencing platform
- what DNA sequencing method and library prep method was used?
- what mapping approach was used?
- how was it established that the sequencing results were authentic, ancient DNA and not any form of contamination (e.g. signs of cytosine deamination at the end of sequences?)
- how was heterozygosity computed to conclude that the sample was "quite heterozygous"?
- genetic diversity of a population does not necessarily lead to many autapomorphies in an exemplar sequence (autapomorphies say something about allele divergence which is a function of time, mutation rate and population size, not about genetic diversity which simply counts the number of different alleles across individuals of a population.  They may well coincide but do not need to necessarily).
--> because all these questions remain, if the ancient genome has not yet been published, this part of the manuscript should be re-assessed.

Overall, I would recommend to deal with the ancient genomics more in a hypothesis testing presentation, for instance something along the lines of: "if established ideas that tomatos were introduced from mexico, our ancient genomes closest sister groups should be more closely related to landraces from mexico than from south america" in the introduction.  Right now, it comes across as "bolted on" to an otherwise historic herbarium and literature study.

Another general remark concerns Aldrovandi:
l 243-244 states that no information of the origin of tomato specimens of Aldrovandi survive, while l 396-402 speaks of "extensive collection of Aldrovandi's manuscript" that might contain more information but that could not be verified. These two bits are in conflict: so what do we really know about where Aldrovandi had his specimens from?  Why could these sources not be verified? Beyond the scope of this study? That would of course be understandable, but it would help to be explicit in the text.

Some more, all minor line by line comments:

l 82 - 95. Somewhat incomplete; especially works of Razifard et al. seem critical here.

l 228. It would be helpful to state where Matthioli spend his days later in life.

l 263. Please indicate whether you suspect the 17 to be the "final number".

l 304. "admire" may be sub-optimal as I suspect Gesner was primarily interested in studying the collections?

l 352 "has no name". This formulations make it seem odd; it would help to be more explicit regarding Matthioli's words, as in l 185-186 (or refer to that bit explicitly.

At various places in the introduction (l 76, 77, 103), early names for the tomato are translated.  It would help to also state them literally in the original language.

Figure 1 and 2 are not particularly aesthetically arranged.
The color coding in Fig 3 is a bit unclear because it is rather small.  Also, it should be made more clear where the new specimen is located (arrow?).


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: SIXTEENTH-CENTURY TOMATOES IN EUROPE: WHO SAW THEM, WHAT THEY LOOKED LIKE, AND WHERE THEY CAME FROM
Review round: 1
Reviewer: 2

Output:
Basic reporting: I consider this contribution to be within the standards required by PeerJ.
In this review I am contributing with my comments and suggestions, which I have also been indicated in the revised document (see attachment)
The article has been written in a technically correct way, although I think some paragraphs are very long and could be summarized.
The texts and bibliographic citations in Latin should be indicated in italics, as well as scientific names. It is necessary to review the entire manuscript, including the Bibliographic list, Figures and Tables.

The authors have conducted an extensive search of the literature published by the first European herbalists in the sixteenth century, as well as of researchers who have investigated the collections of specimens and historical botanical documentation, which contribute to illustrate the initial knowledge of tomatoes grown in Europe.
Some sentences should include the bibliographic citations that support these concepts. See comments in the text:
Line 19 and  55. Could you provide the source to support this statement that first tomatoes were presented to Iberian Kings in Europe?
Line 370. Could you provide a cite for the statement that the Spanish gave the name ‘love apple ’ first to the Mexican tomatillo?
Check the citations in the text, there are missing references in the bibliographic list. For example, Michels, 2020

