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: SYNERGISM BETWEEN CALCIUM NITRATE APPLICATIONS AND FUNGAL ENDOPHYTES TO INCREASE SUGAR CONCENTRATION IN FESTUCA SINENSIS UNDER COLD STRESS
Review round: 5
Reviewer: 1

Output:
Basic reporting: As this is version 4 we only need to deal now with the few remaining issues.
Experimental design: As this is version 4 we only need to deal now with the few remaining issues.
Validity of the findings: As this is version 4 we only need to deal now with the few remaining issues.
Additional comments: It is appreciated that the authors have patiently addressed all that was asked of them. This reviewer respects that and does not wish to ask any more.
There remain now a few (maybe 10) cosmetic issues around the description of the design:
1. On the map (Fig. S1) the titles 'whole plot i .. iii on the top row should be replaced with 'Replicate i .. iii' and the statistical analysis section within M&M revised as follows (see also uploaded PDF with comments): The trials were arranged using a split-split-plot design with a total of 216 plants of F. sinensis, where three Ca(NO3)2 concentrations were randomly assigned to three replicates as main plot treatments, two endophyte statuses (i.e., E+ and E–) were randomly assigned into the main plots as spilt plot treatments, and randomly selected plants were sampled at three times in two strips (two plants each time) as the split-split plot s in two strips (Fig. S1). There were 18 treatments (2 endophyte types × 3 Ca(NO3)2 concentrations × 3 sampling times), with three repetitions for each treatment and 12 plants for each repetition (i.e., 36 plants for each treatment). There was a distance of 22 cm between strips (i.e., endophyte types) and 18 cm between plants within a strip. For spilt plots, plot size was 20 m2 with two surrounding-protection strips and two sampling strips. The strips of plants were 0.5 meters apart from each other.
The headings of the various Tables and the identification of the ANOVA effects in column 1 of the Tables, inconsistently use 'Ca' and Ca(NO3)2. Each Table heading and column1 identifying the ANOVA effects should be carefully checked and made consistent. The identifier should not be 'Ca' because N is also involved in the treatment. At least the identifier should include 'Ca' and 'N', and be consistent across all Table headings, table content and manuscript text.
The titles for the supplementary data files should be edited carefully - there are mispellings of words like replicate and spaces where spaces should not be.


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: STRATEGIES FOR RECOVERY OF IMBALANCED FULL-SCALE BIOGAS REACTOR FEEDING WITH PALM OIL MILL EFFLUENT
Review round: 1
Reviewer: 1

Output:
Basic reporting: Although I am not a native English speaking I consider that the manuscript should be carefully checked by a native English speaker.

The number of references (61) is a little bit high for this kind of articles, although they are adequate to the manuscript topic. However less than 15% are really recent (three last years). The authors could include some more recent references.

Article structure is adequate, and figures and tables supply the required information.
Experimental design: The manuscript topic is within the Aims and scope of PeerJ.

The manuscript clearly defines the research topic and the applied methodology could be adequate although it presents no more than a first step. Experimental design is carried out in batch test and with no more that one feed; this implies that there is no a description of what will occur in a long term test (for example a second feed).

Methodology description is adequate, although there are some specific item as:
-	The determination of first-order kinetic should be explained with more detail (Was the lag phase considered?)
-	Check equation 1. What is "ln"?
Validity of the findings: The main concern of the manuscript is the limited validity of the data as they were obtained in batch test and single test (as is indicated in previous section).

Discussion should be improved as there is no a real comparison among the used strategies. It looks like a mere data exposition, without a real discussion and clear conclusions.

Some specific items are:
-	The self-recovery sludge has very high methane yield and methane production rate. How can this fact be explained if the test was carried out a pH lower that 5.6?
-	Economic evaluation should include a clear comparison of the different strategies.
-	Figure 1. How can be explained that the Acetic acid SMA is lower for the original microbial sludge than for the inhibited sludge? These data should be discussed in the 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: STRATEGIES FOR RECOVERY OF IMBALANCED FULL-SCALE BIOGAS REACTOR FEEDING WITH PALM OIL MILL EFFLUENT
Review round: 1
Reviewer: 2

Output:
Basic reporting: The English and writing style needs to be improved. There are many sentences need to be rephrased. For example:

Line 434-436, Page 19: The pH adjustment with Ca(OH)2 and dilution with 20% v/v of biogas effluent can recovery the acidified and low pH inhibition of POME feeding commercial biogas plant with economically feasible.

Figure 1: There are no error bars in Figures 1. In order to assess the significance of any differences, it is essential to conduct at least duplicate experiments, to provide error bars, and to provide an assessment of statistical significance.

Proper reference should be given for the following statements.

Line 258-260, Page 14: ‘It has been reported that the addition of NaHCO3 resulted in a relatively high level of alkalinity and pH in the digester, which gradually causes specific inhibition on the metabolism and growth of methanogens.’

Line 397-398, ‘The oil palm ash composed of silicon dioxide (58-65%), calcium
oxide (6-7%), and potassium oxide (7-8%)…’

Line 174: Please check the referencing style
Experimental design: No comment
Validity of the findings: Page 14: The hydrolysis constants (kh) in Table 3 should be discussed in the context.

Results and discussion:
This section should be restructured, as there are some repetitive discussion of the results in Page 12-19, especially in the subsection of discussion (page 17-19).
Additional comments: This paper describes the strategies for recovering inhibited biogas reactor feeding with palm oil mill effluent. Overall, the discussion of the results is simple; the authors should provide more justifications for each major finding with references. The manuscript can be published; nevertheless the English, sentence structure and writing style in most of the parts not up to the mark and should be checked thoroughly. Please state clearly the area of improvement/future research area in the conclusion.


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: STRATEGIES FOR RECOVERY OF IMBALANCED FULL-SCALE BIOGAS REACTOR FEEDING WITH PALM OIL MILL EFFLUENT
Review round: 2
Reviewer: 1

Output:
Basic reporting: Article structure is adequate, and figures and tables supply the required information.
The manuscript has been modified according to reviewers´ comments, but there are still some points that could be improved.
Experimental design: Methodology description is adequate, although there are some issues to be considered as is indicated in comments to author.
Validity of the findings: Discussion should be improved to compare the used strategies based the experimental data.
Additional comments: Some general comments:

-     Is the full-scale hybrid cover lagoon reactor (6,000 m3) located in the Prasang Green Power Co., Ltd. palm oil mill? The description of the strategy applied to the full-scale reactor (lines 148 – 151) is not clear enough. The inhibited sludge is sludge from the same reactor? I suppose that the biogas effluent is the effluent from another reactor, but is not clearly indicated.
-	Table 4. Each item should be described. For example, what is the meaning of biogas loss (there is also a biogas production) and why is different for each recovery strategy?
-	Recovery time is not clearly defined in the text.
-	Specific methanogenic activity (SMA) test should be described and discussed more deeply. It is not easy to understand that SMA with acetic acid would be lower than SMA with cellulose as for inhibited sludge and 0,50% w/v Ca(OH)2. The meaning of the different SMA should be discussed.
-	The recovery of AD process is based on methane yield, methane production rate and SMA, but there is no a clear discussion of the meaning of each one of these parameters. The highest methane yield should be not very different for each test, except if there were a clear inhibition. The ratio between the highest methane production rate for each test and SMA will be the same if the SVadded is the same for each test; if SVadded is not the same for each test, the comparison between the highest methane production rate does not supply any useful information.


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: STRATEGIES FOR RECOVERY OF IMBALANCED FULL-SCALE BIOGAS REACTOR FEEDING WITH PALM OIL MILL EFFLUENT
Review round: 2
Reviewer: 2

Output:
Basic reporting: The manuscript has been improved to an acceptable level.
Experimental design: No comment
Validity of the findings: No comment. Amendments have been done accordingly.
Additional comments: No comment. Amendments have been done 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: REVERSINE, A SELECTIVE MPS1 INHIBITOR, INDUCED AUTOPHAGIC CELL DEATH VIA DIMINISHED GLUCOSE UPTAKE AND ATP PRODUCTION IN CHOLANGIOCARCINOMA CELLS
Review round: 1
Reviewer: 1

Output:
Basic reporting: •	The article lacks the use of clear and ambiguous English. There are several grammatical errors through the text. The current phrasing makes comprehension difficult.
•	Background of disease and relevant use of reversine is not clear. Introduction fails to justify why the authors chose to test reversine in cholangiocarcinoma cell lines. Recent literature in the field is missing (for example, 2019 study in colorectal cancer https://www.spandidos-publications.com/10.3892/ijo.2019.4746) and there is no mention of cell cycle related changes in cholangiocarcinoma. Authors should consider explaining why is cholangiocarcinoma a candidate for reversine related G2/M arrest? There is no background on the relation of PI3K/Akt pathway, HIF1a or GLUT1 and autophagy in the introduction section.
•	The structure of the article is well defined. The original western blot raw data .rar file is not accessible or corrupted.
•	The authors defined a hypothesis but have not provided enough evidence to support the conclusions mentioned in the study.

