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: IDENTIFICATION OF AREAS OF VERY HIGH BIODIVERSITY VALUE TO ACHIEVE THE EU BIODIVERSITY STRATEGY FOR 2030 KEY COMMITMENTS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The introduction was structured well and gives sufficient detail to ground this study.
The methods made structural sense as well, but I do have some questions about methodological details (see experimental section) that I think need to be addressed to make sure readers can follow what the authors have done.
The rest of the manuscript is structured well, with appropriate literature cited.

I am not sure if the data shared would be sufficient to replicate the analysis. I generally hope for authors to share their data and analysis scripts, so that others (including reviewers) can properly verify analysis and comment on reproducibility of the analysis underlying a manuscript meaningfully.
Experimental design: I’ve read the methods section three times now and I’m still not sure I understand how the authors have setup their analysis. Some questions that I still have:

Was each 1x1 km pixel of the country used as planning units, or only those that were Natura 2000 sites? I think it was only Natura 2000 sites, but am not sure. Could you clarify that in text?

Starting on line 140 you state: “If a species was included in the Standard Data Form for a given site, it was considered as present in all UTM 1 × 1 km overlapping the respective site.”

Does that mean that if a species was recorded for a Natura 2000 site, each cell of that site was considered as having that species present? To me this is a huge assumption and worrisome. If I think about the fact that some sites are >1200km2, I worry that this could easily be more area than the entire species might occupy. You mention this in the last paragraph of the discussion, but I was hoping you could expand on this in terms of what this would mean for the Zonation analysis as well? I think this would be important to note for reader to fully understand the potential limitations of this data set.

I think making sure readers understand what data was used how is very important to understand both, what the authors did and equally important, what the author’s findings mean.
Validity of the findings: This section is hard for me to comment on, as I did not fully understand the experimental design portion of this review.

I will still comment on two aspects of the work:

1. If the analysis has indeed been conducted at the Natura 2000 site level, I am wondering what the analysis would actually show? If this analysis is restricted to Natura 2000 sites, the analysis can only rank Natura 2000 sites and does not tell us anything about the broader question of whether Natura 2000 sites are effective at capturing high biodiversity areas across Romania. To me, the ranking of existing Natura 2000 sites isn’t that informative. This might very well be my not fully understanding what the authors have set out to do here.

Could you please add text explaining why you think this approach makes sense and what it actually tells us?

If the assumption (and its just my assumption, as I don’t fully understand the methods as currently written) is correct that an occurrence of a species at a site would result in the entire site to be coded as having the species present, I don’t quite understand how the analysis can yield results on high biodiversity areas requiring Zonation analysis. Could you please add some text to help clarify your thinking about this?

2. If the analysis was indeed conducted across the entire country, it would be good to make this clear in methods and also results.
Additional comments: It would be great if you could share the Zonation input files and analysis description/script, so we (reviewers and readers) could replicate your work to help us understand how your analysis was conducted.

Figure 1: Could you make the biogeographic region delineation easier to see, by changing the line color or make them wider? Its hard to make out the different regions at the moment.

Best regards,
Richard Schuster

Research Associate, Carleton University
Email: richard.schuster@glel.carleton.ca


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: IDENTIFICATION OF AREAS OF VERY HIGH BIODIVERSITY VALUE TO ACHIEVE THE EU BIODIVERSITY STRATEGY FOR 2030 KEY COMMITMENTS
Review round: 2
Reviewer: 1

Output:
Basic reporting: -
Experimental design: -
Validity of the findings: -
Additional comments: Thank you very much for carefully addressing all reviewer comments.

I am happy with how you responded to all comments and don't have any further comments and think the manuscript can be published as is.


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: IDENTIFICATION OF AREAS OF VERY HIGH BIODIVERSITY VALUE TO ACHIEVE THE EU BIODIVERSITY STRATEGY FOR 2030 KEY COMMITMENTS
Review round: 2
Reviewer: 2

Output:
Basic reporting: English is sufficient.
Line 78-81 is about Natura 2000 effectiveness.
In 2019, work on this topic appeared: Effectiveness of Natura 2000 areas for environmental protection in 21 European countries (open access). You can find interesting information about the state of Natura 2000 areas in Romania. This is just a suggestion.
The literature references are sufficient.
The article structure is good, as well as all figures, tables, and raw data.
Experimental design: Original primary research within Aims and Scope of the journal - the submission is relevant and meaningful.
Methodology is correctly described (especially after comments from previous reviewers).
Validity of the findings: The results are very interesting in the context of the EU Biodiversity Strategy for 2030 key commitments.
Additional comments: I have read the first draft of the manuscript and the comments of previous reviewers. In my opinion, most of the comments were properly taken into account in the text improvement process.


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: MULTI-DEEP: A NOVEL CAD SYSTEM FOR CORONAVIRUS (COVID-19) DIAGNOSIS FROM CT IMAGES USING MULTIPLE CONVOLUTION NEURAL NETWORKS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Basic reporting
Introduction
The introduction should emphasize more clearly what the advantage of a rapid diagnosis of Covid19 is. The advantages mentioned are a bit vague (to lessen the load on the healthcare organization as well as achieving the finest potential care for infected people). As far as I understand, there is only supportive care available for Covid19 patients apart from experimental treatments, but I’m sure this situation is fluid. Is the advantage to the swab test the speed of getting results? Does this help with isolation protocols and saving resources in case of negative results? Would someone testing negative on the CAD not be put in isolation, knowing that the test does not have 100% sensitivity? Are patients diagnosed based on CT scans alone? Or are CT scans mostly used to assess severity? Do patients with mild symptoms have lung abnormalities? I’m just giving examples of questions, they need not necessarily be addressed, but as a reader I’m not sure I understand what the advantage and relevance of CAD for Covid19 is.
The introduction lists a number of previously developed machine-learning models to diagnose Covid19 from CT scans. There is mention of multiple methods called ResNet50, U-Net++ and others. For a reader in the medical field who is not familiar with the jargon these names mean nothing. Can you give a short explanation of what these are?
I recommend moving the paragraph 2.2 in Materials and Methods to the introduction as a description of what CNNs are.
The language is often awkward. I recommend having the manuscript edited by a native English speaker. Here are some examples:
•	Line 29 COVID-19 is generated initially in Wuhan, China – this sentence should be rephrased to something like: COVID19 was first observed in Wuhan, China
•	Line 120: … “is not investigated”. Do you mean the effect was not investigated in those previous studies?
•	Line 121: This is an incomplete sentence
•	Lines 144/145: is it convolutional layer or convolution layer?
Specific items:
Line 57: Is there evidence for this statement? If yes, please insert reference; if not, please state that this is a speculation
Line 119: Please  define DCNN
Experimental design: Experimental design
Materials and Methods
How was it determined that the images are or are not from Covid19 patients? What was the reference test? I briefly looked at the reference, which is a webpage with a link to the dataset. I could not determine what methods were used to distinguish cases from non-cases. If you have the information, please state it briefly; if you don’t have it then also please comment on the lack of that information.
Furthermore, can you describe the data set of images in terms of severity, e.g. hospitalized patients, a range of severities? Again, if that info is not available, please state so.
Other authors have applied segmentation to distinguish lung from other tissues. Has this been done in your method as well? If yes, where is it described? If no, why not?
Line 264: You mention that you are using 5-fold cross validation. Are the reported performance metrics based on testing with part of the data set aside for this purpose and not used during model training/validation? Or is the entire data set used for training/validation/testing?
Specific issues:
Line 205: here you say the proposed CAD is based on the fusion of multiple CNNs. Then in line 296 you say four pre-trained CNNs are used to detect COVID-19 and non-COVID19 cases individually. Are the CNNs fused or working individually? It seems from the results that they are working individually, so the statement in line 205 needs clarification.
Line 134: is the length and width of CT images unitless?
Line 145: is FC layers = fully connected? Please define at first mention
Figure 2:
What is the pre-processing step in this figure? I don’t see this mentioned in the text.
The resolution of the image should be improved. After reading the paper several times, I’m still a bit confused about your use of the terms stage and scenario. Is the process described a sequence of processes from I to IV or are these different scenarios, i.e. one of the options is chosen?
The diagram looks as if the 4 scenarios are either happening simultaneously or one is picked by the system. In the description of the text, it sounds like all these steps are happening sequentially. Which one is it? The word scenario may not be very good if all steps happen each time; the word stage may be better in this context. Or are there 4 stages and in stage 4 there are 4 different scenarios as mentioned in Line 247 where the scenarios represent increasing numbers of combination of stages?
Table 2: what are the units of the input and output sizes?
Validity of the findings: Results
The first paragraph seems to be a summary of the methods – consider eliminating.
Table 3 and 4: it looks like you have (std) in both table headings but only results in Table 4. Also, time (sec) seems not appropriate in the Table 3 heading.
Discussion
You are mostly summarizing the results in your discussion without critically comparing your work to that of others. Some of the questions your discussion should attempt to answer are:
•	What are the limitations of your study?
•	What is a possible scenario where this algorithm can be used and how will it be implemented? Obviously all patients that get a CT scan for diagnosis of Covid19, but who are those patients? What distinguishes them from those that get a swab test?
•	What are the next steps?
•	The uses you are stating are a bit lofty, i.e. decrease death rates, reduce load on the healthcare organization and improve quality of health management. What is your estimate of the impact this system might achieve? How many more patients will be accurately diagnosed based on the CAD than if this was not available?
•	Do you know what the performance of a trained radiologist is without the help of a CAD (Se, Sp, etc)?
•	Who implements these systems into practice? How do these CAD systems become incorporated into CT software?
Additional comments: The topic of the study is very timely and important and has the potential to affect a large number of patients worldwide. A novel machine learning algorithm is described for the diagnosis of Covid19 pneumonia based on a CT scan that could potentially make important contributions to this field. Your results look promising, but the process needs to be described more clearly. In particular, the four stages or scenarios are quite confusing. Figure 2 should be produced with higher resolution, at the moment it is hard to read. This figure should also be used to more clearly describe how the system works, i.e. what is a stage, what is a scenario, how does the information flow?