The article has been structured in an acceptable format and that allows the monitoring of the results in each of the sections, mainly according to a chronological order.
I suggest that Figure 1, which includes sixteenth-century tomato specimens found in different European herbaria, can be improved to achieve greater sharpness and illumination of the images (e.g. from Fig. 1, I). I also suggest cropping the images (e.g. Fig. 1 J where a part comprises the dark sheets of the book) and also standardizing the image formats (dpi) and sizes of each specimen, trying to achieve a similar aesthetic. I also suggest that the letters of each specimen be placed in the lower right corner.
I recommend the same for Figure 2, which includes the illustrations of the cultivated tomato published in the sixteenth century. In this case it is also desirable to improve the sharpness and lighting of the images, crop the dark parts, standardize the sizes of each illustration and organize an harmonic location of the sheets. In the current Figure the letters have been placed in different places, and I suggest that the letters of each specimen could be located in the lower right corner.
In Figure 3 it is necessary to explain in detail the meaning of colors and codes in each circle. In order to understand this scheme, I suggest to complete Table 3 indicating for each species (S. pimpinellifolium and S. lycopersicum L. var. cerasiforme),the accessions and  corresponding codes, the place of collections, and other relevant data.  This Table may be included as supplement, or indicate where this informtion could be found (previous publications?)
Experimental design: This article presents an interesting approach to elucidate the origin of the first tomatoes grown in Europe. An exhaustive search of the descriptions and illustrations of the sixteen century Herbalists, and the oldest European herbarium specimens was carried out. Through this evidence it is concluded that the first cultivated tomatoes in Europe presented diversity of fruit size, shape, and color. Confusing interpretations of descriptions and illustrations that do not depict tomatoes were also clarified.
Another relevant aspect of this work is the comparison of molecular information obtained from an ancient specimen with a large dataset on genome assemblies of 114 accessions of wild species and traditional cultivars from Latin America. Although a very low percentage (1.2% of the sequenced genome) was obtained from En Tibi anciente tomato specimen, its coverage was sufficiente for comparison and genetic diversity analysis.
Methods and analitical approaches were adecuate and described with suficient details to be replicate.
Validity of the findings: Although, the authors could not elucidate with this approximation the origin of the old specimen cultivated in Europe in the mid sixteenth century and its relationship with a specific geographic area of Latin America, I considered that the greatest contribution of this work is to demonstrate the usefulness of herbarium specimens not only to recover the botanical history but also for modern genomic and diversity studies in tomatoes.
These results are promising for further research of ancient herbarium specimens, exploring old hiden collections, and also encourage to improved DNA extraction techniques.
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: SIXTEENTH-CENTURY TOMATOES IN EUROPE: WHO SAW THEM, WHAT THEY LOOKED LIKE, AND WHERE THEY CAME FROM
Review round: 2
Reviewer: 1

Output:
Basic reporting: Review of "Sixteenth-century tomatoes in Europe: who saw them, how they looked like, and where they came from", submitted by van Andel et al. to PeerJ

This is the second time I see this manuscript, and I am positively struck by the care and attention to detail the authors had in revising the manuscript based on the many comments they received.  In my opinion, especially the integration of the genetic information is much more satisfactory. Overall, I am rather positive about this paper, and the many other adjustments also improved the paper greatly.

Only a few minor comments:

Abstract: Although I am sympathetic toward the idea of rebutting overly speculative claims in the media (regarding reviving this extinct tomato), I don't think it is necessary to do this in the abstract of this paper.

l 255-258.  It strikes me that this phrasing suggest there were recurrent introductions of tomato over a prolonged period of time, due to the use of the tense "has already begun to be imported".

l 289, if there is an estimate of the total number of 16th century herbaria that survive, perhaps in Thijsse 2016,  it would be helpful to include it here, whether you could study it or not.

l 543-544. It recommend to state the bioproject number PRJNA566320 and sequencing read archive number  SRS5407108 to find the associated reads.

lines 609-615, this seems to be better as a paragraph in the same section as the results that Michels 2020 produced, rather than discussion.
Experimental design: no comments
Validity of the findings: no comments
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: SIXTEENTH-CENTURY TOMATOES IN EUROPE: WHO SAW THEM, WHAT THEY LOOKED LIKE, AND WHERE THEY CAME FROM
Review round: 2
Reviewer: 2

Output:
Basic reporting: I consider this contribution to be within the standards required by PeerJ.
In this second review I am contributing with my comments and suggestions, which I have also been indicated in the revised document. Please consider my suggestions in the final version.
The article has been written in a technically correct way, although I consider some paragraphs are very long and could be summarized (see my comments in the manuscript).
In the literature references include the cittation of Miller (1754) and Mathiolli (1583)
Experimental design: Described in the first review.
Validity of the findings: Explained in the first review.
Additional comments: The author responded the questions and observations made in the first review, and incorporate the required changes to improve the final text.
Please consider my suggestions indicated in this second review of the original 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: IMPACT OF DIALYSIS MODALITY CONVERSION ON THE HEALTH-RELATED QUALITY OF LIFE OF PERITONEAL DIALYSIS PATIENTS: A RETROSPECTIVE COHORT STUDY IN CHINA
Review round: 1
Reviewer: 1