---Custom checks – cell line checks – KKU-100, KKU-213A, KKU-213B and MMNK-1 are cholangiocarcinoma cell lines.
Experimental design: •	The research is within the Aims and scope of the journal.
•	The study identified the clear need for novel therapies in cholangiocarcinoma and has attempted to fill the gap. However, the authors failed to clearly justify the relevance of blocking G2/M phase of cell cycle and inducing autophagic cell death in cholangiocarcinoma. The authors should consider explaining the concept clearly.
•	The investigation is conducted in a random manner. The experimental design is not clear. The method description for cell proliferation and cell cycle assays is not clear.
•	In line 59, the cell lines are cultured in a DMEM media. Since the changes are associated with ATP differences in the untreated and treated cells, the author should explain the use of low-glucose or high-glucose DMEM.
•	In line 106-110, the range of dosages tested is very few. Since the highest dose tested does not result in 100% death, the dose quoted as IC50 is not true IC50.In Figure 1, 48 hour graph fails to show 100% cell viability at the lowest dose, does this mean that the control or vehicle wells were different for all the time points? The method for drug dose response assay is not clear. In the method section, 5 dosages are tested, whereas in the figure authors are showing only 4 dose points? How was the background correction done? For MMNK-1, the graph shows a clear reduction of % viability. There is no p value or statistical evaluation of the results.
•	In line 113-117, what is the ploidy level of these cell lines? How did they infer if the peaks of cell cycle are all from the same ploidy? Did they sort for nuclei? Why is G0-G1 phase not shown in the cell cycle plots? The authors should consider showing cell cycle differences at different time points. Depending on the doubling rate of both of these cell lines, the effect of a cell cycle inhibitor would be better understood in a time dependent manner. Is the G2/M arrest reversible or irreversible? A major predictor of G2-M checkpoint is CDK1 and cyclin A. Authors should consider showing other cell cycle checkpoint regulator proteins to validate the flow-cytometry based G2-M arrest.
•	In figure 2C – for cleaved caspase 3 both the cell lines are showing different bands. Cleaved caspase 3 (CC-3) is depicted by a single and not an array of bands. Authors should comment on the CC-3 western blot image for KKU-213A. No statistics shown for western images.
•	LC3BII/I blot is not clear. LC3BI band is not visible in the untreated cells, would this be linked to stress in the untreated cell lines? The authors should consider commenting on the autophagy state in the untreated cells and test other autophagy related markers like beclin1A or SQSTM1. The .rar file with all the original blots is not accessible.
•	Authors have not tested the cell lines from a chemotherapy point of view so conclusions based on these speculations are too far-fetched.  The authors have identified MMNK-1 as a normal cholangiocyte whereas it is an immortalized cholangiocyte cell line.
Validity of the findings: •	The rationale and benefit to literature is not clearly stated. The statistical evaluation of cell proliferation and western blot images is missing. The conclusions made are not limited to supporting results. Several results are tied in with literature based speculations and too far-fetched.
Additional comments: Cholangiocarcinoma is a lethal disease and there is an urgent need for novel targeted therapies to improve patient outcome. I commend the authors for their attempt to propose a novel therapy for the treatment of cholangiocarcinoma. The overall approach used is clear and relevant however it lacks a well-defined thorough experimental analysis.
The data set is limited with the use of three cholangiocarcinoma cell lines and an immortalized cholangiocyte cell line. The cellular proliferation assays are not clearly defined and lack statistical power. Authors have used the proliferation assay data set to justify the use of chemotherapy-resistant cholangiocarcinoma; however, there is no evidence to support this hypothesis. Although the results are compelling, the cell cycle assays are limited to a single time point and do not display the sub G0 population or ploidy levels. Generally, these cell lines are slow growing and any cell cycle related changes should be analyzed based on their doubling rate. There is clearly an induction of apoptosis but it’s relation to autophagy is not clear. The most important issue is that the untreated cell line also has a LC3BII band but no LC3BI and a lack of statistical analysis. This could be improved by supplementing with other autophagy markers like beclin1A or SQSTM1.
Generally, the study is interesting and is worth publication but it needs to be majorly revised for clarity and statistical analysis.


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: REVERSINE, A SELECTIVE MPS1 INHIBITOR, INDUCED AUTOPHAGIC CELL DEATH VIA DIMINISHED GLUCOSE UPTAKE AND ATP PRODUCTION IN CHOLANGIOCARCINOMA CELLS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript is generally clearly written and structured according to the standard format. The data in the figures and tables are well organized and presented.
However, there are a few points that could be improved:
1.The image for GAPDH is missing in Figure 3B, although it is mentioned in the figure legend.
2.Magnification is missing in Figure 3A. In addition, it would be easier for the reader to see the changes if images from higher magnification would be included.
3.The introduction could be expanded to give more information to the reader. The knowledge gap in the field could be further described. The topics studied in the manuscript, such as autophagy/ autophagic cell death, apoptosis, cell cycle arrest could be briefly introduced and the markers, which are being measured in this study, could be very shortly presented.
Experimental design: The research questions are well defined in the introduction and the experimental design of the study is generally good.
However, some points would need further clarification:
1.Glucose uptake assay: From the Methods section (lines 88-93), it is not very clear if glucose concentration in medium or glucose uptake was measured. There are 2 different kits provided by BioVision: Glucose Uptake Colorimetric Assay (catalogue number K676) and Glucose Colorimetric Assay (catalogue number K686), with which different parameters can be measured. Including the catalogue number of the kit and a brief description of how the assay was done, would clarify exactly which parameter was studied.
2.Figure legend for Figure 2: It is mentioned that the results shown in this figure were obtained from 2 independent experiments. For a better statistical significance of the results, at least one more independent experiment should be included.
3.Table 1: How the data on IC50 were calculated and obtained should be explained in detail in the Methods section.
Validity of the findings: The conclusions of the study are generally well stated.
There are a few points, which need to be addressed:
1.Figure 3B and line 127: An increase in the protein levels of LC3-II does not mean there is induction of autophagy. It means there is accumulation of LC3-II and autophagosomes, which could be caused BY increased production of LC3-II (as a consequence of induction of autophagy) OR BY inhibition of fusion autophagosome/autolysosome (which results in inhibition of autophagy). To differentiate between the 2 situations, measurement of autophagic flux could be performed (for example LC3-II Western blot in the presence of lysosomal inhibitors, such as bafilomycin A1; Klionsky D et al, Autophagy, 2016).
For clarification, the comments in the text could be changed or measurement of autophagic flux could be performed.
2.Results, Discussion, Figure 4: Throughout the text, there are statements on causal links, which are not supported by experimental results, such as: ‘Reversine induced autophagy and apoptosis via inhibiting PI3K/AKT signaling pathway’ (line 127). There are no data showing that the reduced PI3K/AKT signalling is the cause for reversine-autophagy induction. For this, analysis of the effect of modulation of PI3K/AKT on reversine-induced autophagy should be performed. This type of statements could be presented as opinions, speculations or hypothesis.
Additional comments: In this manuscript, the effects of reversine on different cholangiocarcinoma cell lines are investigated. Cell viability and markers of cell cycle, autophagy, apoptosis and insulin signaling are evaluated. The results indicate that reversine has an antiproliferative effect on these cancer cell lines. Thus, the findings of this study could be potentially interesting in the future for developing novel strategies for the treatment of cholangiocarcinoma.


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: REVERSINE, A SELECTIVE MPS1 INHIBITOR, INDUCED AUTOPHAGIC CELL DEATH VIA DIMINISHED GLUCOSE UPTAKE AND ATP PRODUCTION IN CHOLANGIOCARCINOMA CELLS
Review round: 1
Reviewer: 3

Output:
Basic reporting: The English language was clear unambiguous most of the time. Nevertheless, improvement is needed, especially in the introduction section. The introduction and background were very shallow scientifically. The authors talked about the unsatisfactory results of chemotherapy on unrespectable CCA. However, the authors did not detail, which chemotherapy specifically showed unsatisfactory results. The authors said the proposed treatment worked on multiple cancers but did not explain if these pieces of evidence were obtained from the laboratory or human studies (line 44-46). Also, the authors did not talk about the primary resistance mechanism. This is very important to show the gap in the knowledge in treating CCA. Also, this gap in the knowledge will put Reversine in the potential filling of this gap. As of now, the importance of Reversine in treating unrespectable CCA is not clear in comparison with the already available chemotherapies. Figures are relevant. However, some figures are not well labeled or not high quality.
In figure 2 A, color legends for the cell cycle phase were not clear. In figure 2 C, the quality of the western blot was not high. This was clear in multiple rows, including cleaved caspase-3, Bax, and Bcl-XL. In figure 3 A, the cell pictures were not clear: the type of scope used, the magnification have not been mentioned. The vacuoles of autophagy were not clear as well. In figure 3 B, the western blot's quality was not good as well, especially on the CLUT3. In addition to all of that, the authors did not talk about the current study's limitations.
Experimental design: The study is within the aim and the scope of this journal. However, the research question was not well defined. The authors did not identify the gap in knowledge and how this study research question will fill this gap. The investigation was acceptable with some significant concerns. Western plot experimental quality was weak and inconclusive. Some of the experiments have only repeated two times, which at least should be repeated in triplets. Although the authors used a negative control cell line of the immortal cholangiocytes, positive control was not used in this study using a resistant model to Reversine. Also, the authors used the vehicle but did not compare the standard treatment for CCA. Finally, this is just in vitro study of treatment of effect on cell survival, which is not enough and should be accompanied by in vivo treatment study to see if this treatment is working on these cell lines or patient-derived xenograft models on animals. The methodology could be improved to help in the reproducibility of these findings in other labs. For example, cell cycle analysis in lines 72-76 needs more details here and supplementary material, especially concerning the flow cytometry analysis and gating. Also, the authors decide to use four micromolar as the treatment dose for the subsequent experiments after the viability assays. However, it was not clear why this number. It was not clear why the authors continue the experiments only on two cell lines instead of all the three available.
Validity of the findings: The western plot quality was questionable and not helpful in drawing valid conclusions. The methodology was not novel. The conclusions are very firm for study depends only on in vitro results. Some of the studies' replicates need to be increased to at least three times. The authors found the treatment was effective against the chemo-resistant cell line (KKU-100). However, no clear explanations of why this happened and what is the possible explanation for such a phenomenon. The second sentence in the conclusion section (line 214-215) was written in a convincing tone that needs to be softened, especially that the authors did not take the opportunity to compare this proposed treatment to the standard chemotherapy for CCA, separately or in combination.
Additional comments: The study is a good start point to start building a substantial manuscript using more replicate, positive controls and additional experiments on animals. The authors should work more on the gab of the knowledge, its importance and how this new treatment will help in filling this gap. Also, authors should compare this treatment to the current standard chemotherapy for CCA.