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: MULTI-DEEP: A NOVEL CAD SYSTEM FOR CORONAVIRUS (COVID-19) DIAGNOSIS FROM CT IMAGES USING MULTIPLE CONVOLUTION NEURAL NETWORKS
Review round: 1
Reviewer: 2

Output:
Basic reporting: There are a number of concerns about the manuscript, including the following:#

1) The manuscript has some merits, but there are too many grammatical and typo errors throughout, but especially in the Introduction. This detracts from the paper. Some examples are given below. This should be fully addressed.

2) Line 1: Title of paper – this should be  “… diagnosis of …”.
3). Line 29  Abstract. Change ‘generated’ to ‘originated’.
4). Line 50-53. Include the AUC value for your method as part of the metrics.
5). There are a number of factual inaccuracies and speculation. E.g. in Line 57. The authors are speculating about the origin of SAR-COV2 (i.e. from bat). This is inappropriate as this has not been conclusively proven.
6) Line 61: it is incorrect to say that WHO declared COVID-19 a pandemic on 11 March. It was actually at the end of January 2020. Note also that the correct phrase is that it was declared a pandemic. It was stated as a  pandemic is incorrect phrase.
7). Line 129: This is an incomplete statement. If you mean to say that Table 1 should be inserted here, then say so, otherwise delete line. E.g. (Please insert Table 1 here).
8). Line 138-139. Similar comments to Line 129 above.

9) Line 175: AlexNet is one of the primaries of CNN. This does not make sense. Unclear what is meant by this.
10) Line 216 See comments to Line 129 above.

11). Line 203-215 Section 2.3 You should give a good overview of your system here. Explain clearly the role of each block and how the overall system is used to achieve the detection of COVID-19.

12). Line 304 See previous comments.
13). Line 315-318.  These are where the figures are to be inserted. If so make this clear (e.g. Please insert table 4 here)
14) Line 366- Section 6 Discussions. The English in this section need attention.
15) Line 394. See previous comments on insertion of figures and tables.

20.	Line 315-318.  These are where the figures are to be inserted. If so make this clear (e.g. Please insert table 4 here)
Experimental design: 1).. The authors have given a good review of the literature in the Introduction. However, in Line 120-121 the authors appear to be saying that the main issue with existing methods is that the effects of combining multiple CNNs on classification/diagnosis has not been investigated, together with the need to reduce the feature space. Given that these are the main contributions and novelties in the paper, the authors should expand these points in both the Introduction and in the Discussions.

2).. The CT shown for COVID-19 and non-COVID-19 are quite distinct. Why do we need the complicated structure of proposed in your methods to distinguish between them?

3). Section 2.31 Why is it appropriate to use these particular pre-trained models, given that the classification task in COVID-19 and in the original task for these CNNs are different?

4). Line 266-273 Section 3.2 Augmentation. What are the final sizes for the datasets (COVID-19 and non-COVID019 sets) after augmentation and what is the rationale for settling for these.
Validity of the findings: 1). In Table 1, one of the best models has performance metrics of AUC = 0.996, Sensitivity of 98.2% and specificity of 92.2%.  Some of the existing methods appear to perform better than your method in some of the metrics. A comment on this should be included in the Discussions and Abstract.

2). Your method uses publicly available CNN models, including those developed by commercial companies. You should discuss the implications of this in clinical practice.

3) Line 341. Please give the AUC to allow effective comparison

4). Line 403-407. According to Table 1, it appears that some of the existing methods have better or comparable performance metrics than MULTI-DEEP. What are then the benefits of MULTI-DEEP?

5). There should be comments on the novelties/contributions of the study in relation to existing methods in the Discussions.

6). It is unclear how the outcome of the study can be translated into clinical practice, given that some of the CNN tools are owned by a commercial company and the issue of confidentiality of patient information. This point should be discussed.
Additional comments: The software codes for the study should be made publicly available.


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: MULTI-DEEP: A NOVEL CAD SYSTEM FOR CORONAVIRUS (COVID-19) DIAGNOSIS FROM CT IMAGES USING MULTIPLE CONVOLUTION NEURAL NETWORKS
Review round: 2
Reviewer: 1

Output:
Basic reporting: There are still some issues with English in the manuscript. I've pointed out a few instances, but there are a number of awkward phrases or grammatical errors.

Line 52: I recommend changing the wording to “the WHO encouraged efforts to reduce the spread of the infection, many countries…”
Line 55: “led to a tremendous increase in the need for hospital beds and necessary medical equipment”
Line 56:” Moreover, doctors and nurses are at high risk of infection.”
Line 63: “ the sensitivity of the RT-PCR test is inadequate resulting in more infections through test negative patients.”
Line 67: “precisely at the center of the pandemic area” – what do you mean with this phrase? The center of the pandemic is hard to pinpoint and is all around the world but also constantly shifting.
Line 72: use “advantage” instead of “privilege”.
Line 73: “deducing a more favorable illustration” – I don’t understand this phrase
Line 80: “help radiologists in giving an accurate diagnosis based on medical images”
Line 96: “DL methods are more appropriate for detecting covid-19 from CT images” – do you mean more appropriate than a radiologist? Or should they be used to enhance the radiologist’s diagnostic capabilities?
Line 155, , 174: convolution layer – you said in your rebuttal it was convolutional layer – please correct.
Line 168: “In the literature, these networks have not yet been used to detect and classify COVID-19”
Line 177: “AlexNet CNN was initially trained with ImageNet data…”
Line 197: “to lower the computation cost”
Line 402 “Discussion” instead of “Discussions”
Line 403 Delete the word “Nowadays” – it means “at the present time, in contrast with the past” Suggest changing the wording to “COVID-19 has led to the greatest worldwide health crisis in the recent past.”
Line 438 – 450 This section is a repeat of your methods and has already been explained at length before. Please delete. Figure 5 is part of the results and should be described in the results section.
Line 458 Your missing a digit, should be 33.765 s
Line 505 I wouldn’t use “inadequate” – use “relatively small”. Inadequate implies that you really didn’t have enough images to do the work
Experimental design: Your narrative of the design would be a lot easier to read if you presented it in the past tense as a description on how you developed and tested the system. The use of the present tense throughout your description implied to me that the entire process is part of MULTI-DEEP, when in reality, as I understand, it is scenario 3, which maximizes accuracy and computational speed. Please describe the development of the system in past tense instead of describing what you did in the present tense. E.g., line 221: “In the end-to-end DL classification stage, four CNNs of different architectures were constructed with the images available in the dataset. Each of the CNNs performed the classification and diagnosis procedure individually. In deep feature extraction, valuable features were extracted from each CNN separately…”
I still found the term “stage” confusing in your narrative, as it implies that subsequent stages depend on previous stages. I would prefer a term like “component” or “approach” to enhance clarity.
Validity of the findings: Line 507: I think you mean that you want to expand the model so that it can correctly diagnose other types of pneumonia while at the moment it can only distinguish COVID-19 from non COVID-19 cases. If so, please change the wording accordingly.