Output:
Basic reporting: Introduction appropriately outlines the topic, provides good context for the study with adequate referencing. The results are properly reported, including tables. The discussion and conclusions are clear. I appreciate that the authors acknowledged limitations.
My only recommendation here is that the authors edit the language using professional services as English language should be improved. This is specifically the case for the Instruction section where most sentences in lines 91-96 are really difficult to follow / understand.
Experimental design: This is an original research that contributes new and interesting knowledge on the impacts of home modality conversion on the HRQOL. The research topic is within the scope of the Journal. The methodological approach is extensively described with details. The design is correct and all conducted analyses appropriate. My only two requests here are:
-the authors should provide power analyses or other justification that the sample they used was sufficient to detect the effects, I appreciate that the authors listed sample size as one of the limitations of the study, however power calculations should also be provided
-the authors conducted a lot of tests  and comparisons, and I would expect some corrections to be applied to the p-values (for example Bonferroni corrections) to assure that the effects reported were not the results of so many comparisons conducted not necessary a true effect
Validity of the findings: This is a novel research with sound methodology and robust data. Conclusions are well stated, limitation are listed and the benefit to the literature as well as medical practice are stated.
Additional comments: I believe this is a very interesting study providing new and interesting knowledge on the impacts of different types of home dialysis on dialysis patients' quality of life. I recommend this study for publications, given the revisions listed above are done.


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: DEVIL IN THE DETAILS: HOW CAN WE AVOID POTENTIAL PITFALLS OF CATS REGRESSION WHEN OUR DATA DO NOT FOLLOW A POISSON DISTRIBUTION?
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: There are no experiments.  This is a purely theoretical paper.
Validity of the findings: No comment
Additional comments: Review of the manuscript entitled « Devil in the details : how can we avoid potential pitfalls of CATS regression when our data do not follow a Poisson distribution? » by Zoltan Botta-Dukat

This manuscript applies my CATS regression to my method of decomposing the proportional contributions of local trait-based selection and meta-community affects during community assembly.  In so doing, it represents an important advance that allows for a generalization of the method to many different types of sampling distributions while still being computationally feasible, and it corrects for bias in the original formulation.  Furthermore, the R code should, after some modification, make the method available to any ecologist that is familiar with R.  I did not identify any major errors in logic or in the statistical details and the text is generally well written.  The following points are minor corrections and suggestions that will further improve the manuscript.
First, a minor correction:  It is important to clearly state that the original CATS model is based on the maximum entropy formalism of Jaynes, which is a type of Bayesian model.  The derivation comes from the notion of information entropy and its maximization given linear constraints (here, constraints on trait means or other moments) and does not make any distributional assumptions.  It is true that the parameter estimates are identical to those obtained by maximizing the likelihood of a generalized linear model with a Poisson distribution (or, incidentally, given a multinomial distribution). It is untrue that CATS is “equivalent” to such a GLM because the maximum entropy version of CATS does not make – or require – the additional assumptions concerning distributions.  However, this is a minor point because, in any empirical study, it is possible and reasonable to justify particular distributional assumptions (Poisson, negative binomial etc.); doing this will improve parameter estimation and allow the full strength of generalized linear models to be used. I have two suggestions for improvement of this manuscript.
First, it is essential that ecologists who are not experts in statistical theory be able to apply the method.  Therefore, the author should carefully explain the R code by adding lots of comment lines (#) so that users can easily follow the steps and the R code.
Second, although the simulated data sets are useful and needed, it would be good to also present the full analysis of your method using an empirical data set.  For instance, (Baastrup-Spohr et al., 2015) present data on many local plant communities (quadrat samples) growing in Alvars.  It would be very useful to many ecologists  if you could arrange to have their data and present the full analysis on this data.

Baastrup-Spohr L, Sand-Jensen K, Nicolajsen SV, Bruun HH. 2015. From soaking wet to bone dry: Predicting plant community composition along a steep hydrological gradient. Journal of Vegetation Science, 26: 619-630.


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: DEVIL IN THE DETAILS: HOW CAN WE AVOID POTENTIAL PITFALLS OF CATS REGRESSION WHEN OUR DATA DO NOT FOLLOW A POISSON DISTRIBUTION?
Review round: 1
Reviewer: 2