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: REVERSINE, A SELECTIVE MPS1 INHIBITOR, INDUCED AUTOPHAGIC CELL DEATH VIA DIMINISHED GLUCOSE UPTAKE AND ATP PRODUCTION IN CHOLANGIOCARCINOMA CELLS
Review round: 2
Reviewer: 1

Output:
Basic reporting: There is a significant improvement in the manuscript. The text is clear and unambiguous. The background and introduction are well written.
Experimental design: The authors have addressed all previous concerns in the experimental design in the revised manuscript.
Validity of the findings: No comments.
Additional comments: The revised manuscript addresses all major concerns and is in a good shape to be published now.


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: REVERSINE, A SELECTIVE MPS1 INHIBITOR, INDUCED AUTOPHAGIC CELL DEATH VIA DIMINISHED GLUCOSE UPTAKE AND ATP PRODUCTION IN CHOLANGIOCARCINOMA CELLS
Review round: 2
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The comments and questions were addressed and included 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: DIFFERENTIAL RELIEVING EFFECTS OF SHIKONIN AND ITS DERIVATIVES ON INFLAMMATION AND MUCOSAL BARRIER DAMAGE CAUSED BY ULCERATIVE COLITIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Safe and effective treatments are needed for ulcerative colitis. In this manuscript, the authors have investigated shikonin (SK) and its derivatives as treatments for ulcerative colitis. They have employed a DSS-induced IBD in vivo mouse model and performed comprehensive studies to determine the effects of SK derivatives and understand the underlying mechanisms for the efficacy of shikonin.
-Overall, the article is well-structured and the experiments are well-controlled. The authors have provided sufficient background information in the introduction and shared raw data.
-Rationale for testing COX-2 and iNOS levels needs to be added in the results section in lines 233-239, since that is where COX-2/iNOS is being investigated first.
-Reference is needed to support statements in lines 234-236.
-Abbreviations need to be defined at first mention. For example, ‘DSS’ appears first in the abstract, but is described only in the Methods section. Many other abbreviations are used, but not defined. For example, MPO, iNOS, NLRP3, ASC, ZO-1, VCAM-1.
-The English language should be improved in certain sections of the manuscript, particularly the abstract, lines 136-139, 143-147, 152-154, 159-160, 215. I recommend the authors to use English editing services for this manuscript.
Experimental design: The authors have defined the research question and their research is relevant and within the scope of the journal.
-The ‘Figure titles’ are missing and need to be added. There are no Figure Legends and the symbols * and # used in the Figures to denote statistical significance are not described anywhere in the text.
-Were the cytokine levels determined in the supernatants of cultured colons (line 220) or serum (line 225)?
-In Fig 7, how was the ‘average optical value’ determined? Needs to be added in the Methods section.
Validity of the findings: -In the quantification of Western blot data, what is % relative band intensity?
-Fig 1C: The Y-axis shows % body weight of the original; however, the Y-axis title does not represent that.
-Fig 1F: Is the spleen weight in g or mg?
-Fig 3: The levels of claudin do not look different between DSS and DSS+treatment groups. The conclusion ‘SK and its derivatives at 25 mg/kg can restore the expression of these epithelial TJ proteins’ should be specific since not all TJ protein levels are affected by the SK derivatives.
-Fig 4A: The Western blot results for ASC are not clean.
For IL-1β, the bands for ‘DSS’ and ‘DSS+mesalazine’ appear to be of similar intensity, yet there is a significant difference in the band intensity in Fig 4B. Are the values used to plot Fig 4B correct?
-Fig 8A: IκBα and pIκBα bands are barely visible.
-The authors write that ‘Such results were consistent with those found in histological examination, in which the inflammatory cell infiltration extent in colonic tissues was less than that in SK- and AK-treated animals and naphthoquinone-, acetyl-SK- and β, β-dimethylacryl-SK-treated mice’ (lines 299-302). However, they have not looked at inflammatory cell infiltration specifically. Hence, this statement should be modified.
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: DIFFERENTIAL RELIEVING EFFECTS OF SHIKONIN AND ITS DERIVATIVES ON INFLAMMATION AND MUCOSAL BARRIER DAMAGE CAUSED BY ULCERATIVE COLITIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: In this manuscript Han et.al have performed multi-faceted analysis of to evaluate various aspects of IBD with the aim of identifying safe, mild, durable and effective drugs to cure IBD. The authors have shown evidence of pharmacological efficacy of SK on ulcerative colitis against inflammation and mucosal barrier damage. While this is an interesting study, there are some key issues in the manuscript, which if addressed, would make the manuscript stronger.

1.	The title and the conclusion suggest that shikonen (SK) and its derivatives are effective against ulcerative colitis. However, majority of studies support role of only SK as an effective pharmacological therapy but not all the other derivatives. The title and conclusion are therefore not entirely in keeping with the results.
2.	The language of the title is not very precise. Precise language like “Relieving effects of shikonen on inflammation and mucosal barrier damage caused by ulcerative colitis”, might help the readers understand what the paper is about more clearly.
3.	In reference to lines 194 and 195, it is unclear what the authors are trying to convey by saying “SK and its derivatives strengthened the prevention of colon from DSS-induced the histological damage”.
4.	Figure 8 A and C: The labeling on top of the figures do not coincide with the lanes of the gel on they are supposed to coincide with. Also, the readers would be able to analyze the figures better if better images for panel IkBa and p-IkBa were provided.
5.	In line 218, “When the circulating leukocytes were recruited into the colon, pro-inflammatory mediators...”, “were” (highlighted) should be replaced with “are”.
6.	In the background section of the abstract, the abbreviation DSS is used without stating what DSS stands for. Although stated later in the paper, it would be more beneficial for the readers if the abbreviation was explained before using it.
Experimental design: Naphthoquinones are commonly known to cause oxidative DNA damage and increase in reactive oxygen species (ROS) production. As SK is a naphthoquinone, as stated in the paper, it would give the readers more clarity about the by-stander and off-target effects of SK on neighboring cells, if the effect of SK treatment on ROS production and resultant oxidative DNA damage was evaluated.
Validity of the findings: In reference to line 215, it would be more beneficial to the readers if the authors are more precise about reporting which derivatives specifically show an effect on levels of which specific proteins, rather than making a generalized statement like “SK and its derivatives reduce the level of NLRP3 inflammasome activation in colonic tissues “.
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: DIFFERENTIAL RELIEVING EFFECTS OF SHIKONIN AND ITS DERIVATIVES ON INFLAMMATION AND MUCOSAL BARRIER DAMAGE CAUSED BY ULCERATIVE COLITIS
Review round: 2
Reviewer: 1

Output:
Basic reporting: I thank the authors for addressing my concerns in their revised manuscript. There are some more concerns that need to be addressed before publication.

There are still several typos throughout the manuscript and some awkward word usage. For example, in lines 223-224 -However, changes of caspase-1 and IL-1β were significantly “receded” by SK and naphthoquinone.

The added part ‘All animal experiments and welfare were treated in strict accordance with the relevant Guidelines for Care and Use of Laboratory Animals of Nanjing University and approved by the Animal Ethical and Welfare of Nanjing University (IACUC-1909002). All efforts were made to minimize the animals’ suffering and to reduce the number of animals used’ is already present in the original text and therefore, needs to be removed.
Experimental design: No comment
Validity of the findings: Fig 3. Legend is missing and needs to be added.

Fig 5. The ** and ## do not appear to be at the correct place in this figure. There is a clear difference between the control group and the DSS group and yet there is no ** on top of the DSS bars in the graphs. Besides, SK significantly reduces levels of pro-inflammatory cytokines and increases IL-10 and yet, there is no ## on the DSS+SK bars in the graphs.

Authors have described statistics as follows: * p<0.05, ** p<0.01 and # p<0.05, ## p<0.01. The greater the observed difference, higher the statistical significance and lower the p value. So assuming * p<0.05 and # p<0.05 is correct, is ** p<0.01 and ## p<0.01 correct? From the figures, it is clear that the observed difference is greater for ** compared to * and ## compared to #.
Additional comments: In the abstract, SK increased the ‘anti-inflammatory’ cytokine IL-10. It is mentioned as ‘inflammatory’ cytokine IL-10.