Conclusions

You should add that MULTI-DEEP should undergo further field testing before it can be employed by radiologists. Also, it will likely have to undergo regulatory approval by health authorities before its implementation into hospitals.
Additional comments: Thank you for putting a lot of effort into the revision of the manuscript. I have a few more comments at this point.


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: MULTI-DEEP: A NOVEL CAD SYSTEM FOR CORONAVIRUS (COVID-19) DIAGNOSIS FROM CT IMAGES USING MULTIPLE CONVOLUTION NEURAL NETWORKS
Review round: 2
Reviewer: 2

Output:
Basic reporting: The English has improved, but there is still a need to still proof read the revised manuscript.
Experimental design: This is satisfactory.
Validity of the findings: This is satisfactory.
Additional comments: The authors have addressed most of my concerns.
However, it is recommended that a further proof reading of the manuscript is undertaken.


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: EXPLICATING ANTI-AMYLOIDOGENIC ROLE OF CURCUMIN AND PIPERINE VIA AMYLOID BETA (AΒ) EXPLICIT PATHWAY: RECOVERY AND REVERSAL PARADIGM EFFECTS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The current study by Manap et al. identifies numerous biological pathways that are differentially regulated in Aβ amyloid-induced SH-SY5Y cells by treatment with the individual compounds Curcumin, Piperine, or a combination of both. This study builds on their previous work (Manap et al., 2019) where they showed that Curcumin and Piperine act in synergy to confer protection from Aβ toxicity. Now, the new results provide a comprehensive gene expression profile comparison via microarray analysis between Aβ amyloid-induced SH-SY5Y cells and the three treatment conditions. The results suggest that these compounds act on multiple pathways of relevance to Aβ toxicity including production, trafficking and clearance of amyloids, as well as downstream effects on neuronal health and function. The authors mine existing literature to provide potential mechanisms by which some of these gene expression changes may confer protection from disease progression. For the remaining significant differentially expressed genes are suggested as potential starting points for further exploring unknown mechanistic details of the disease.
The study has included ample references and background literature to support their conclusions. However, there are multiple areas for improvements (outlined below) that would enhance the general readability and accessibility of the manuscript.
Experimental design: The question is well defined and how the results address it are stated clearly, including identification of potential new avenues to address existing knowledge gaps. The Methods are also described in sufficient details.
Validity of the findings: Overall it is a well-designed study with appropriate methodology and analysis, including statistical significance of the results. A large part of the study relies on summarizing existing literature for theorizing on their own findings but they also provide several new potential avenues for research into Aβ toxicity, e.g. the ‘novel’ genes ITPR1, GSK3B, PPP3CC, ERN1, APH1A, CYCS and CALM2.

There are a few concerns to be addressed:
i.	The Microarray analysis identifies APOE as a gene whose expression is significantly reduced by Curcumin treatment. In the Discussion (Lines 452-468), the authors discuss in detail the role of the APOE*ε4 allele in risk for AD disease and also mention “We demonstrated that APOE*ε4 gene expression was upregulated in the group treated with Aβ alone, which may increase the risk for AD” (Line 459). However, it is not clear how the authors arrived specifically at the ε4 allele in their results. The microarray results do not suggest the specificity of the allele being upregulated, and APOE allele specificity is also typically unknown in cell lines including SH-SY5Y.

ii.	Line 525: “We demonstrated from our study that the expression of CREB was downregulated in the group treated with Aβ”. In this line and a few other places, it appears that the authors are describing gene expression changes due to Aβ induction alone. However, a control condition for cells without Aβ induction was not included in the study so this is confusing. Can this be clarified?
Additional comments: Major:
i.	The overall writing can be improved for better clarity and conciseness. It is currently very wordy and written in a casual and inconsistent writing style which makes it a bit difficult to read and understand in several places. Some sentences are very long whereas some are extremely abrupt and confusing. Examples: Lines 326-330, 582-585, 589-590.
ii.	The Discussion section is a good effort in summarizing existing knowledge about cellular pathways involved in Aβ toxicity and where the genes identified can the current study may be potentially exerting their effect. However, it is very lengthy and in multiple places it feels like summaries of other papers e.g. Line 335 -346 appears to summarize just Yu et al., 2005.
iii.	Figure 12 is practically identical to Figure 3 from Xu et al., 2014. While the reference has been cited appropriately in the figure legend, it only serves to provide the background for how PICALM may be relevant. There is no reason to reproduce a published figure from another study where no new information or insight is being added. Similarly, Figure 13 summarizes the findings of Russel et al., 2012 without advancing existing knowledge.

Minor:
i.	The current version has individual experiments as individual figures, more like a thesis structure. For example, Figures 2 and 3 both demonstrate the presence of Aβ in the cells and involve microscopy images. These could be easily combined into a single figure as separate panels. Similarly, Figures 7, 8 and 9 could be panels in a single figure or at least two could be combined.
ii.	The labels of Figure 5 are too small to read.
iii.	Lines 406-409 give the slightly misleading impression that these are hypotheses tested in the study. It should be stated more appropriately as background knowledge that is relevant to and supported by the new findings.
iv.	The first two sections under results are written like Materials and Methods. Please considering describing these better and with appropriate subtitles indicative of the result being reported.
v.	Errors: Line 436 – I believe ‘components’ should be replaced by ‘substrates’. The current sentence is very tricky to understand. Also, PCA is “principal component 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: EXPLICATING ANTI-AMYLOIDOGENIC ROLE OF CURCUMIN AND PIPERINE VIA AMYLOID BETA (AΒ) EXPLICIT PATHWAY: RECOVERY AND REVERSAL PARADIGM EFFECTS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The authors elucidated gene expression profiles in a AB-42 induced neuroblastoma cell line in response to circumin and piperine treatment. Overall, it is a comprehensive study identifying putative genes that might contribute to the development of AD.

The authors were careful to reveal the shortcomings of microarray analysis with respect to false positives and negatives in their introduction. They might want to consider to reiterate the same in the Discussion.
Experimental design: Research question is well defined and putative hits analysed thoroughly. Future work in primary neurons is required to confirm the validity of these findings as well as elucidate the mechanistic details of the molecular pathway/s.
Validity of the findings: 1. The authors might consider explaining why pretreatment of the cell line with the compounds was performed prior to addition of AB.
2. Micrarray analysis of control neuroblastoma cells pre treated with circumin/ piperine/circumin +piperine might be a critical control and might be considered
3. Downregulation of RAB 5B was observed in all 3 treatment groups in response to AB addition. Downreguation of RAB 5 will lead to severe defects in intracellular protein trafficking ultimately leading to cell death. Did the authors observe any cell death over time?
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: EXPLICATING ANTI-AMYLOIDOGENIC ROLE OF CURCUMIN AND PIPERINE VIA AMYLOID BETA (AΒ) EXPLICIT PATHWAY: RECOVERY AND REVERSAL PARADIGM EFFECTS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The revised version of this manuscript has been significantly modified by the authors based on the recommendations made in the original review. The overall writing has been improved and a lot of the extra details have been trimmed to make it somewhat more concise compared to the original version.
While most issues have been taken care of appropriately, I have a few specific suggestions for improvement as outlined below.
Experimental design: N/A
Validity of the findings: N/A
Additional comments: Thank you for addressing most of the original concerns suitably. There are three minor areas where this paper could still improve further to meet the publishing quality of PeerJ:

1. Abstract: It would be really helpful to have a compact and focused abstract outlining just the key findings of the study, in context; and excluding all superficial detail.

2. The PICALM figure (now Figure 9) as pointed out before, is practically identical to a figure from Xu et al., 2014. If it is considered essential to the current study, please make sure to cite the original source in your figure legend and say something like 'adapted from'.

3. The formatting of the figures could be improved. For example, all panels in Figure 2 look exceptionally stretched out horizontally. Please ensure that images are resized properly maintaining scaling in both dimensions. Also, in Figures 4, 6 and 7, the details and text in the figure are almost impossible to read. Please fix these for the final version.