Output:
Basic reporting: This paper expands the framework of Shipley 2014 and Warton 2015 to non Poisson distributions, and as such should represent a useful contribution (empirical abundance data are rarely Poisson distributed, and other types of data such as cover are definitely not, as the author points out). It would perhaps be even more useful to have a modelling framework that allows for more complex designs (eg hierarchical), as for example dispersal and environmental filters may act at different spatial scales. The author has brought together various results from the statistical literature and this in itself was interesting to read.
l.228-9: “Using the classic R2 as a measure of explained variance is suitable only in OLS regression when a normal distribution is assumed”. I don’t understand the assumption of a normal distribution – what matters is how it is defined/calculated (see Kvålseth 1985), and then interpreted depending on eg what is predicted/explained (Cox and Wermuth 1985). See Gelman et al. (2019) for some additional discussion and definition. The different definitions of R2 (as for example when you compare your equations 15 an 25) are discussed eg in Kvålseth for different models.
l. 239 “Although this procedure is correct, it […] results in slightly different values for each run”. I would not see this as a problem if the different values reflect the uncertainty of the estimate (as in eg a Bayesian R2, Gelman et al. 2019). R2 or IC are estimates and understanding the associated uncertainty is useful (it is often larger than people would believe).
l. 332: “Unfortunately, this adjustment is valid for only Gaussian distributions”. Again I do not think this is correct. If you read Fisher (1925) the argument is purely geometrical, and there is no assumption of a normal distribution as far as I could see. The adjustment (usually called after Ezekiel, not sure exactly why) has been evaluated on small samples in different papers, most recently Hittner (2020) I think.
For the whole discussion on R2, it is not impossible in the linear case to get for example that R2(trait)+R2(offset)- R2(trait; offset) is negative (that is the whole model – trait and offset – explains more than the sum of each model). This is called suppression I think (see eg Bertrand and Holder 1988) – not common in a multidimensional setting but not impossible (we encountered it in this paper: Randin et al. 2009). I don’t know if that is possible with other definitions of R2 in nonlinear models.
Details:
L212 «for a for Poisson…” remove for before Poisson

Bertrand, P. V., & Holder, R. L. (1988). A Quirk in Multiple Regression: The Whole Regression can be Greater Than the Sum of Its Parts. Journal of the Royal Statistical Society. Series D (The Statistician), 37(4/5), 371-374. doi:10.2307/2348761
Cox, D. R., & Wermuth, N. (1992). A comment on the coefficient of determination for binary responses. American Statistician, 46(1), 1-4. doi:10.2307/2684400
Gelman, A., Goodrich, B., Gabry, J., & Vehtari, A. (2019). R-squared for Bayesian Regression Models. American Statistician, 73(3), 307-309. doi:10.1080/00031305.2018.1549100
Hittner, J. B. (2020). Ezekiel’s classic estimator of the population squared multiple correlation coefficient: Monte Carlo-based extensions and refinements. The Journal of General Psychology, 147(3), 213-227. doi:10.1080/00221309.2019.1679080
Kvålseth, T. O. (1985). Cautionary Note about R2. The American Statistician, 39(4), 279-285. doi:10.2307/2683704
Randin, C. F., Jaccard, H., Vittoz, P., Yoccoz, N. G., & Guisan, A. (2009). Land use improves spatial predictions of mountain plant abundance but not presence-absence. Journal of Vegetation Science, 20(6), 996-1008. doi:10.1111/j.1654-1103.2009.01098.x
Experimental design: Not relevant here except perhaps for the 3 examples used that are described with enough details
Validity of the findings: The conclusions are clear and point to limitations of previous modelling analyses and how the proposed analyses correct for these issues.
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: USING INVEST TO EVALUATE WATER YIELD SERVICES IN SHANGRI-LA, NORTHWESTERN YUNNAN, CHINA
Review round: 1
Reviewer: 1

Output:
Basic reporting: English can be improved in some parts of the text by using synonyms instead of repeating them.

Authors could be more eclectic in their references.

Notes about the figures are in the PDF annotations.
Experimental design: Há detalhes deixados para trás nos materiais e métodos e estão nas anotações do PDF.
Validity of the findings: no comment
Additional comments: Understanding which factors affect water yield in a sensitive and ecologically important region is a very interesting and pertinent subject, especially in the current moment of convergence of the effects of climate change.

The study is simple and easily replicable to other locations, which is relevant to science. Furthermore, it shows that, although there are weaknesses and uncertainties in InVEST, it can indeed be used to quantify, temporally and spatially, the water yield in a given territory.

Additionally, it is also interesting to understand what to do with the information generated in terms of the management and conservation of the territory in the study area. The issue of tourism and the prioritization of protection in areas with high water production was briefly explored. In this sense, a window of opportunity opens to be explored within the context of the present work, praising not only the use of InVEST but also the use of its outputs.