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: DIFFERENTIAL RELIEVING EFFECTS OF SHIKONIN AND ITS DERIVATIVES ON INFLAMMATION AND MUCOSAL BARRIER DAMAGE CAUSED BY ULCERATIVE COLITIS
Review round: 3
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: I thank the authors for addressing my concerns in the revised 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: REPRODUCING COUNTRY-WIDE COVID-19 DYNAMICS CAN REQUIRE THE USAGE OF A SET OF SIR SYSTEMS
Review round: 1
Reviewer: 1

Output:
Basic reporting: (1) Line 25, 'One of the natural first approach to such...' should be 'One of the natural first approaches to ....'
(2) The meaning and derivation of R0 in this model should be discussed.
(3) Conclusion can be strengthened by adding some future directions and open problems.
Experimental design: (4) Line 65, why this can be easily checked to see if the data satisfies the logistic curve is not straigtforward.
(5) Regarding the system, what would happen if a feedback introduced like Global stability of disease-free equilibria in a two-group SI model with feedback control? I suppose the numerical results will be better especially for Spain as there is a clear feedback factor.
(6) In the tables 1 & 2, the meaning of these parameters should be recalled directly in the tables themselves.
Validity of the findings: (7) The work is based on numerical simulations. However, it should be mentioned that alalytical exact solutions can be derived. The solutions can be worked out via the Lie algebra method as shown in A Lie algebra approach to susceptible-infected-susceptible epidemics, Analytical solution for an in-host viral infection model with time-inhomogeneous rates. I understand that an exact solution is beyond the scope of this paper but a remark would be useful.
(8) In Figure 1, how do you fit these data? Are the curves polynomials?
(9) The resuls for Russia are interesting. There is inconsistency between ascending part of the outbreak for circles, which exhibit a transition in dynamics within the week from April 16 and April 30. More explanations are appreciated.
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: REPRODUCING COUNTRY-WIDE COVID-19 DYNAMICS CAN REQUIRE THE USAGE OF A SET OF SIR SYSTEMS
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1)	The overall language of this paper in terms of grammar, sentence structuring and typos should be improved to ensure that an international audience can clearly understand your text. Some examples where the language could be improved include:

•	 Line-18: edit (challenges of public health)
•	 Line-22: edit (the effectiveness of applying)
•	 Line-25: edit (first approaches to such modeling)
•	 Line-27: edit (which can also be supplied)
•	 Line-28: edit (exposed persons (E))
•	 Lines 28-31: kindly restructure the sentence.
•	 Line-31: remove (at the)
•	 Line-45: edit (reproducibility could be restored)
•	 Line-49: edit (known as the logistic substitution model)
•	 Line-59: restructure sentence (As the source of data, there was chosen the repository, which belongs to the projects “Our world in Data”)
•	 Line-59: edit (This data was downloaded)
•	 Line-61: edit (growth and saturation parameters)
•	 Line-64: edit (component transforming)
•	 Line-68: edit (Figure-1: …..persons infected by COVID)
•	 Line-69: edit (accuracy for state of the art)
•	 Line-70: edit (effects of outbreaks exceed)
•	 Line-76: edit (do not reproduce the dynamics accurately;)
•	 Line-78: edit (with the root mean)
•	 Line-79: edit (the solid red line)
•	 Line-94: edit (demonstrates a more)

2)	Data source is shared, and figures meet the basic requirements.
3)	I feel that the paper in its current form lacks context. There is not much relevant background and adequate number of references are lacking.
Experimental design: It can be interpreted that in past author has already published another paper that contradicts the findings from the current one and this is mentioned in his notes. The author has loosely tried to justify this paper by highlighting data limitation during previous research.

When you are trying to contradict your own research in such a short period of time, then you need to really put a good effort to justify that. The current paper is short and seems to be done in a rush. It feels that only the model has been re-run on a larger and more updated dataset and conclusion is made based on the results.

With COVID-19 data still being updated – I will highly recommend the author to verify and tabulate his findings by running the results on top 10 countries that are leading in total cases/ deaths.
Validity of the findings: Although the results may seem to make sense, but the level of effort put on this paper doesn’t give enough confidence to agree with it.
Additional comments: •	The paper in its current form needs a major re-structuring with efforts on context, language and justification.

•	Best wishes – hope you come back with a quality contribution.


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: REPRODUCING COUNTRY-WIDE COVID-19 DYNAMICS CAN REQUIRE THE USAGE OF A SET OF SIR SYSTEMS
Review round: 2
Reviewer: 1

Output:
Basic reporting: n/a
Experimental design: n/a
Validity of the findings: n/a
Additional comments: The author has addressed most of my questions. Therefore, I am glad to recommend it for publication in PeerJ.


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

Input:
Title: REPRODUCING COUNTRY-WIDE COVID-19 DYNAMICS CAN REQUIRE THE USAGE OF A SET OF SIR SYSTEMS
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: In the manuscript, the author is highlighting the importance of using multi-logistic decomposition of countrywide COVID-19 cases to demonstrate its utility in delineating outbreak time phases and spatially distinct outbreaks. The following are my key comments and concerns regarding the study.
1.	Conceptual advancement: I fail to agree on why in the first place the author thought that a simple SIR model and its analytical solution in terms of the Verhulst logistic equation can simply fit well on countrywide data. SIR models inherently assume the population is homogeneous (i.e there are not metapopulations). While a country (however small) will certainly abide by this assumption. Hence, the author shows through its analytical results that multi-logistic decomposition is a better fitting option is not a new finding. I would suggest the author change the narrative of the study. Rather than showing that multi-logistic decomposition is a better option than simple logistic decomposition (obvious result), I would rather ask the author to explore if multi-logistic decomposition can help decipher key epidemiological events/characteristics in a countrywide outbreak. The author briefly describes how the multi-logistic decomposition of Russian COVID data can help understand the outbreak in various cities and regions. Hence, understanding the number of phases and determining how many phases (sub SIR models) could be the key parameter to understand the epidemiology of COVID19 in a country. Furthermore, I also do not understand why the author is fitting SIR compartmental model rather than the SEIR model which is what most of the COVID19 models are based on.
2.	Writing style: The author needs to majorly restructure its manuscript. I would recommend differentiating methods and results and bringing in all introduction related writing into the first section. Please see the attached pdf file with comments on it for more details. Furthermore, I also understand the manuscript has been written from an analytical perspective, but the author of PeerJ will be mostly epidemiologists and biologists. Hence, I would recommend including details related to all data transformations with explanations in the methods section. For example, the Fisher-Pry plot needs a detailed explanation of its utility and interpretation. Other tiny things such as figure subplots numbering and citing the correct figure subplot would significantly improve the manuscript reading experience.
3.	Log let software: Looks like most of the model fitting is done using Log let the software, but details are missing to reproduce the results presented in the manuscript. Please provide all step-by-step details about analyses conducted in the manuscript (either in the main text or in supplement).


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: REPRODUCING COUNTRY-WIDE COVID-19 DYNAMICS CAN REQUIRE THE USAGE OF A SET OF SIR SYSTEMS
Review round: 3
Reviewer: 1

Output:
Basic reporting: The author has put up a good effort on improving the manuscript.
Experimental design: Restructured paper in its current form along with more citations and detailed discussion looks good.
Validity of the findings: Although the research in itself in not novel but it definitely contributes with its content and case study for the academic audience.
Additional comments: Good work!


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: REPRODUCING COUNTRY-WIDE COVID-19 DYNAMICS CAN REQUIRE THE USAGE OF A SET OF SIR SYSTEMS
Review round: 3
Reviewer: 2

Output:
Basic reporting: The author has addressed all my comments and concerns.
Experimental design: N/A
Validity of the findings: N/A
Additional comments: The author has addressed all my comments and concerns.


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

Input:
Title: BIOCHEMICAL CHARACTERIZATION AND INHIBITION OF THERMOLABILE HEMOLYSIN FROM VIBRIO PARAHAEMOLYTICUS BY PHENOLIC COMPOUNDS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The study by Vazquez-Morado et al evaluated the biochemical and inhibition parameters of the thermolabile recombinant hemolysin (VpTLH) Vibrio parahaemolyticus

--The study highlights an important observation, but the organization of the manuscript is poor. Overall, the manuscript is poorly written, many typos at multiple places and inconsistent use of tenses.
Experimental design: Experimental design and methodology is good, well described, and with appropriate controls.
Validity of the findings: The conclusions drawn from the study are good and relevant to the prospective implications in the field.
Additional comments: Authors should work upon improving the language of the paper.


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: BIOCHEMICAL CHARACTERIZATION AND INHIBITION OF THERMOLABILE HEMOLYSIN FROM VIBRIO PARAHAEMOLYTICUS BY PHENOLIC COMPOUNDS
Review round: 1
Reviewer: 2

Output:
Basic reporting: For the most part, the article are written clearly. The biggest issue I have is with the rendering of the symbols (like micro, beta, etc). A lot are rendered simply as boxes. Please have this corrected, and be sure that no rendering error exist in the final version.