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: EXPLICATING ANTI-AMYLOIDOGENIC ROLE OF CURCUMIN AND PIPERINE VIA AMYLOID BETA (AΒ) EXPLICIT PATHWAY: RECOVERY AND REVERSAL PARADIGM EFFECTS
Review round: 2
Reviewer: 2

Output:
Basic reporting: My concerns have been addresses satisfactorily.
Experimental design: NA
Validity of the findings: NA
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: GENETIC IDENTIFICATION AND HYBRIDIZATION IN THE SEAGRASS GENUS HALOPHILA (HYDROCHARITACEAE) IN SRI LANKAN WATERS
Review round: 1
Reviewer: 1

Output:
Basic reporting: I have read and reviewed the article entitled 'Genetic identification and hybridization in the seagrass genus Halophila (Hydrocharitaceae) in Sri Lankan waters' and overall find the manuscript well written in a professional English tone with only a few punctuation and spelling errors (line 29 - period instead of common after Kurniawan et al.; line 55 - space after 1991, Lines 83-85 & elsewhere - consistency in spacing between value and uL; . Line 198 and 207 - Use or no use of hyphen with 'H. decipiens-like'; Line 259 - misspelling of H. stipulacea; etc.)

The referenced literature is largely sufficient to frame the study, although it is unclear where all referenced measurements from Vera et al. (2014) are sourced from. For example, I don't believe in Vera et al. (2014) the blade width reported but appears in Table 1 here, unless measurements were approximated from the herbarium press photograph. The authors may want to consider other morphological measurements for H. stipulacea from the Caribbean for which several are available before and after Vera et al. 2014. If not included in the table, they could be discussed after Line 207-210.

The structure, figures, and tables are professionally prepared. One suggestion is to remove the decimal place on the numbered axes of PCA figure as they seems unnecessary and somewhat distracting.  The scale used on the PCA also seems to bunch up values along the X-axis. I suggest re-scaling the Y-axis to -/+ 100,000 only and then stretching the figure to be taller.

Also, please include latitude/longitude grid/markings on Figure 1 (map of Sri Lanka) for readers unfamiliar with this geographic region. A regional map/insert would be helpful as well.  What is the significance of the coding in Figure 2 - MA5, MA18? Kindly explain in legend. I find the in-tree sample labels on Figure 3 and Figure 4 very hard to read - the font size is very small. Can this be improved upon? Perhaps color coded by sampling location/source?

The paper is self-contained with relevant results presented and discussed.
Experimental design: The research is original and fits with the aims and scope of PeerJ. Furthermore, it is quite timely given increasing interest in this seagrass genus across its members' native and non-native ranges as outlined in recent review papers.

The research question is clear, but no explicit hypothesis is stated. Perhaps one could be crafted and included in lines 58-60. Inclusion of statistical hypotheses may be appropriate for strengthening the case of potentially novel/cryptic/hybrid Halophila species/plants found in Sri Lanka.  These could be framed around the degree of genetic or morphological differentiation among species and tested via appropriate inferential statistics.  Not critical for the paper, but why were morphological data transformed (lines 120-122; lines 166-167) for the PCA?  What was the justification to transform instead of using straight values?

Most methods used are well-vetted and described with sufficient detail (the exception would be the cloning/testing for hybrids which I lack expertise to judge).
Validity of the findings: Findings seem valid and well-supported.  The contribution of new knowledge on Halophila seagrasses in Sri Lankan waters is also very much appreciated.

One additional note - I am not familiar with the angiosperm literature on hybridization and what metrics are typically used to justify plant hybrids in the same genus, but it appears to me the authors have sufficiently explained this in the discussion. I defer to other reviewers’ more knowledgeable on the topic. As they state in lines 260-263, I encourage the authors to investigate this novel discovery.
Additional comments: No comment.


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

Input:
Title: GENETIC IDENTIFICATION AND HYBRIDIZATION IN THE SEAGRASS GENUS HALOPHILA (HYDROCHARITACEAE) IN SRI LANKAN WATERS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The ms is written well, and you give a great background and wrote a nice introduction. It is great that you give good background about the resolution of ITS. Please do the same for rbcL. As many readers my have no knowledge on details of molecular identification, consider to also add information on what these markers are (i.e. internal transcribed region, which…). Please also provide some details, on why you chose the 3 assessed sites and if they are representative of much of the surrounding area.

References are appropriate.

The first 3 figures are great. The phylogenies are so big that they are difficult to read. Please consider how you can improve them. Can you eg use symbols instead of individual names? Not all writing on the PCA will be readable. Do you need all ticks with writing? Do you need to report eg 5000.0? Increase font size of writing of component 1 and 2. Table 1 was cut off for me, so can’t comment on entire table. The summary states that there are 6 figures and 2 tables, but I can only see 5 figures and 1 table.

The sequences derived from cloning have been submitted to GenBank and accession numbers are given (I have not checked if they are accessible), others are given in supplementary. I thank you for providing the raw data, however your supplemental files need to be referenced to in the main ms and needsmore descriptive metadata identifiers.
Experimental design: The research question is well defined & meaningful. You make a very good case about the relevance of this work to record biodiversity and distribution of species in Sri Lanka.

The methods are appropriate, especially for an initial study that may lead the way to phylogenomic studies. The parameters collected are also valuable, but I am curious also about flowering rates. Did you observe flowers at any site?
Validity of the findings: The conclusions are well stated and confirmed by the data. Make sure you repeat in your conclusion the content of the following sentence from the abstract, as that might be of considerable interest for some: H. major (Zoll.) Miquel is reported for the first time from Sri Lanka
Additional comments: Abstract L15: Taxonomic
Intro
L23: Do you mean ALL seagrass spp?
L38: Needs ref here
M&M
L79: Please give refs for primers
Results
L157: It DOES in fact indicate that, yes.


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: GENETIC IDENTIFICATION AND HYBRIDIZATION IN THE SEAGRASS GENUS HALOPHILA (HYDROCHARITACEAE) IN SRI LANKAN WATERS
Review round: 1
Reviewer: 3

Output:
Basic reporting: The literature related to seagrass communities and biodiversity in Sri Lanka is very scarce. Here authors apply molecular markers - bar coding markers - to demonstrate that the species diversity is actually larger than previously presumed, especially in the Halophila genus.

Overall, the manuscript is well written and, following what I consider a major revision, should be accepted.
I would ask that the authors refer to Les et al. 1997, 2006 and Procaccini et al. 1999 (Structural, morphological and genetic variability in Halophila stipulacea (Hydrocharitaceae) populations in the western Mediterranean) – all of which are super relevant to the study here.
The authors could have wrapped up the paper with something relating the results to future biodiversity conservation efforts in the region…this is important for explaining the broader impacts of this work.
Lastly, the authors totally neglect to discuss the relevance of their methods in the new age of next-generation sequencing (NGS) which could be of particular relevance to DNA barcoding of genetically close species (e.g., DOI: 10.1111/1755-0998.12236, DOI: 10.1007/978-1-61779-609-8_2). In this respect, the methods used in this paper are, by now, “ancient” and in my eyes are history and should not be encouraged…

Clear, unambiguous, professional English language was used throughout this manuscript.
Intro & background are written well.
Authors do neglect some basic papers that are very closely related to their study - examples including papers by Les et al. 1997, 2006 and Procaccini et al. 1999.
Literature referenced can be greatly improved – I have mentioned where…many issues.
Books are not referenced properly…revise the formatting. Missing important papers
article structure, figures, tables - are in order.
Experimental design: The exp design is ok - but raw sequences have not been uploaded in public banks (e.g., NCBI).
Validity of the findings: Building on from the latest report that showed that Sri Lanka had 14 seagrass species as of 2017 (Udagedara et al., 2017), in the present study, based on genetic analyses, the authors show that some plants previously considered H. ovalis based only on morphological differences), are actually H. major (Zollinger) Miquel (1855), which is a new species record for Sri Lanka (thus, bringing the species list to 15). These results support the notion that relying only on morphological measurements, could lead to underestimating seagrass biodiversity due to morphological plasticity.
Authors also point out to the existence of potential hybrids that have both H.ovalis and H. major ITS sequences. On the other hand, authors show that the rbcL chloroplast gene failed to resolve species boundaries in the genus Halophila.
This study demonstrates again the complexity of the H. ovalis species boundaries in particular, and the morphological plasticity found in Halophila in general.

Overall, the manuscript is well written and following revision should be accepted.
I would ask that the authors refer to Les et al. 1997, 2006 and Procaccini et al. 1999 (Structural, morphological and genetic variability in Halophila stipulacea (Hydrocharitaceae) populations in the western Mediterranean) – all of which are super relevant.
The authors could have wrapped up the paper with something relating the results to future biodiversity conservation efforts in the region…
Lastly, the authors totally neglect to discuss the relevance of their methods in the new age of next-generation sequencing (NGS) which could be of particular relevance to DNA barcoding of genetically close species (e.g., DOI: 10.1111/1755-0998.12236, DOI: 10.1007/978-1-61779-609-8_2).
Additional comments: Abstract:
L 6-7: “…we incorporate genetic identification with morphological examination to reveal the identity of Halophila plants in Sri Lankan waters.”
Since this work was already done, use past simple throughout the text - incorporated
Sri Lankan waters – all around the island, or in one specific region ? why not “…in southern and northwestern Sri Lankan waters”

L 16-17: “…the low resolution of genetic markers, further comparative phylogenomic approach might be needed to solve species boundary issues in the genus Halophila”
Here the authors already admit one of the major flaws of their markers that in these days are considered very old, with very low resolution.