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: USING INVEST TO EVALUATE WATER YIELD SERVICES IN SHANGRI-LA, NORTHWESTERN YUNNAN, CHINA
Review round: 1
Reviewer: 2

Output:
Basic reporting: Water yield is an important issue in ecosystem services. This paper uses several input data layers for the Integrated Valuation of Ecosystem Service Tradeoffs (InVEST) model to estimate the water yield of Shangri-La City in Southwestern China from 1974 to 2015, and obtained important results. The paper is well written in general, and I have added my comments/corrections in the PDF document. The paper has a clear structure, sufficient literature references, and relevant discussions. The figures are also of high quality. I suggest that the authors add a small map (location of the study area in China) to Figure 1 so that readers can easily understand the geographical location of the study.
Experimental design: The experimental design is appropriate.
Validity of the findings: The conclusions are well stated. The limitations are also discussed.
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: USING INVEST TO EVALUATE WATER YIELD SERVICES IN SHANGRI-LA, NORTHWESTERN YUNNAN, CHINA
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: Just one point regarding the impacts of tourism on the topic of discussions does not seem consistent to me. More details in the attached .pdf.
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: USING INVEST TO EVALUATE WATER YIELD SERVICES IN SHANGRI-LA, NORTHWESTERN YUNNAN, CHINA
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors have addressed my comments and the other reviewer's comments. I think the paper can be accepted for publication.
Experimental design: The experimental design is appropriate.
Validity of the findings: The findings are valid.
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: GENOME-WIDE IDENTIFICATION AND EVOLUTION OF WNK KINASES IN BAMBUSOIDEAE AND TRANSCRIPTIONAL PROFILING DURING ABIOTIC STRESS IN PHYLLOSTACHYS EDULIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript ' Genome-wide identification and evolution of WNK kinases in Bambusoideae and transcriptional profiling during abiotic stress in Moso bamboo' is an interesting a well-designed study that could shed light on the role of WNK kinases in Bambusoideae, and in WNK kinases evolution in the plant kingdom.
Experimental design: As far as I know, the manuscript is relevant and up to date, and despite being an article similar to others already published, addressing the same target in other plant species, the results are unprecedented for moso bamboo. However, I think that this study would need some improvement.
Validity of the findings: -First of all, you ignored the role of WNK kinases in circadian rhythms, which was mentioned in the introduction. I think you should consider circadian rhythms when designing the abiotic stress responsing experiment. The treatment time should be at least up to 48 hours.
-I am a little confused about the sentence in lines 323-324 “we identified 41 WNK genes in five species of Bambusoideae, 324 including 11 PeWNK genes in Moso bamboo.” Does the sequences of 11 PeWNK genes in Moso bamboo different from those in the other four species? It is better to compare the sequences of WNK genes, which one is the same and which one is different.
-the reference should be uniform. I find some of the Journal names are the abbreviation some are not.
-the word “isolate” is not proper in lines 86, 96, 145…….
- it is preferred to have a table or a figure to introduce the name of WNK genes in five species. Such as the species name of BamWNKs, GanWNKs, OlaWNK and RguWNKs.
-why was only PeWNK1 analyzed in Figure4 and 5?
-line 229-230 “In this study, the genes with the two-fold difference compared with the control were considered differentially expressed.” should have reference.
Additional comments: -I would also suggest thorough English proofreading since I found several misspellings and confusion regarding the use of adverbs, nouns, and adjectives. I am not a native speaker and I know how hard could be to write in English, and I am not requiring perfect English but the most correct possible.

Below you can find some of the errors in your MS

1.	LINE 13 change “plays” to “play”
2.	Line 18 “cis-elements” the cis should be italic
3.	Line 35 “Arabidopsis thaliana”, line 75 “Phyllostachys edulis”, line 81-82， line94-95，line 97-99 ………… should be italic
4.	Line 78-80 the reference format should be uniform.
5.	Because you don’t have any wet experiments in your MS. I think more details should be added in the Method parts of lines 129-135. It is hard to understand the Abiotic Stress process for readers
6.	Line 124: you should say “The PeWNK gene expression in different tissues”
7.	Space should be added between the number and the unit. Line 131, 306,……
8.	The general rule is to capitalize the first letter of the first word in a title or heading, the first letter of all other words in a title or heading except conjunctions, articles, prepositions of fewer than four letters, and the “to” in infinitives.


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 IDENTIFICATION AND EVOLUTION OF WNK KINASES IN BAMBUSOIDEAE AND TRANSCRIPTIONAL PROFILING DURING ABIOTIC STRESS IN PHYLLOSTACHYS EDULIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1. Title: I think that “Moso bamboo” should be “moso bamboo”?
2. Line 132: 1uM should be 1μM.
3. Line 207: I think GnWNK1 should be GmWNK1.
4. Latin names of species should be italicized.
5. English editing is needed. Please check the spelling and grammar through the whole manuscript carefully. I suggest that the paper should be polished by native English speakers.
Experimental design: 1. Line 129: Please add the source background of plant materials. What are the bases for selecting reagent concentration and treatment time in this section?
2. Figures: In figure 3, Why did you choose HUMAN_WNK1 for alignment? I think the figures should be better reorganized in the revised manuscript.
3. In this manuscript, 41 WNK genes were identified from five bamboo species, but only the WNK genes in moso bamboo were studied in detail. Why? Is the stress resistance of moso bamboo stronger than the other four bamboo species?
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 IDENTIFICATION AND EVOLUTION OF WNK KINASES IN BAMBUSOIDEAE AND TRANSCRIPTIONAL PROFILING DURING ABIOTIC STRESS IN PHYLLOSTACHYS EDULIS
Review round: 1
Reviewer: 3