Here are the lines where I catch these symbol rendering errors:
Line 94, 95, 129, 437, 495, 546, 547, 554, 562

Other than the rendering error, there are a number of minor changes to the text that I would suggest:
- Line 65: not controlled to uncontrolled
- Line 84: delete "that" in "and also that participate in cell"
- Line 141: please correct the grammar for "and determined the kinetics Michaelis-
142 Menten parameters using p-nitrophenyl laurate (PNPL) as substrate"
- Line 154: gen to gene
- Line 158: platted to plated
- Line 181: precipitated to precipitate or precipitant
- Line 188: Niquel to Nickel
- Line 192: "to a constant non-bounded protein". I don't understand what this means. Please clarify
- Line 197: sequential to sequentially
- Line 199: please rephrase: "by overnight each" to make the sentence clearer
- Line 203: delete "by the" in "... measured by the spectrophotometrically..."
- Line 221: established to "established protocol"?
- Line 336: >50 mg lecithin. Is this supposed to be 50 microgram?
- Line 540: please correct "specie"
- Line 552: anti-body to antibody
- Line 557: caffeic to caffein
- Line 570: excepting to except for
Experimental design: The experiments are well designed, with numerous biophysical techniques used to purify the protein, find the rate of its enzymatic and hemolytic activities, and understand the interaction of the VpTLH with flavonoids through molecular modeling. The authors do a great job putting all the experiments together into a coherent study.
Validity of the findings: I have a few questions that I have regarding the data that hopefully will help improve the next interation of the paper:
- Line 279-280: "The data were analyzed with the dose-response model in Prism-5 software". Are there parameters that need to be inputted for the model to be built? Can the author provide more detail?
- Line 337-338: "A quantitative hemolytic activity assay showed that 2-10 ug of purified VpTLH elicits 76-85 % relative activity (100% hemolysis was tween-20)." Can this data be presented in the paper?
- For all figures with residual activity, is it possible for the magnitude of the rate at 100% activity be written next to the figures?
- Line 373: "VpTLH has the highest Vmax and turn over compared with other TLH". The Vmax, km and kcat is around an order of magnitude greater than the other TLH measured. Has the author ever tried measuring the other TLH in the lab to compare the values to the literature? If this is not difficult to do, it will greatly add confidence in the result. An order of magnitude increase is very significant.
- Table 2 and Figure 5 comparison. The result on Table 2 does not seem to agree with Fig. 5. At around 5 uM, the flavonoids have decreased the residual activity to 20% or less in Fig.5, but the IC50 values here and Table 2 results show that at 5 uM, the inhibition is just at 50% or less. Am I missing something?
- Line 535 to 539: Can the author comment on the fact that the protein is thermolabile, but during the purification of the protein, they undergo denaturation and renaturation. Does the author know approximately what percentage of the enzyme is active after renaturation?
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: BIOCHEMICAL CHARACTERIZATION AND INHIBITION OF THERMOLABILE HEMOLYSIN FROM VIBRIO PARAHAEMOLYTICUS BY PHENOLIC COMPOUNDS
Review round: 1
Reviewer: 3

Output:
Basic reporting: None
Experimental design: None
Validity of the findings: Data supports the observations and conclusions drawn.
Additional comments: In this manuscript, the authors evaluated the biochemical and inhibition parameters of the recombinant VpTLH using enzymatic and hemolytic assays and determined the molecular interactions by in silico docking analysis. They concluded that flavonoids inhibit both enzymatic and hemolytic activity of thermolabile hemolysin from Vibrio Parahemolyticus. The manuscript is well written and the experiments designed well with proper controls and supports the observations and conclusions drawn with solid data. I have no major concerns with the manuscript and have few suggestions below.
1.	The manuscript is difficult to read. The authors must use separate paragraphs to describe distinct experiments and ideas. This applies to the majority of the manuscript from Introduction to Discussion.
2.	Figure 5, the graphs are confusing, please use different colors for the graphs.


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: DOES DROSOPHILA SECHELLIA ESCAPE PARASITOID ATTACK BY FEEDING ON A TOXIC RESOURCE?
Review round: 1
Reviewer: 1

Output:
Basic reporting: The language needs some revision, as pointed out in my specific comments. The scholarship needs substantial additions, as I have pointed out in my specific comments. The structure of the Introduction needs to be expanded, and the structure of the Methods and Results don’t align. The figures need clearer captions in places. The authors did supply the raw data.
Experimental design: This is an observational study. The research question is defined, but hypothesis lacks rationale and scholarly context. How the work contributes to field is not completely clear because of lack of scholarship and reporting of what is already known. The design of the observational study is appropriately rigorous. However, the description of methods needs some revision, as I have stated in some specific comments.
Validity of the findings: The study is valid, but replication is limited. Consequently, the small sample sizes make this study questionable as a stand-alone paper. I suggest adding two components: 1) the unpublished results described in the Discussion section (lines 187-189) and/or 2) an expansion of the hypothesis posed here. The latter is critical for revising the manuscript. By truly explaining the hypothesis (or alternative hypotheses), this could be more of an idea paper inspired by previous experimental results combined with the modest yet important observational data reported here already. The scientific soundness is fine, but the contribution is modest.
Additional comments: This study provides rare observations about the natural history and tritrophic ecology of Drosophila seychellia, which has attracted interest among evolutionary biologists and geneticists because of its exception ecological specialization. The main field observation reported here, that D. seychellia was not found to be parasitized while a congener using the same fruit at different stages was found to be parasitized, is quite interesting even if based on a small sample of observations. While interesting, the small sample size limits the contribution of this study and manuscript. I suggest bolstering these field observations with either an expansion of the conceptual framework beyond what is typical for a data paper or combining these observations with additional empirical results. Some interesting unpublished results cited in the Discussion section would seem to be a good choice.

In its current form, the manuscript suffers from various problems in presentation of ideas, methods, results, and discussion. These issues need to be addressed before the manuscript can be considered for publication. I have provided many suggestions for revisions in my specific comments below.

Specific comments
Lines 12 and 38. Replace “specialized” with “specializes.”

Lines 27-28. This is a matter of style, so not necessary. However, I suggest replacing “speciation and to be a key factor underlying the diversity of insects Ehrlich and Raven (1964)…” with “evolutionary diversification of insects (Ehrlich and Raven 1964; …).”

Lines 33-34. The sentence starting with “One of the pioneer species…” is not clear to me. Rephrase to explain what you mean.

Line 47. Remove the comma. It is not grammatically correct.

Lines 47-49. Replace “provides this species with a reproductive advantage by being able to access the food source during an earlier time in the fruit’s development” with “provides this species with a unique ecological niche among coexisting and potentially competing Drosophila species.” I think this is the important point, and the details of the niche could be added to this point.

Line 50. Add an apostrophe (D. sechellia’s).

Line 52. Cite addition references here.

Lines 57-58. What is the rationale for this hypothesis? There are many species of insects that feed on toxic foods and still experience substantial mortality from parasitoids. I suggest you consider how host-specific the parasitoids are. In fact, the host range of the parasitoid observed in this study has been studied by Schlenke and coauthors, so there is relevant information to cite. According to a meta-analysis by Zvereva and Koslov 2016, Ecological Monographs 86: 107-124, chemical defenses of herbivorous insects are generally effective against generalist predators but not parasitoids and specialist predators. Generalist parasitoids show a wide range of responses, so it is plausible to hypothesize that toxic hosts might be defended against generalist parasitoids.

Line 59. In this paragraph, it is worth discussing related work in which fruit flies gain resistance against parasitoids from their potentially toxic diet (Milan et al. 2012, Current Biology 22: 488). I suggest summarizing this study because it involves the parasitoid species found here and gives a precedent for your hypothesis. There is also work on geographic variation in anti-parasitoid resistance in Drosophila by Kraaijeveld and colleagues (late 1990’s and early 2000’s). There is also relevant scholarship on ecological immunology that mostly deals with insect herbivores outside of Drosophila (e.g., reviewed by Singer et al. 2014, Integrative and Comparative Biology 54: 913).

Line 75. Provide more details about these other cases of Drosophila lacking melanotic encapsulation ability. What are their ecological characteristics? Do they feed on toxic substrates? This is all relevant context for this study.

Line 102. Elaborate on your hypothesis providing your rationale. This should probably go earlier, when the hypothesis is first described.

Line 112. First describe the study system. What is already known about the natural history? Or would this be the first study to look at the natural history of tritrophic interactions in this system?

Line 116. Replace “of” with “off.” To correct the grammar, replace “A total of “ with “In total,” at the beginning of the next sentence.

Line 118. On what basis were these categories determined? Did they look or smell different over these time periods? As described, the classification seems arbitrary.

Line 120. Be more specific. The time of exposure could be an important factor determining parasitoid access.

Line 125. For ecological clarity, I suggest referring to this part of the study as a survey of fruit-feeding Drosophila in the same community. Later you could refer to it as the community survey.

Line 127. Replace “were” with “was” for proper grammar.
Lines 131-132. Justify the exclusive use of morphological characters for the fruit fleis when you used DNA barcoding on the parasitoids.

Line 137. Replace “characterization” with “community survey.”

Line 140. Where are the results from the other fruit baits? Only the noni fruit data are shown in Table 1 and described in the Results section. If the passage in the Discussion mentioning the loss of data due to animals and weather caused there to be no data from the broader fruit bait survey, then don’t describe this survey in the methods of this paper. As it stands, the methods create the expectation that all of the results from them will be reported.

Figure 1 caption. Label all the panels with letters to you can refer to them unambiguously. “Column” is not clear enough. “Circles” is misspelled. Give the species name of the wasp. Last sentence: replace “is” with “are.”

Figure 2 legend and caption. If “alcohol” could be replaced with “ethanol” it would be clearer.

Line 150. I suggest adding “ecological” to the list of types of studies.

Line 156. This paragraph should be expanded so you can explain your hypothesis and how these results relate to it in greater detail.

Line 174. Describe these studies further. It’s important to know the circumstances under which L. boulardi can develop in D. sechellia. Did the flies feed on noni fruit or artificial diet in this experiment? If the former, the present results suggest that the parasitoid avoids ovipositing in flies eating ripe fruit. If the latter, resistance might occur either through reduced parasitoid attack or reduced parasitoid survival in toxic hosts.

Line 180. The topic (first) sentence here suggests the paragraph will be about parasitoids promoting evolutionary diversification of their hosts. Either change the first sentence or make the paragraph about that topic. The existing content of the paragraph should probably be moved to the previous paragraph following my comment there.

Lines 187-189. This is very interesting! Why isn’t this experiment/result included formally in this paper?