Introduction:
L 20: “habitat” – maybe “habitats”.
L 19-21 – this is a very simplified and limited description on the services and functions of seagrasses. Also, in my eyes there are much better and newer citations that could be used here in addition to Short et al. 2011..nothing was even mention about their estimated value…you don’t need to repeat things that we’ve read many times, but at least cite other papers….

L 21: “has been shown that seagrass habitat disappeared worldwide at a rate of 110 km2 per year…”
Change “disappeared” to “declined”…
L 23: Short et al. (2011) suggested 72 species of – suggested THAT….
If you use Short here, use 2-3 different ones above so as not to use the same one….more relevant here.
L 24-25: red list of International Union for the Conservation of Nature (IUCN) – change to “Red List of ….”. before you go on explain what is the Red List Category Criteria for at least the genus of Halophila, “while most species of Halophila are not in danger, and seem to be proliferating, there are some species within this genus that listed as very vulnerable….

L 27: change “or unclear characters” to “characteristics”
L 30-31: there is no Den Hartog et al. 2006 in the reference list. There is a “Den Hartog, C., & Kuo, J. (2007)” which means it is not 2006 and it is not “et al.”
Please get to know Les et al. 1997, 2006.
Are you sure you want to go with “sections” ? why not “clades” ?
Why not introduce a figure showing the different morphologies of the different family types ?

L 51: In addition, Udagedare et al. (2017) have…
How is this “in addition” ? this is a totally new paragraph/topic…

Methods:
L 64 – so add info explaining that these are shallow or even intertidal plants
Add GPS info for each location
L 116-118 : “…for morphological measurements consisting of lamina width, lamina length,
117 distance from intramarginal vein to lamina margin, cross-vein angle, and number of cross-veins…” – so why not add another figure/insert showing an example of where does one find these parts on the leaf?
This will allow others to follow…

Results:
L 138-139: “…which have complicated phyllotaxy and can clearly separated from species having simple phyllotaxy in section Halophila, except H. australis”.
This seems to fit the discussion more than the results section – either way, I would suggest adding a reference to support this conclusion.

L 156-158: Since rbcL lacked genetic variation among the different Halophila species, the unresolved phylogeny may indicate that rbcL cannot resolve species boundaries in Halophila.
This seems to fit the discussion more than the results section – either way, I would suggest adding a reference to support this conclusion – have other studies, in other organisms, pointed out to the low resolution of this genetic marker ?
L 168 - change “is” to “was”.

Discussion:
L 191-4: This morphological plasticity could lead to underestimating seagrass
biodiversity in our recent survey of the Gulf of Mannar, Puttalum Lagoon, and southern Sri
Lanka (Ranahewa et al., 2018; Ranatunga & Pethiyagoda, 2015; Gunasekara, 2017) by
mistakenly identifying H. major as H. ovalis.

This is not unique to seagrasses. Bring examples of other cases where ITS-2 had a stronger resolution than morphology – see zooxanthellae clade diversity for example in papers by Todd C. LaJeunesse. Same story

L 203-204: “…rbcL chloroplast gene, which failed to resolve species boundaries in the genus Halophila…” – bring other examples of lower resolution of this marker gene – you are not the first case…

References:

Please go over one by one – many mistakes, many different formats for example:

Den Hartog, C., & Kuo, J. (2007). Taxonomy and biogeography of seagrasses. In Seagrasses:
biology, ecology and conservation (pp. 1-23). Springer, Dordrecht
-	who are the editors? how many pages in book ?
-
L 290-292: Kuo, J., KANAMOTO, Z., IIZUMI, H., & MUKAI, H. (2006). Seagrasses of the genus Halophila Thouars (Hydrocharitaceae) from Japan. Acta Phytotaxonomica et Geobotanica, 57(2),129-154
Why the capitol names ??? Use same format for all papers.

336-337 Short, F. T., Moore, G. E., & Peyton, K. A. (2010). Halophila ovalis in the tropical Atlantic Ocean. Aquatic Botany, 93(3), 141-146.
All latin names s

Figures:
Fig 1 – missing arrow north, add coordinates on left and bottom sides
Fig 2 – so why not add arrows to the morphological you used for the measurements (at least on one species ) ?
Legend of Fig 3 seems to be double/confusing / something wrong there
Not clear which sequences were the out group…


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: CLINICOPATHOLOGICAL FINDINGS AND IMAGING FEATURES OF INTRADUCTAL PAPILLARY NEOPLASMS IN BILE DUCTS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The review has a clear aim and is well written. There are a few grammatical concerns that I think may need some attention.

Line 69: mentions the word rattan and I the word may not be in the appropriate context as it seems the intent was to use it as an adjective.

Line 80:  The word 'were' may need to be removed from the sentence "The first two groups were belonged to benign tumors."

Line 90: telephone visits may be an option instead of "telephone ways"

Lines 111-112: The following sentence needs to be reworded, as it does not seem to flow.
"In this study, patients showed typical massive jelly-like mucus by biliary drainage before surgery or intraoperative biliary tract."

Lines 116-118: Sentence needs to be reworded as it does not flow, needs attention to terms like 'electronic duodenum' and 'by below the' and 'full filled with full' and word slime should not start with capital S.

"The  electronic duodenum showed a common bile duct duodenal fistula by below the duodenum  above the duodenal papilla, about 1.0 cm in diameter, full filled with full of jelly-like Slime  (Figure 1C)."

Line 125: the word 'were' should be removed from the sentence
"with malignant tumors were died"

Line 128: perhaps consider revealed the following factors

"revealed that prognostic factors"

Line 156: consider adding the word 'with' so that it reads as often presents with instead of "IPMN-B often presents multiple lesions."

Lines 157-158:
the word accumulate does not flow with the sentence, not sure if they meant to indicate involvement of bile ducts in the hilar region
"When bile duct tumors inside and outside the liver  accumulate bile ducts in the hilar region often indicate an enhancement of tumor infiltration"

Line 169: consider changing word slowly to slow so that it reads as 'slow transition' instead of  "slowly transition from adenoma"
Experimental design: The research question is well defined and is an interesting concept as it is relatively rare. The study design was a prospective one and the length of time as well as number of patients seemed adequate for a statistically significant yield. Methods used to perform statistical analysis were outlined well.
Validity of the findings: The article was well written with a clear aim and straightforward study design and the authors gave a concise discussion and conclusion as well. The methods that they used to perform their analysis show that their results were significant and valid. Conclusion recaptured their aim and tied the overall study findings together.
Additional comments: This is a good study and interesting to read. There are only a few minor grammatical concerns I had.


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: CLINICOPATHOLOGICAL FINDINGS AND IMAGING FEATURES OF INTRADUCTAL PAPILLARY NEOPLASMS IN BILE DUCTS
Review round: 1
Reviewer: 2

Output:
Basic reporting: We read with interest this paper, which reports cases of intraductal papillary neoplasm of the bile ducts- It addresses a relevant topic, especially given the growing attention towards this form of rare biliary cancer.

The manuscript is quite well written and organized.
Figures and tables are comprehensive and clear. The introduction explains in a clear and coherent manner the background of this study. Despite this, English language could be improved, since some parts makes comprehension difficult. The manuscript would probably benefit from English language editing.
Experimental design: No comment
Validity of the findings: The results of the study are coherent with literature data. The discussion includes articles of historical importance in this setting. Therefore, we recommend to include results and references related the following papers:

- Rizzo A, Frega G, Ricci AD, Palloni A, Abbati F, De Lorenzo S, Deserti M, Tavolari S, Brandi G. Anti-EGFR Monoclonal Antibodies in Advanced Biliary Tract Cancer: A Systematic Review and Meta-analysis. In Vivo. 2020 March-April; 34(2): 479-488. doi: 10.21873/invivo.11798.

- Nakanuma Y, Kakuda Y, Uesaka K. Characterization of Intraductal Papillary Neoplasm of the Bile Duct with Respect to the Histopathologic Similarities to Pancreatic Intraductal Papillary Mucinous Neoplasm. Gut Liver. 2019 Nov 15;13(6):617-627. PMID: 30982236 . doi: 10.5009/gnl18476.
Additional comments: We believe this article is suitable for publication in the journal although minor revisions are needed.