Output:
Basic reporting: The paper entitled “Genome-wide identification and evolution of WNK kinases in Bambusoideae and transcriptional profiling during abiotic stress in Moso bamboo”by Liu identified 41 WNK genes in five bamboo species and analyzed the gene evolution, phylogenetic relationship, physical, cis-elements, conserved motifs, various abiotic stresses, PeWNK genes expression levels.  In this regard, the new reported results would be very useful in future functional genomic studies of Bambusoideae WNK genes.The work is suitable for the journal and the results are interesting.
However, as written, the paper needs major revision. I have the following suggestions that the authors may want to consider when revising the manuscript:
Abstract is not concise, and the main idea of the paper is not clearly visible: the long introduction about only the “WNK” of the object of the study; the results are not summarized in appropriate way and the conclusion is too ambitious.
Experimental design: First mandatory action is the through revision of the English grammar. There are many instances in which the English is very poor and compromises understanding what the authors want to say due to the amount of formatting errors that exist (lack of spaces, names without italics, etc. etc.) . For instance, the sentence of the abstract”While overexpression of AtWNK9 increases drought tolerance through the ABA signaling cascade, nine WNK(1-9) were identified in rice and showed differential transcriptional regulation for various abiotic stresses such as heat, cold, salt, and drought”. I strongly recommend the authors to find a native English speaker to proofread the manuscript.
Materials & Method section. The methodology part needs to be reframed and the authors should provide the bioinformatics part along with the parameters used for the same. Also, check basic rules for botanical publishing. The genus and species names should be written with italics in the text. For instance, “At the same time, other WNK genes of the Bambusoideae were isolated from draft sequences of the herbaceous diploid bamboo species O. latifolia and R. guianensis, and the tetraploid and hexaploid woody species G. angustifolia and B. amplexicaulis [24].” Also,in the part “Expression Analysis of PeWNK Genes in Response to Abiotic Stress”, what is the basis for selection“ 25% Polyethylene glycol (PEG), 200mM Sodium chloride (NaCl), 1uM Abscisic acid (ABA) and 1mM Salicylic acid (SA) nutrient solution” ? and also for just only “for 3 h and 24 h, respectively”?
Validity of the findings: Results section.Figures in the Results section should be rechecked (e.g. lack of the legend in Fig. 1, etc. etc. ) , what’t the difference for the evolution of specific domains in WNKs between the diploid and polyploid bamboo species? All the N-terminal domain and C-terminal domain are the same, respectively? Are there some conseverd palnt WNK motif in C-terminal domain?
Discussion should be more focused on the interpretation of the results. Here, the results were presented again without a clear connection with the previous studies and the importance of the present study.
The conclusions section, as written, is only a plain summary of the results. Instead, it should additionally state the significance of results and what are your thoughts/ perspectives for the field given the results.Reference list should be rechecked and corrected according to the journal style.
Additional comments: There are several ways how this paper can be improved.
For example, you can add the phosphorylation sites of protein kinase PeWNK .And also, the chromosomal level of Moso bamboo (temperate tetraploid woody bamboo) as a whole-genome sequence had been reported. You can add the map of the distribution of PeWNK gene on chromosomes if you can.
Did you calculate any conservative sequence analysis in all plant WNKs family or the selected five Bambusoideae species? Are there any amino acids that are conserved Lysine residue in the active center?
Do you see any correlation between the diploid and polyploid bamboo species for the evolution of WNK family ?


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 IDENTIFICATION AND EVOLUTION OF WNK KINASES IN BAMBUSOIDEAE AND TRANSCRIPTIONAL PROFILING DURING ABIOTIC STRESS IN PHYLLOSTACHYS EDULIS
Review round: 2
Reviewer: 1

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


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

Input:
Title: GENOME-WIDE IDENTIFICATION AND EVOLUTION OF WNK KINASES IN BAMBUSOIDEAE AND TRANSCRIPTIONAL PROFILING DURING ABIOTIC STRESS IN PHYLLOSTACHYS EDULIS
Review round: 2
Reviewer: 2