Line 193. Remove the comma.

Line 200. How so? This conclusion was never explained.

Line 209. There is a typo: it should be “conducting.”

References. Give the full reference for Andrade Lopez (2015). I gather this is a published thesis, so there should be a university and it should be identified as a thesis. The journal title in Farine et al. has a typo.


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: DOES DROSOPHILA SECHELLIA ESCAPE PARASITOID ATTACK BY FEEDING ON A TOXIC RESOURCE?
Review round: 1
Reviewer: 2

Output:
Basic reporting: In general, the study is fairly clearly presented. There are a fair number of instances of awkward language and some errors, but this would not be too difficult to clean up with a close reading of the manuscript with attention to my comments below (general comments).

Overall structure is fine and the background sufficient. The overview of previous work examining D. sechellia’s lack of parasitoid resistance, both experimental and through comparative genomics was very interesting and well presented.

Figure 1 and 2 seem unnecessary. All the data in figure 1 was previouly published and explained in the introduction. It does not seem necessary to reproduce it here as a figure.
See comments below regarding Figure 2.
Experimental design: I appreciate the limitations the authors had, but 28 noni fruit seems a pretty small sample size for this sort of study, and just 9 wasps from two rotting fruit (a stage that hosted both Drosophila species) is not strong evidence of a pattern.

It seems possible that wasps don’t parasitize larvae of either Drosophila species until the fruit is rotting due to use of odors in host location and perhaps physical barriers (e.g., fruits are too hard). Still this would be consistent with the idea that using ripe but not rotting fruit provides some enemy free space.

It is not entirely clear what the artificial baits were for. Just to see what might be attracted?  To compare attractiveness of the baits/substrate use? Are the only results from this that, yes, the two species of Drosophila were found in the baits? Was there a difference in attraction to different baits between the species?  Were D. sechellia attracted to the papaya or banana? Was the other species attracted to the noni fruit bait?

It would have been more revealing to allow the Drosophila in the baits to be exposed to parasitoids to assess parasitism rates. Even better would have been to present wasps with host larvae of each Drosophila species and allow those larvae to develop on various substrates and assess parasitism rates. This would be key in assessing if the fruit toxins actually harm the wasps or if they are simply attracted to rotting fruit due to the volatiles or whatever
Validity of the findings: The authors conclude from a limited sampling study that because they reared parasitoids from only rotting fruits that are less frequently fed on by D. sechellia, that feeding on ripe nona has relaxed parasitoid pressure and resulted in diminished physiological defenses. This is logical, and I tend to think the author’s story is likely, but it is a bit of a leap based on the limited evidence they present. Just 9 wasps from two rotting fruit (a stage that hosted both Drosophila species) is not strong evidence.

“D. malerkotliana, was abundant in unripe noni and also present in the rotting stage but completely absent from the ripe stage.” – But this is just 4 fruit!

If typical development time of the wasp is four weeks, why did they emerge after seven weeks?

Are the noni toxins toxic to the parasitoid wasps?

Are the wasps attracted to ripe noni fruit?

Are wasps able to penetrate the ripe fruits?

Why were no D. malerkotliana in unripe fruits parasitized if toxin levels are low?
I find the hypothetical curves in Figure 2 very unconvincing. What is the basis of these curves? Show the data. Also, the pie charts are misleading because the low number of experimental units = fruit is not apparent.

If the author did a study and found that noni extract is harmful to adult parasitoid wasps, why isn’t that study described and include in this paper instead of referring to unpublished results? Still, those conditions appear artificial and did not involve D. sechellia.
Additional comments: p. 1
while closely-related species (delete “the”, this article (“the”) is used many times in the manuscript in places where it is not necessary and sounds awkward)
L. 26 I would say parasitic organisms
L. 28 refs should be in ()
Radiation followed by host shifts? Do you mean the opposite? And it’s unclear how the listed factors facilitate radiation. (radiation misspelled, enemy free space)
What is meant by “traces left in the genome”?

The authors say that [genome] sequencing of Drosophila has allowed comparative analyses but they don’t mention any such analyses or the insights they have provided.

…a small proportion of adults also found in other substrates(?) Adults aren’t found in substrates. Same applies to D. simulans.

Adults on substrates?

P. 2

L. 55 driving trait loss

L. 56 apply to

L. 57 with protection from attack

L. 59 significant mortality factor?

L. 61 In this process, the parasitoid egg...

L. 63 it should be stated that the hemocytes become melanized

L. 72 infection of which wasp species?

L. 80 associated with

P. 3

L.104 Cousin or Couisin?

L. 113 drosophilid

We collected noni fruit that had fallen from [or off] the plant,

L. 131 Drosophilidae is not italicized.

P. 4

Table 1 names need a space, e.g., D.malerkotliana

Figure 1. These results were previously published, I do not think they need to be placed in a figure such as this here. That said, check the legend for spelling/grammatical errors.

Figure 2 is also not necessary, and is actually misleading. The hypothetical curves are just that, hypothetical. If they are based on data, show the data. The pie charts are total numbers of insects, but this is misleading because fruit sample sizes were much lower.

P. 7

L. 152 This could cause a relaxation…

L. 157 feed and breed


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: DOES DROSOPHILA SECHELLIA ESCAPE PARASITOID ATTACK BY FEEDING ON A TOXIC RESOURCE?
Review round: 2
Reviewer: 1

Output:
Basic reporting: Generally good. The language still needs some minor revision, as pointed out in my specific comments. One of the figures needs some revision. I didn’t see raw data supplied by the authors. Did I miss it?
Experimental design: The research question is defined, and the hypothesis is now described with much more scholarly context (perhaps a bit more than needed, but it’s fine). The design of the observational study is appropriately rigorous. The design of the newly added lab experiment is sound. However, the description of statistical methods still needs some revision, as I have stated in some specific comments.
Validity of the findings: The study is valid, and the additional scholarship and lab experiment were helpful in validating it further. The overall weight of the evidence from this study and others make a more compelling case than could be easily understood from the previous version of the manuscript.
Additional comments: This study provides rare and valuable observations about the natural history and tritrophic ecology of Drosophila seychellia, which has attracted interest among evolutionary biologists and geneticists because of its exceptional ecological specialization. The main field observation reported here, that D. seychellia was not parasitized while a congener using the same fruit at different stages was parasitized, is quite interesting even if based on a small sample of observations. I am pleased to see that this observational study is bolstered with more discussion of the previous scholarship and a laboratory study that was previously only mentioned in passing. The evidence suggests the ripe noni fruit deters parasitoids because it is toxic to them (according to the lab study).

The revisions in this version were very helpful. A minor revision based on the comments below should make this manuscript ready for publication.

Specific comments

Line 25. Delete “drastically” because there isn’t enough evidence to support it.
Lines 33-34 (and perhaps elsewhere). Follow a conventional order when listing in-text citations (usually by year from oldest to newest).
Line 35. “Affects mating in pea aphids”
Line 42. “Has been described”
Line 44. “Dietary specialist”
Line 50. Cite reference to support this statement.
Line 53 and in bibliography. Lanno et al. 2018
Line 59. Remove the comma between “toxicity” and “provides.”
Line 61. Add a comma between “species” and “we.”
Line 71. Each is a singular subject, so the verb should be “is” instead of “are.”
Line 75. “Herbivorous insects”
Line 85. Remove the comma.
Line 87. Remove the quotation marks around “foreign body.”
Line 123. I don’t see the need to use the metaphor of “machinery” when you can just say that they have the genes, but they no longer function properly due to limited expression in response to parasitoid attack.
Lines 127-129. I suggest making the distinction here between direct toxicity of noni fruit and toxicity of fly larvae which have fed on noni fruit. Both are possible at this point, and later evidence is presented for the former possibility.
Line 154. UC San Diego (not San Diego University)
Line 163. The sample size of 4 replicates is important. It is not clear to me if this is the number of experimental units treated as independent replicates in the statistical analysis. I would like to see an explicit description in the methods.
Line 170. Move parenthesis: Salazar-Jaramillo et al. (2014)
Line 188. Don’t capitalize drosophilid.
Line 194. Specify the type of alcohol (ethanol?).
Line 197. Give the institution for Prof. Cariou.
Lines 204-207. The repetition of the hypothesis and methods is not needed. Delete this paragraph.
Lines 212-213. These P-values seem pretty small for a sample size of 4 (see comment above), so I’m wondering about the unit of replication in the statistical analysis versus the experiment.
Line 217. The wording here could be clearer: “left unparasitized.” I think you mean that fewer hosts were attacked by parasitoids in the noni medium.
Lines 226-227. In this case, it would be clearer to say “Our survey of the Drosophila community” because you just mentioned flies and wasps.
Line 230. “Was also present”
Line 233. Don’t capitalize or italicize drosophilid.
Line 237. Clearer to say “parasitoid oviposition occurred during the overripe stage.”
Fig. 1 B. The title has “parasitization” misspelled.
Fig. 1 caption. Describe the figure so the reader can interpret it without repeating the methods. Your Fig. 2 caption is a good example of what to do.
Fig. 1 and caption. The graphical depiction of data suggests a contingency test was used, whereas the methods say a GLM was used. The figure should reflect the comparison made in the analysis, and thus should have error bars in panel B.
Line 243. “Evolution of dietary specialization”
Line 248. “Immunological resistance against parasitoids”
Line 263. “Infested” not “invested.”
Lines 264-267. Don’t repeat the methods here. You can just say that the legal restrictions limited the research to an observational study with limited sample sizes.