The main strengths of this paper are that it addresses an interesting and timely question and provides a clear answer, with some limitations. Linguistic revision and the addition of other references are highly suggested


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: CLINICOPATHOLOGICAL FINDINGS AND IMAGING FEATURES OF INTRADUCTAL PAPILLARY NEOPLASMS IN BILE DUCTS
Review round: 1
Reviewer: 3

Output:
Basic reporting: There are numerous basic English language errors:

In the first title page, the author name "su lai liu" appears twice is not capitalised.

In the second title page, the author name "Yu jiang" is not capitalised.

In the abstract, the word "with" should be removed after "a worse prognosis"

In line 51, "significant expansion and obstruction in clinical practice" is poorly written. I suggest "significant expansion and obstruction of the bile ducts".

in line 66 "yellow staining of the skin and sclera" should simply be 'jaundice'.

line 69 "soft rattan" is not a medical term. What does this mean?

line 70 "electronic imaging" is not a medical term. What does this mean? Is this meant to be endoscopy?

line 90 "Recurrence was defined as the recurrence of jaundice and imaging...confirmed recurrence" is incorrectly phrased. It should state "Recurrence was defined as both the recurrence of jaundice and new lesions on imaging."

line 116: "electronic duodenum" what does this mean?

line 120: "4 patients" should be "Four patients"

line 125: the word "were" should be removed

line 134: "and intrapancreatic a subtype)"- what do these words mean? They do not seem to make sense.

line 139: "B- ultrasound"- what exactly does this mean?

line 148: "were complained" should be "complained of"

line 148: what does "head biliary calculi" mean?

line 148: there should be no full stop after the word "calculi"

Several issues with structure:

The methods section contains a large amount of descriptive information and percentages that belongs in the results, not the methods (e.g. from line 70 to 74)

Line 161 to 167 belongs in the results, not in the discussion. Also, R0, R1, and R2 resection are never defined.

Line 137 to line 142 is repeating what has already been said in the results, and should thus be deleted (except for the information about ultrasound findings, which should be moved to the relevant paragraph in the results).
Experimental design: The statistical methods section is inadequate: it needs to state that categorical data was also compared using frequencies expressed as percentages, and compared with chi-squared testing.

The criteria used to differentiate the three subgroups of histological findings should be described. Was there a validated international system that was used?

The survival analysis methodology is flawed in line 121 onward. It is not stated if the survival curve is for disease free survival or overall survival. I assume it is disease-free survival, as two elderly patients with benign tumors died and were excluded from the survival curve. However in line 124 the authors state they are analysing "overall average survival time", in which case the elderly patients should not have been excluded (assuing they were excluded for non-cancer related deaths). Assuming then that the authors are actually analysing disease free survival, it is scientific convention to not exclude such deaths from survival Kaplan Meier curves, but censor them and mark the censored events as ticks or dashes on the curve.
Validity of the findings: Many unclear aspects to the results:

"MST of benign IPMN-B patients was not reached" is a confusing way to express the better survival of benign patients in the abstract and the end of the discussion. Their MST should be mathematically estimated to provide comparison to the malignant group. Alternatively, proportions of survival at a set time-point (e.g. the median follow-up time, or the one-year survival rate or three-year survival rate) can be compared via chi squared testing between groups.

Furthermore, in line 124, it is stated that regarding the benign group, "the overall average survival time of this group was 62.1 ± 5.1 months". How did the authors calculate this number, yet could not provide the MST in the rest of the manuscript? What do they mean by "average" survival time? Similarly, why do the authors provide the one-year survival rate and three-year survival rate of the malignant group, but not the benign from in this paragraph?

in line 155 "(date not shown)" is confusing. Do the authors mean "data not shown"? And if so, why have the authors chosen not to include this useful data? If thy have constructed a ROC curve why not include it?
Additional comments: There are too many basic errors in scientific writing, and incomplete results and statistical analysis, to render this publishable.


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: CLINICOPATHOLOGICAL FINDINGS AND IMAGING FEATURES OF INTRADUCTAL PAPILLARY NEOPLASMS IN BILE DUCTS
Review round: 2
Reviewer: 1

Output:
Basic reporting: Nothing to add
Experimental design: Nothing to add
Validity of the findings: Nothing to add
Additional comments: Improved by recent changes we suggested.
We recommend acceptance of the article.
We congratulate the authors and we thank the Editor for the opportunity to revise this interesting 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: CLINICOPATHOLOGICAL FINDINGS AND IMAGING FEATURES OF INTRADUCTAL PAPILLARY NEOPLASMS IN BILE DUCTS
Review round: 2
Reviewer: 2

Output:
Basic reporting: All the previous English language errors I commented on have been corrected.

However, the new sentence "R0 is a complete resection under a microscope, R1 is a residual under a microscope, and R2 is a residual under the naked eye" is poorly written. It should be rephrased "R0 is complete microscopic resection, R1 is residual microscopic disease and R2 is residual macroscopic disease".
Experimental design: The survival analysis methodology in line 134 onward remains highly flawed and the authors have provided a totally unacceptable answer as to why they decided to remove 2 elderly patients who died with benign tumours from the overall survival analysis.

The authors have replied to me that, when these patients were included, "the one-year survival rate of the benign group was 84%, meanwhile, the one-year survival rate of the malignant group was 88%, which was not in line with the objective development of the disease".

However, with these patients excluded, the the one-year survival rate of the benign group was 94%, which I assume the authors feel is "in line" with the objective development of the disease.

But this is scientifically improper. Scientists and researchers cannot just deliberate exclude some of their collected data when they feel that these data do not fit a hypothesis! If the data do not fit a hypothesis, report this and explain it! (The obvious explanation here is that this study numbers are so small that 2 patients' deaths make a proportionally large impact.)
Validity of the findings: 1) The information about this study's patients Lines 167 to 174 should be in the result section, not the discussion section.

The actual numerical data about CEA and CA242 levels between the two groups should be included in the manuscript, as well as the ROC curve that the authors presented to me in their rebuttal. After all, the abstract and conclusion mention CEA and CA242 extensively: this focus on CEA and CA242 needs to be adequately reflected in the manuscript.

2) where it is stated that "median survival time of benign IPMN-B patients was not reached" the authors should include the average follow-up time of these patients.
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: CLINICOPATHOLOGICAL FINDINGS AND IMAGING FEATURES OF INTRADUCTAL PAPILLARY NEOPLASMS IN BILE DUCTS
Review round: 3
Reviewer: 1

Output:
Basic reporting: Nothing to add. The paper has been improved by recent changes.
Experimental design: Nothing to add.
Validity of the findings: Nothing to add.
Additional comments: Nothing to add. As I stated in the last revision I made, I believe the article should be accepted as is.


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: CLINICOPATHOLOGICAL FINDINGS AND IMAGING FEATURES OF INTRADUCTAL PAPILLARY NEOPLASMS IN BILE DUCTS
Review round: 3
Reviewer: 2

Output:
Basic reporting: Now acceptable.
Experimental design: Now acceptable.
Validity of the findings: Now acceptable.
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: GLOBAL RNA EDITOME LANDSCAPE DISCOVERS REDUCED RNA EDITING IN GLIOMA: LOSS OF EDITING OF GAMMA-AMINO BUTYRIC ACID RECEPTOR ALPHA SUBUNIT 3 (GABRA3) FAVORS GLIOMA MIGRATION AND INVASION
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript from Vikas Patil et al. reports the loss of editing of gamma-amino butyric acid receptor alpha subunit 3 (GABRA3) favors glioma migration and invasion using global RNA editome landscape. Specifically, in this study, the authors carried out a comprehensive analysis of RNA sequencing data of normal brain, lower grade Glioma (LGG) and glioblastoma (GBM) samples from publicly available datasets. They provided evidence to understand the distribution of RNA editing events across different regions of the genome, the comparison of editing events between normal, LGG and GBM samples, the regulation of ADAR enzymes in gliomas and the differential editing events between the different types of gliomas. Among them, a total of 5 out of the 63 pathway-related genes, including GRIA2, GABRA3, GRIK2, NOVA1 and GRIK1, were found to be carrying missense type editing from their analysis. Hence, they performed some experiments confirming these findings.

Overall, it would be good for the authors to revise the manuscript by a native speaker.