Output:
Basic reporting: The paper entitled “Genome-wide identification and evolution of WNK kinases in Bambusoideae and transcriptional profiling during abiotic stress in Moso bamboo”by Liu identified 41 WNK genes in five bamboo species and analyzed the gene evolution, phylogenetic relationship, physical, cis-elements, conserved motifs, various abiotic stresses, PeWNK genes expression levels.  In this regard, the new reported results would be very useful in future functional genomic studies of Bambusoideae WNK genes.The work is suitable for the journal and the results are interesting.
Experimental design: The methodology part  is much better than before and provide the bioinformatics part along with the parameters used. Also, check basic rules for botanical publishing. The genus and species names should be written with italics in the text.
Validity of the findings: Here, the results were presented again with a clear connection with the previous studies and the importance of the present study. The conclusions section, as written,  state the significance of results and what perspectives for the field given the results.
Additional comments: There are several ways how this paper can be improved.
For example,the genes names should be written with italics in the text.
Reference list should be rechecked and corrected according to the journal style. like "Transcriptome data of 26 different tissues of moso bamboo were obtained from the NCBI Short Read Archive database (SRX2408703) [19] "
The tile "PeWNK genes response to circadian rhythms" should be  capitalized  in some words.


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: THERAPEUTIC TARGETING OF ARID1A AND PI3K/AKT PATHWAY ALTERATIONS IN CHOLANGIOCARCINOMA
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: This study is highly flawed in many respects, the most obvious one being the incredibly high doses of MK-2206 used (10-150uM, and 30uM for the western blots). This is 10-1000 times higher than most publications. At this dose, there are too many off-target effects to be believable. Other issues are lack of clear PI3K pathway activation after shARID1A, weak "sensitization" to MK-2206 after shARID1A (Fig. 5C-D), and lack of connection between the experiments and the EPHA2 loss finding. Additionally EPHA2 is not really a canonical PI3K member.
Validity of the findings: Conclusions are based on faulty study design, see above.
Additional comments: No comment.


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

Input:
Title: THERAPEUTIC TARGETING OF ARID1A AND PI3K/AKT PATHWAY ALTERATIONS IN CHOLANGIOCARCINOMA
Review round: 1
Reviewer: 2

Output:
Basic reporting: In this paper, with the analysis on large-scale patient data, the authors reported the correlation of ARID1A mutation and PI3K/AKT pathway in cholangiocarcinoma (CCA), which would be useful for understanding the current therapeutic outcome and developing future targeted therapy. This is an innovative and interesting study. From the perspective of academic criticism, several technical concerns need to be addressed to further improve the quality of this manuscript, as appended below.

Missing citation: Line 74, 76, 78, 85, 93, 105, 259, 261, 263.

Gramma issue and ambiguous sentence: Line 74, 81, 93, 110-113, 113-115.

The introduction is a stack of information rather than a logical story. The cancer genetics and molecular background of ARID1A and PI3K/AKT pathway need to be sorted and summarized. As the author is trying to tell a therapeutical story, the state-of-art CCA targeted therapy should be the focused point of the introduction. Please re-organize the language and information accordingly.
Experimental design: Fig. 1 and Fig. 2A are hard to read due to the resolution and font size. It is reasonable to truncate or split up the figure and put those genes that were not highlighted in the story in a supplementary figure.

A gene map in the main figure or a detail description about the mutation hotspot and common mutation types of ARID1A would be helpful to better understand the story.

The gene expression profile after shRNA knockdown needs to be shown.

The quantitative analysis of the western-blot results needs to be included as part of the Fig 4A, 5A, 5B.
Validity of the findings: The insignificant shortening survival rate in the ARID1A-PIK3CA mutations and ARID1A-LAMA1 mutations need to be explained after result description.

As mentioned in the introduction, ARID1A expression has been shown to have dynamic regulation on cancer progression in different types of cancer (Sun, Xuxu, et al. "Arid1a has context-dependent oncogenic and tumor suppressor functions in liver cancer." Cancer cell 32.5 (2017): 574-589.). It would be interesting to look into the ARID1A mutation and expression pattern at different stage of CCA with the existing patient dataset and visualize the data in a stage-dependent way.