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: GENOMIC DIVERSITY OF ESCHERICHIA COLI FROM HEALTHY CHILDREN IN RURAL GAMBIA
Review round: 1
Reviewer: 1

Output:
Basic reporting: The study describes the diversity of E. coli collected from healthy children in the rural Gambia between 2007-2011. The authors have shown commensal E. coli differ in their sequence types, phylogenomic groups, virulence, and antibiotic resistance genes. The manuscript is well written. The bioinformatic analysis is excellent, and the data presentation is very well done. Figures and tables are appropriate and informative. A drawback is the low recovery rate of E. coli isolates from frozen stocks.
Experimental design: The authors have carried out an extensive bioinformatic analysis to find the genomic relatedness among the isolates.
Limitation of the study:
E. coli isolates could be recovered only from a total of 21 of the 66 stool samples. Isolates could not be recovered from 9-13 years old frozen stool samples for the remaining 45 samples. Hence, it is not sure if whatever E. coli strains recovered if it reflects true diversity.
Currently, it is not clear if five colonies per sample if sufficient to capture the diversity of E. coli. The recovery rate could influence the analysis of the true diversity of E. coli in the samples. I agree that it is not possible to assure complete diversity even from fresh stool samples. This limitation should be clearly mentioned, discussed, and conclusions (number of STs/individual, immigration, etc.) should be made accordingly.
Validity of the findings: The conclusions should be considered with the limitations due to low recovery issue. The observed diversity could represent only be a fraction of the diverse E. coli population existed.
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: GENOMIC DIVERSITY OF ESCHERICHIA COLI FROM HEALTHY CHILDREN IN RURAL GAMBIA
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript is clearly written in English and easy to read. It cites references appropriately. Figures and tables are appropriate and nicely done.
Experimental design: The research question of this manuscript is clear and well defined. The research seems to be performed properly. Generally speaking methods are properly described however there are a couple of analyses where more detailed methodology is required (see "General comments for the author").
Validity of the findings: Dear Dr. Souza,

I have read in detail the manuscript entitled “Genomic diversity of Escherichia coli from healthy children in rural Gambia” submitted by Foster-Nyarko et al. I enjoyed reading this paper and I find it to be a great contribution to our understanding of the nature and mechanisms driving the genetic diversity of strains isolated from healthy infants from a particular geographical area. I will be happy to recommend this manuscript for publication in PeerJ after my comments are addressed.
Additional comments: Major comments

Abstract:

The abstract suggests that the main goal of this study is to evaluate the mechanisms that generate the within-host diversity of E. coli isolated from healthy children from rural Gambia and it doesn’t mention anything about the accessory gene content findings. I suggest the authors to include some of the virulence and antimicrobial genes and plasmid findings in the abstract otherwise it would be convenient to reconsider the title of the manuscript.

Methods:

I encourage the authors to explain in detail how the migration and within-host evolution events were defined and determined using the SNP data since I find it unclear and it is crucial for this study. I would also like to know more details about how the SNPs where detected, the quality control steps done (including quality score values) and an explanation of why they used different algorithms to do it. Also for accuracy, I request the authors to include the software, their versions and parameters used to process the whole genome raw data and assembly.

I didn’t find reported the access number of the whole genome sequences obtained in this study neither in the main text nor in any table.

Results:

I am confused about how the within-host diversity results section is reported. According to the introduction (Lines 67 to 68) the formation of clouds of intra-clonal diversity refers exclusively to the within-host evolution processes therefore 2 or more within-host evolution events reported in Table 2 are present in just 3 individuals (within-host evolution events column) and not in 13 as it is written in the main text. There is an expanded comment about this below. Please make sure to clarify these results since they are crucial. It might have been a typo. I suggest to report also in the main text the number of immigration events to avoid confusions.

Also, I would have liked to see more details about the “Within-host diversity” section since the authors make special emphasis on the sources of within-host genomic diversity in the abstract, introduction, discussion and conclusions.  In addition to other comments (see below) this section would be highly improved if the authors:

-	Calculate the nucleotide diversity from the core-genome alignment for all the isolates and by phylogenetic group.
-	Estimate if the proportion of immigration events and within-host evolution changes varies per phylogenetic group (discuss these results) and,
-	Evaluate the fitness effects of the mutations by obtaining dN/dS ratios.

Discussion:

I would have liked to read about the role of the ecological competition and cooperation among strains (mentioned in the introduction) as well as some of the host traits as factors that determine that different E. coli strains thrive in the hosts.

Also, it would contribute to the discussion if the authors discuss the role of different genetic mechanisms such as point mutation, homologous recombination and, horizontal gene transfer in the generation of the genomic diversity found in their isolates. Although the authors analysed SNPs, it has been demonstrated that homologous recombination is responsible for the generation of a proportion of the core genome diversity (see González-González et al., 2013. Hierarchical clustering of genetic diversity associated to different levels of mutation and recombination in Escherichia coli: A study based on Mexican isolates. Infection, Genetics and Evolution 13: 187-197.)

Supplementary files:

Make sure all the supplementary files are properly cited and numbered in the order they appear in the text.

Minor and specific comments

Introduction:

It would be very good to provide more details about the The Global Enteric Multicenter Study. Although the authors include the references so the reader can go and learn, it would be useful to have a small description about this strain collection to contextualize the results better.

Line 56: I like the ecological mechanisms mentioned and I would like the authors to expand on which epidemiological analyses there are thinking about.

Methods:

Line 86: Explain why ten samples were unavailable for the study.

Line 105: Mention the kit used to prepare the DNA libraries to be sequenced.

Line 107: Although the authors included a list (Supplementary file 5) with the individuals that were sequenced twice, it would be nice to mention the actual number of isolates in the main text.

Line 109: Number supplementary files as they are mentioned in the text.
Supplementary file 5, should be Supplementary file 1 because is the first supplementary file to be mentioned in the text. Also, specify the units of total length (I suppose is “bp”) in Supplementary file 5 Column E.

Line 114: I would like to see a bit more details on this section. I suggest to include the programs (and versions) and parameters used to do the quality check and genome assembly. The authors mentioned that these steps were done as in DeSilva et al., 2016 but I find it more convenient that the reader can obtain this information from the manuscript.

Line 115: Specify if there were any changes in the default when running Snippy.

Line 128: Include MLST in parenthesis after Multi-locus sequence typing.

Line 132: Include cgMLST in parenthesis after core-genome MLST.

Line 158: Include what does cgST stands for in parenthesis.

Line 141: Please include the quality control steps (and the strains used as reference) followed when using CSIPhylogeny tool to detect the SNPs. Also include the read mapping, SNP quality and SNP depth quality scores used.

Line 140: Shouldn’t be “the whole genome sequences” of the strains sequenced twice instead of “duplicate sequence reads”? Duplicate sequence reads normally refer to those raw reads that are sequenced twice by the sequencer and that can be the product of extra PCR cycles during the library prep.

Line 143: Which two steps? Shouldn’t be two different algorithms or software to detect SNPs instead? I suggest that the SNPs detection have its own subsection therefore you can expand more in details about parameters and quality checks used. Also, in this same paragraph the authors can explain in detail how did they differentiated between migration and within-host events. Dixit et al. 2017 describe nicely all this.

Line 151: Explain which software or algorithm was used to calculate the co-occurrence of AMR genes.

Line 164: Do you mean all the pathogenic and commensal E. coli strains from the GEM database, including samples from other countries such as Kenya, India, etc? Clarify and include the number of strains.

Results:

Line 181: I would prefer Table 1 to be a supplementary file.

Line 183: Refer to Supplementary information 2 at the end of  “yielding 88 isolates”.

Line 185-186: Besides mentioning that the strains were scattered over all the eight main phylogenetic groups of E. coli, include a more quantitative description about their distribution taking into account all 88 isolates. For example, one way could be to report the proportion or percentage of isolates that belong to phylogenetic group A out of the 88 isolates and so on with the other phylogenetic groups.

Lines 195-197: What does variant refers in this context? What do you mean with SNP cloud? Define. I am confused about how the way these results are reported. According to the introduction (Lines 67 to 68) the formation of clouds of intra-clonal diversity refers exclusively to the within-host evolution processes therefore the within-host evolution events reported in Table 2 are present in just 3 individuals (within-host evolution events column) and not in 13 as it’s written in the text. I am a bit unclear about how the immigration events explain the three quarters of the observed variation calculated according to the information of Table 2. Please make these results as clear as possible. Also report how many SNP differences where detected between the two whole genome assemblies of the same isolate.

Line 201: How did you determine that two or more isolates belonging to the same ST present in the same host are the results of an immigration or within-host evolution event (For example host H-2 in table 2, all 5 isolates belong to the same ST9274)?

Line 208: I suggest moving Table 4 to supplementary information.

Line 211: Could you elaborate more why to focus on ST38? Mention in the main text the message conveyed on each panel of Figure 5.

Line 215: Mention which clinically relevant classes of antibiotics are these.

Line 213: Expand on the results about the encoding putative virulence factors. For example, mention the most prevalent putative virulence factors or you can mention the main categories included in Figure 2 and if they distribute across all the samples, etc. Also test if there is a statistical association of particular virulence factors with ST and phylogenetic group.

Line 220: How large are these plasmids? Include approximated size.

Line 240: Nice! I find this very interesting.

Tables and Figures:

Table 1. Can go to supplementary information

Table 2. I suggest to change “Genotype number” for “Colony or isolate number”. I find colony number more appropriate since for instance, all the colonies isolated from particular individuals belong to the same ST or have the same genotype (Invididual H-2, H-28, etc.).

Figure 5. Panel A. I guess the size of the circle represents the number isolates per clade. Specify this in the figure caption. I assume that persistent strains refer to commensal strains. I suggest to change “persistent” for “commensal” to be consistent with the main text.  Make sure that the explanation about the red highlights used to draw attention to a particular isolate in panel C is said for all panels because for example in the figure caption corresponding to Panel A and B, there is no explanation about these red highlights. Also, you say that the geographical locations where isolates were recovered are displayed in Figures 4A-C and Figure 4 correspond to the plasmid content results.

GEMs consent form file. This form is not filled out. I though the authors had obtained the samples from the GEMS collection directly and that they didn’t have to collect them. If this is the case, then, I question the need to include this black consent form.


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

Input:
Title: GENOMIC DIVERSITY OF ESCHERICHIA COLI FROM HEALTHY CHILDREN IN RURAL GAMBIA
Review round: 2
Reviewer: 1

Output:
Basic reporting: The manuscript is clearly written and structured. It also cites relevant references in the field. The figures are self-explanatory and with a nice design.
Experimental design: I feel satisfied of how the authors expanded on how they performed molecular and bioinformatic analysis. Also, the authors present clear aims and their results filled a knowledge gap.
Validity of the findings: Dear Dr. Souza,

I have read in detail the new version of the manuscript entitled “Genomic diversity of Escherichia coli from healthy children in rural Gambia” submitted by Foster-Nyarko et al. and I find satisfactory the way they authors incorporated and addressed my previous comments. I recommend this new version of the manuscript for publication in PeerJ.
Additional comments: No additional comments


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

Input:
Title: A NOVEL SIMILARITY SCORE BASED ON GENE RANKS TO REVEAL GENETIC RELATIONSHIPS AMONG DISEASES
Review round: 1
Reviewer: 1

Output:
Basic reporting: With regard to two diseases, for a list of genes they are associating with these two diseases, when the ranks of these genes are considered in the association with the diseases, the author has proposed to use the geometric mean of the reciprocals of the ranks as the basic unit to measure the similarity between the two diseases by summing up all of the geometric means. The measurement is termed as SimSIP. The new similarity measurement was tested on simulation datasets, and also on real gene expression datasets from TCGA. The method was compared with WeiSumE*, OrderedList, FES0.01 and EucD to demonstrate its superior performance in the identification of similar diseases. The author also reported associated genes and pathways with colorectal cancer.
Experimental design: Comments: The manuscript is well written and organized; the novelty of the ideas is not that strong; the performance evaluation is well designed and discussed.
Validity of the findings: Suggestions:
1.	I don’t understand why equation \sum^n_{i=1}(sqrt{1/a_i})^2 is popped up in the second paragraph of  page 3. Why did you need this sentence? Please clarify.
2.	Ranking methods for genes are less discussed in the method section. In fact, the SimSIP results are very sensitive to the ranking lists of the genes. Which ranking methods should be recommended? Please clarify.
Additional comments: See above.


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

Input:
Title: A NOVEL SIMILARITY SCORE BASED ON GENE RANKS TO REVEAL GENETIC RELATIONSHIPS AMONG DISEASES
Review round: 1
Reviewer: 2

Output:
Basic reporting: Generally, the paper makes a good impression mainly connected to the experimental study section. However, the theoretical part raises many questions. First of all, the motivation according to the implication of the new connection measure is very blurred. Computanchilay, a vector composed from the inverse ranges, provides more reliable results, but it is entirely not understanding why.  An explanation given in rows 115-118 is very abuse and does not offer any useful information about why such a measure performs better in comparison with the standard inner product.
The paper is written in a deplorable manner. For example, what does it mean:
All empirical sizes shown in Supplementary 158 Table S1 are around the significance 0.05 and are well controlled. I feel that the paper provides exciting results, but it very hard to understand it from the text.
Experimental design: Provided in a good manner
Validity of the findings: I thing that more solid comparison has to be done
Additional comments: Generally, the paper makes a good impression mainly connected to the experimental study section. However, the theoretical part raises many questions. First of all, the motivation according to the implication of the new connection measure is very blurred. Computanchilay, a vector composed from the inverse ranges, provides more reliable results, but it is entirely not understanding why.  An explanation given in rows 115-118 is very abuse and does not offer any useful information about why such a measure performs better in comparison with the standard inner product.
The paper is written in a deplorable manner. For example, what does it mean:
All empirical sizes shown in Supplementary 158 Table S1 are around the significance 0.05 and are well controlled. I feel that the paper provides exciting results, but it very hard to understand it from the text.


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

Input:
Title: A NOVEL SIMILARITY SCORE BASED ON GENE RANKS TO REVEAL GENETIC RELATIONSHIPS AMONG DISEASES
Review round: 2
Reviewer: 1

Output:
Basic reporting: The basic findings stated in the manuscript have not been changed after the revision. The authors have well addressed my questions and concerns.
Experimental design: The experimental design is sound.
Validity of the findings: The performance evaluation can be trusted.
Additional comments: The revised version of the manuscript has reached a very high standard. I recommend to accept the paper for publication in PeerJ.


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

Input:
Title: A NOVEL SIMILARITY SCORE BASED ON GENE RANKS TO REVEAL GENETIC RELATIONSHIPS AMONG DISEASES
Review round: 2
Reviewer: 2

Output:
Basic reporting: The current presentation arrears to be sufficiently appropriate, but intensive proofreading is required.
Experimental design: The experiments are designed  well
Validity of the findings: Experiments validity is appropriate
Additional comments: It would help if you improve the English style


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: HEALTH STATUS OF POLYCHRUS GUTTUROSUS BASED ON PHYSICAL EXAMINATION, HEMATOLOGY AND BIOCHEMISTRY PARAMETERS IN COSTA RICA
Review round: 1
Reviewer: 1

Output:
Basic reporting: It is a very good paper, with an adequate experimental design wich offers new and valuable  biological and physiological information about a non well know lizard specie from Central America.
However, need some corrections or adequations in order to be a paper of excellence
The literature provided is appropriate and sufficient.
Experimental design: It is a good research paper, with a very good experimental design that fall into the scope of the journal
Validity of the findings: The paper offers valuable  biological and physiological information about a non well know new lizard species.Statistical analysis is well designed
Additional comments: Materials and methods
Lines 150-151:  How much animals were bleed twice ? (Caudal and  jugular vein) This procedure can affect the hemogram and some biochemical analytes  as a CPK or AST. In this case, the sample was processed or discarded?and in the case that the sample was processed, the results of this duplicated samples  were included?
Results Lines 215-216
The erythrocytes and thrombocytes shapes and dimensions were measured ? Because in the text the authors write” Both shape and appearance of erythrocytes and thrombocytes were similar to those reported for other reptiles.” Please, it is necessary to clarify this point with a brief descriptions of erythrocytes and thrombocytes  morphology, shape  and appearance.

Discussion,Lines 286-292
Please clarify the point  about the validity of the ratio Heterophiles: Lymphocytes as a tool to assay the stress, taking in mind that H:L ratio is based in species wich have heterophiles as a the predominant white blood  cell. In stress condition  a decreased lymphocyte count occurs( lymphopenia) and appears a concomitants heterophilia . In this cases, wich the animals exhibiting  linfocytes as a predominant leukocytes, is not clear the adequate use of this ratio as an health status evaluation tool.  A lot of studies suggest that H : L ratios can be used to assess glucocorticoid levels and stress in reptiles; however, it is  point out that less work has been conducted in the reptilian taxon than in others vertebrates.
Figure 2.c: The cell described as an eosinophile is not this type of leukocyte .The eosinophile have reddish,round and regular sized granules, well visibles with the stain,and the nucleus is eccentrically located. In the picture i can see a round cell, with a unique and central nucleus and a lot of colorless vesicles, without granules. Probably is another type of blood cell , but ,in this case , unfortunately not an eosinophile.  Please verify the picture and  the stain.


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: HEALTH STATUS OF POLYCHRUS GUTTUROSUS BASED ON PHYSICAL EXAMINATION, HEMATOLOGY AND BIOCHEMISTRY PARAMETERS IN COSTA RICA
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: The submission was clearly defined the research question. The study method was conducted rigorously and to a enough technical standard. Methods was described with sufficient information to be reproducible by another researchers.
Validity of the findings: All underlying data have been provided; they are robust, statistically sound, and controlled. Conclusions are well stated, linked to original research question and limited to supporting results.
Additional comments: Dear Authors,
I have gone through your submission about the hematology and some biochemistry parameters of Polychrus gutturosus in Costa Rica. Unfortunalety, there are limited data on baseline data of healty parameters in many herptile species. The present study will be fill the gap in this subject for Polychrus gutturosus. However, I noticed there are some minor point which need revise or remove.  I indicate my suggestion on text. In my opinion, the article should be accepted if the authors make some revisions, minor enough that I would NOT necessarily need to re-review it.


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: HEALTH STATUS OF POLYCHRUS GUTTUROSUS BASED ON PHYSICAL EXAMINATION, HEMATOLOGY AND BIOCHEMISTRY PARAMETERS IN COSTA RICA
Review round: 1
Reviewer: 3

Output:
Basic reporting: Approved. See specific comments, suggestions, recommendations in PDF review. Specific changes recommended on several figures and tables.
Experimental design: Approved. See comments and general recommendations in PDF review.
Validity of the findings: Approved. See comments in PDF review.
Additional comments: See notes embedded in review of PDF.
Overall well put together study and paper. See comments and suggestions in PDF review.