The mutation status of ARID1A has also been suggested to influence the outcome of immunotherapy (Li, Jing, et al. "Epigenetic driver mutations in ARID1A shape cancer immune phenotype and immunotherapy." The Journal of clinical investigation 130.5 (2020): 2712-2726.).  It would also be interesting to explore the ARID1A mutation-related therapeutic outcome in the CCA patient dataset.
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: THERAPEUTIC TARGETING OF ARID1A AND PI3K/AKT PATHWAY ALTERATIONS IN CHOLANGIOCARCINOMA
Review round: 1
Reviewer: 3

Output:
Basic reporting: Supharada et al analyzed the public database of cholangiocarcinoma (CCA) patients and identified genetic alterations in the ARID1A gene. The authors subsequently performed functional studies by knockdown and overexpression of ARID1A in cell lines to show increased sensitivity to AKT inhibitor (MK-2206). The authors concluded that ARID1A deficient CCA tumors are dependent on PI3K/AKT pathway and may be more vulnerable to AKT inhibition.
Experimental design: Overall, the study is well designed. The authors performed studies using multiple CCA cell lines which is ideal for robust research findings.
Validity of the findings: Results:
1- Fig 4A and B: Please provide full western blot images in the supplementary figures. Additionally, please include dose-response curves for the cell lines instead of reporting IC50 values.
2- Fig 5b: The western blot for ARID1A doesn’t provide sufficient evidence for ARID1A knockdown. Please include cleaner blots.
3- Fig 5c-f: The effect of ARID1A knockdown on AKT inhibition is minimal in the dose-response curves. Please explain why the cells were treated for only 24h with MK-2206. It may be better to treat cells for a longer time to see a more robust effect.
4- Fig 6: The authors report significantly higher apoptotic cells at 30 uM drug concentration. Is such high micromolar concentration clinically meaningful? Additionally, MK-2206 may have non-specific effects given that IC50 for AKT inhibition is less than 100nM.
5- Fig 7: Again, 30 uM drug concentration is probably not clinically meaningful. Since there is a variable amount of pAKT in shN vs shARID1A cells at baseline, the authors should report the ratio of decrease in pAKT levels with and without the drug. Overall, changes in pAKT levels with ARID1A knockdown and AKT inhibition are relatively small.
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: THERAPEUTIC TARGETING OF ARID1A AND PI3K/AKT PATHWAY ALTERATIONS IN CHOLANGIOCARCINOMA
Review round: 2
Reviewer: 1

Output:
Basic reporting: Thank the author for the efforts. The manuscript is good for publication now.
Experimental design: good
Validity of the findings: good
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: THERAPEUTIC TARGETING OF ARID1A AND PI3K/AKT PATHWAY ALTERATIONS IN CHOLANGIOCARCINOMA
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors have addressed all my concerns in their revised manuscript.
Experimental design: The authors have addressed all my concerns in their revised manuscript.
Validity of the findings: The authors have addressed all my concerns in their revised manuscript.
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: FILTERED MUD IMPROVES SUGARCANE GROWTH AND MODIFIES THE FUNCTIONAL ABUNDANCE AND STRUCTURE OF SOIL MICROBIAL POPULATIONS
Review round: 1
Reviewer: 1

Output:
Basic reporting: In general, the writing and presentation of the data and information provided from the results of this study is quite good, but the information about the importance of using filtered mud is not clearly legible. The observed variables are also quite good with the data display  informative enough. Something new from filtered mud has not appeared much in the discussion.
Experimental design: The experimental design of this study is not following statistical rules. Where if there are 4 treatments, accordingly, it should be at least 5 replications or 5 groups (in the case of RCBD). The way to determining replication is based on the formula t(n-1) > 15.  While in this study the replications were only 3.  As a result, the conclusions of this study are not valid according to design error.
The reasons for making comparisons in treatment are also not explained so that if implemented in the field, it is somewhat less suitable.
Validity of the findings: The design used does not meet the correct rules, because experimentally design, that determining the test or group is based on the formula t(n-1) > 15, so the minimum number of tests or groups used is 5, so statistically this result is not yet can legitimately conclude the results. The reason for using comparison treatments as in this study must have a basis. Because when applied in the field, it is very difficult to apply 3 parts of filtered mud with 2 parts of soil. Even though research in greenhouses is in pots, the implementation must also be considered when applied. And must also follow the rules of research. How to grow sugarcane in pots is also not explained. More details can be seen in the annotated manuscript.
The results of the study have been displayed well, but they are still often blocked because the tests used are not enough. The discussion is often less relevant and extends to organic materials in general, even though filtered mud of sugar cane has distinctive and different properties from organic materials used by other researchers. In general, a lot of discussions refer to the effect of the application of organic matter to the soil, there is no specificity of the organic matter you add. Why FM2 organic matter is lower has not been widely discussed, why, maybe because the sample is less representative. Moreover, how is it related to the higher FM content, the enzyme activity increases, while the plant performance is the opposite.  The inconsistency of the results of diversity indices may be due to improper design.
Additional comments: In addition, the discussion is not in-depth and refers to the treatment material used so that the discussion extends to other matters which are quite unrelated to the material discussed in this study.