The bioinformatics analyses are quite thorough and complete, and overall this reads like a very high-quality RNA sequencing report. However, additional data should be performed in the experimental section for the validation of bioinformatics works. As the materials are available in this research, I would recommend performing the additional analysis of RNA and protein levels of GABRA3 gene in T98G cell line as well. Moreover, it would be good to see how GRIA2 function, as it is one of interesting finding in this research that has been previously reported to be essential for the functioning of the normal mammalian brain as the authors stated.
Experimental design: The transfection of DNA plasmids into cell lines for the overexpressing experiments should be obviously described. For reference and repeatable in other studies, the concentration of plasmid DNA and experimental condition should be stated.

The cultural condition for RNA extraction should be described in the materials and method section. Also, please add the information of the primers designed for Real-Time PCR using in this research.

Please add the description for protein experimental work in this research.

Figure 8: Please provide the figure for cell staining of vector transfection as well.

Please obviously state in the manuscript of how many replications has done throughout laboratory experiments.
Validity of the findings: The data and RNA sequencing seem sound, I would strongly suggest performing additional experiments though as mentioned above.
Additional comments: There are errors typing throughout the manuscript. Please carefully double check in revision version.


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: GLOBAL RNA EDITOME LANDSCAPE DISCOVERS REDUCED RNA EDITING IN GLIOMA: LOSS OF EDITING OF GAMMA-AMINO BUTYRIC ACID RECEPTOR ALPHA SUBUNIT 3 (GABRA3) FAVORS GLIOMA MIGRATION AND INVASION
Review round: 1
Reviewer: 2

Output:
Basic reporting: see below
Experimental design: see below
Validity of the findings: see below
Additional comments: The paper “Global RNA editome landscape discovers reduced RNA editing in glioma “reports interesting work on abnormal RNA editing in human glioma.
They used sequences from 5 independent datasets for their studies. This included two human glioma databases (TCGA and CGGA) taken from independent patient cohorts, each of which included a cohort of lower grade glioma tissue, and a second cohort to GBM tissue.   Finally, the results were validated with a fifth “cohort” of sequence from a cadre of glioma cell lines.   After filtering for likely SNPs, sequence from each of the five databases were independently compared to sequences from normal human brain to find differentially edited sequences.  The results were robust, in that 72-85% of all edited events were concordant between the TCGA and CGGA datasets.
The authors perform an extensive characterization of editing events, include introns/exons, alu-repeats vs non-alu repeats, etc.  Personally, I found this portion a little overwrought with 2 figures and 5 supplementary figures.  However, this is perhaps a matter of taste since there are not journal -specific restrictions of figure count
The authors then analyzed abnormally edited sequences from glioma tissue and found that reduced editing was much more frequent than increased editing. Furthermore, they showed that the vast majority of glioma-specific hypo-edited sequences were shared between low grade and high-grade gliomas.
critique about figure 4: My version of figure 4 seems to be a very low-resolution screen shot from the authors computer.  It is impossible to see any of the details when zooming in on the figure.  This should be replaced by a higher quality version.
They then perform a gene ontology analysis, which identified 14 pathways common to all 5 datasets. Further analysis found a total of 63 abnormally edited transcripts common to all datasets, but only five of these sequences included a missense change in the codon portion of the transcript.  Interestingly, all but one of these genes is involved in glutamate/GABAergic neurotransmission.
The work then finishes up with a more in-depth analysis of GABRA3 (GABA Receptor α3 subunit).  As previously reported (Ohlson et al., 2007; Rula et al., 2008) most α3 in adult brain is fully edited.
Critique -GABRA3 is expressed in neurons, but it is unclear to me how much is expressed in glia.  It would be good for the authors to test whether some of these differences could be confounded by a high proportion of neurons in the control tissues.  If this is impossible in this setting, they should at least discuss this as a potential pitfall, and how it could skew their results.
Critique- Figure 7 is confusing.  Figure 7A shows the medians as being below the point scatter.  If this is because of many sequences with 0% editing, this should be made more clear.    Similarly, regression line in 7D has a positive slope, but the line is clearly flat by eye.  It seems that this line includes all the transcripts with an editing ratio of “0”, which have a very wide scatter.  If so then the regression is based on widely scattered points at “0” which have a slightly lower median GABRA3 expression at the left end of the line, and the invariant GABRA3 expression on the right (editing ratio = 0.2 – 1.0).
They then try to test the significance of these findings by transfecting 2 GBM cell lines with low endogenous GABRA3 expression.  One set is transfected with an unedited version, the other is transfected with a version to recapitulate the edited version.
Critique- it seems the authors simply used the relevant genomic sequence to make their ‘unedited” version.  However, there is nothing to prevent editing once in the cell.  In figure 8E, they report overall GABRA3 mRNA expression, but not whether the mRNA from “unedited” cells expressed a significant amount of edited GABRA3 mRNA.  If I am mistaken, and the authors modified the sequence to be “uneditable” please state this in the paper.  If not please either measure % editing in those cells, or at least discuss the possible confound.
They also report that the edited version seems to express a higher amount of protein, but it seems to be based on a sample size of “2”. This is not adequate, especially given previous work showed that gabra3 editing actually reduces protein expression, which may contribute to the developmental down regulation of this protein in perinatal brain (Daniel et al., 2011)
Finally, the authors show that GABRA3 expression/editing may be an important determinant of GBM lethality, by measuring tumor migration and invasion rates. This could be the most interesting and compelling part of this entire project.  However, technical details about how the assay is performed and quantified are sorely lacking.  Indeed, it is unclear whether the data in Figure 8A-D were derived from a single round of transfections, or multiple experiments.  This section requires much more explanation of the technique and explanation of the results.

More general critiques
	The extremely poor English and ubiquitous typographical errors make the paper essentially unacceptable (in my opinion) at this point without extensive edits.  For example, the section titled “editing ratio calculation” is riddled with missing words and improper verb usage.  Moreover, multiple instances of typographical errors, such as “GBARa3” and “uneditided” are found throughout the manuscript, figures and tables.
It is unclear if the authors are familiar with the previous literature about GABA signaling in brain tumor.  Personally, I think the discussion would be improved by including mention some of previous work on the effects of gabra3 editing on protein expression/function in recombinant systems, as well as overall GABAergic signaling in gliomas.


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: GLOBAL RNA EDITOME LANDSCAPE DISCOVERS REDUCED RNA EDITING IN GLIOMA: LOSS OF EDITING OF GAMMA-AMINO BUTYRIC ACID RECEPTOR ALPHA SUBUNIT 3 (GABRA3) FAVORS GLIOMA MIGRATION AND INVASION
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript “Global RNA editome landscape discovers reduced RNA editing in glioma: Loss of editing of Gamma-Amino Butyric acid Receptor Alpha subunit 3 (GABRA3) favors glioma migration and invasion” quantifies and describes the RNA editome in various types of gliomas.

Overall, the manuscript conforms to the structure recommended by the journal viz. Introduction, Materials and Methods, Results, Discussion, Conclusions. Barring few typos, the overall English comprehension and grammar of the article is good and attains standards of a scientific article. However, the figures are poorly presented and need considerable work to make them of publication quality. I also found the method section needs to improve by adding more details and explanation for each procedure. Further, I would appreciate if the authors could provide scripts used for the analysis in order to be transparent as per journals motto.

 Below are specific points:

1.	Please maintain consistency in the text. For e.g. GABRA3 is written a few places and sometimes GBARA3 is mentioned in the text. Please correct wherever applicable.
2.	Mention the full-form at the first occurrence of an abbreviation. Example, GBM in the background section of the abstract
3.	Include a proper citation to the datasets used for the study. It would be very helpful for the readers following up on your study.
4.	Although the introduction section does a good job of providing the relevant background of the key phenomenon and methods, it lacks proper motivation behind the study. Also, it does not provide any rationale to study the RNA editing of GABRA3 gene. This section can be improved by including the knowledge gap underlying RNA editing and gliomas. Also, refer to specific studies looking at GABRA3 RNA editing. Such information is important and will help to understand the relevance of the present study. Further, the reference to the historical development of RNA editing technologies is unnecessary instead a proper motivation and original research question of the study can be highlighted.
1.	In the abstract, “median survival of grade IV glioma” should be rephrased to something like “median survival of patients with…”
2.	Please mention any accession number for the 45 Glioma cell lines downloaded from the CCLE-cgHub. Publicly available datasets are constantly been updated. For future studies, it would be important to refer back to the exact samples used in this study. Similarly, mention the GEO accession IDs for data downloaded from Chinese Genome Glioma Atlas.
3.	Please cite the tools used mentioned in the RNA editing pipeline, for example, BWA-aligner, Picard, GATK profiler etc.
4.	Introduce 1000g and ESP6500 datasets with proper reference.
5.	The figures in general needs improvement. For many figures, the font size is very small and the font type is inconsistent. Increase the font size and maintain the same font type for all figures.
Experimental design: 1.	Please provide a justification for using editing ratio = 0.2 (line 124) as a cutoff for classifying an RNA editing event. Presently it sounds very arbitrary. One way to decide the cutoff would be to use the distribution of all editing ratios.
2.	The approach explained in the subsection Differential RNA editing of the Materials and Methods section is not clear. It is unclear the utility of calculating “editing difference”. It is also unclear what was the input to the Mann-Whitney Wilcoxon test? A standard way to refer to this test is to either use Wilcoxon test or Mann-Whitney U-test.
3.	The criteria for deciding differential RNA editing - “absolute difference between average RNA editing of tumor samples versus control brain samples is greater than 0.2” is arbitrary. Please provide justification.
4.	Another way of deciding differential RNA editing is by using Ficher’s exact test as used in the paper - https://www.pnas.org/content/116/6/2318#sec-10
5.	Line 204, There is no C-to-U data shown in figure 2D. If you meant C-to-G edits, please correct accordingly in the main text.
6.	Line 214, Please justify why 10 number of reads is defined as high confidence. A better way would be to plot a distribution of reads mapped to the editing events and choose 90th or 95th percentile as the cutoff.
7.	Please explain the procedure used to calculate the p-value for the figure 2E and similar supplementary figures.
8.	Also, for figure 2E and similar supplementary figures, it would be important to see the probability density function to see the distribution of editing ratio values.
9.	In figure 4A and 4B, the x and y-axis are not labeled. Please replace with a high-quality version of these two figures. Include the color legend on the figure apart from the text. The color bar legend is also unreadable. I couldn’t judge the results mentioned in this graph as there is no information.
10.	In figure 6B, the x-axis is missing, therefore, the results cannot be interpreted.
11.	The term “pathway-related” is ambiguous. Replace with a suitable alternative.
12.	In figure 8F, ¬¬¬quantify the amount of protein on the gel. Even though there is a mention of 39% increase there is no quantification provided.
13.	Line 296, typo “tis”
14.	Figure 7D, there is a discrepancy between the R-squared value shown on the graph and mentioned in the main text (line 283). Please replace with the correct value. Also, it should be R-square and not just “R” (line 283).
15.	For the regression shown in figure 7D, explain which method was applied to calculate the p-value.
16.	In figure 7A and 7B, mention legend for the horizontal red bar. It does not look like the median value.
17.	For figure 7A, B, C, what was the procedure used to calculate the p-value.
18.	Line 296, mention citation for PolyPhen-2, SIFT, PROVEN and MutationAssesor.
19.	Please mention what are the potential candidates that were highly edited in the normal brain compared to gliomas. It would be important for future studies.
20.	The discussion section lacks to highlight the additional knowledge gained by performing this study. For example, it is a well-known fact that there is a high frequency of RNA edits in Alu repeats and the same is found in the study. Try to highlight the relevance of this observation in the context of gliomas.
21.   In figure 6E, it is unclear how were genes with missense editing were obtained. Please mention the source or method used to obtain them.
Validity of the findings: 1.	The data set used in the study should be better described (see Basic Reporting section)
2.	Mention statistical test for each figure
3.	Link to the data set should be included. In which repository sequencing datasets are made publicly available (e.g. SRA)?
4.	For further comments refer section Experimental design
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: GLOBAL RNA EDITOME LANDSCAPE DISCOVERS REDUCED RNA EDITING IN GLIOMA: LOSS OF EDITING OF GAMMA-AMINO BUTYRIC ACID RECEPTOR ALPHA SUBUNIT 3 (GABRA3) FAVORS GLIOMA MIGRATION AND INVASION
Review round: 2
Reviewer: 1

Output:
Basic reporting: In this revised manuscript, the authors have addressed all my concerns raised in the previous review report, therefore this version is ready for publication.
Experimental design: All good
Validity of the findings: All good
Additional comments: No additional comments


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

Input:
Title: GLOBAL RNA EDITOME LANDSCAPE DISCOVERS REDUCED RNA EDITING IN GLIOMA: LOSS OF EDITING OF GAMMA-AMINO BUTYRIC ACID RECEPTOR ALPHA SUBUNIT 3 (GABRA3) FAVORS GLIOMA MIGRATION AND INVASION
Review round: 2
Reviewer: 2

Output:
Basic reporting: see attached critique in PDF format
Experimental design: see attached critique in PDF format
Validity of the findings: see attached critique in PDF format
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: COMPUTATIONAL PERSPECTIVES REVEALED PROSPECTIVE VACCINE CANDIDATES FROM FIVE STRUCTURAL PROTEINS OF NOVEL SARS CORONA VIRUS 2019 (SARS-COV-2)
Review round: 1
Reviewer: 1

Output:
Basic reporting: The main idea of the work is to propose epitopes from the SARS-CoV-2 which can act as vaccines, using viral proteins, using computational methods.
The English language is well written and clear in all the manuscript. The authors addressed the main problem and ideas in the introduction section, as well as used appropriate references. On the other hand, I have some questioning described below:

•	Lines 49 – 51: Please update WHO numbers.
•	Lines: 79 – 80: Do you have any information about what is this variation? Please include some paper talking about it.
•	Lines 87 – 91: Could you better describe ACE2/RBD importance in SARS infection here? I have bee reading many papers targeting its importance for viral pathogenicity, as well as drug development. Dong et al. (2020) just hypothesize about ACE2 role in viral infection, and I think you need more examples to confirm this sentence. Please complete your sentence with other references.

Figures 1, 5 and tables are well-representatives in most of them, except in the cases below:

-Figures 2 and 3: should be completed with 2D interaction maps and re-docking experiments.
-Figure 4: due to bad resolution I can't read coverage results.

Computational predictions of possible epitope activity were validated by docking studies with crystallographic structures from PDB. In this case, I found some flaws that are described in the experimental design.

I will recommend the paper publication only with major revisions, that will be described in the next sections.
Experimental design: Comments about experimental design are described below:

•	Lines 126 - 128: VaxiJen server uses physicochemical proprieties in its analysis, instead of peptide alignment. Did you consider using another tool with alignment mode? Why VaxiJen is a better choice? Please, include an advantage to use it. Additionally, indicate here in methods the threshold that you considered.
•	Lines 143 – 144: Please include the default parameters you used.
•	Lines 162 – 163: Please include a reference for this statement.
•	Lines 168 – 173: Too simple description of the determination of population coverage. Please, include more details about this part of the methods.
•	Lines 185 -188: Too simple method description.
•	Lines 189 – 195: What criteria did you use to select the best epitopes for modeling? What comes first in those characteristics that you considered (antigenicity, conservancy, etc.). Please, be clearer on what was more important to select your best epitopes.
•	Lines 196 – 202: Is the Autodock Vina the best option for peptide docking? I think there is a problem considering this program instead of other peptide docking programs (Cluspro, Haddock, DockThor, etc.) and/or Autodock 4 as well, giving better evaluation results. In the case of Autodock Vina, its algorithm is more restricted for huge molecule evaluation, as well as its results, could not reflect several characteristics in peptide interaction, such as protonation states of the amino acids, charges and solvent accessibility for example. In this case, this could be a problem to represent the better pose of the molecule inside the protein of interest. I suggest you choose another program that uses a peptide docking approach, in order to validate this part of your work.
•	Line 202: What Pymol version? Cite Pymol.
•	Line 205: in Autodock Vina the program considers Affinity energy, which is a little different from binding energy. Change the term.
•	Line 207: Setting up the grid box as default is not the best choice. Depending on the type of binding site that you have, it will require smaller or bigger boxes.
•	Lines: 227 – 230: Wrong terminology. I think the authors wanted to say physicochemical instead.
I would like to do some considerations/questioning about the methods section:

1)	The authors used complete or partial protein sequences? Please include this information in the methods section.
2)	As you are proposing a vaccine using protein sequences for computational predictions, I strongly recommend that you include variability analysis using sequences from different viral strains. From what region of the world the sequences that you used came from? They are consensus sequences from different countries or from just one country?
3)	I recommend you include a sequence alignment for each protein group, or you can align just the region that you used as epitopes for each protein type
Validity of the findings: This work presented important findings of new peptide sequences that can be used for vaccine development. On the other hand, there are computational validations and other explanations that must be included. The study didn't say how many sequences for each protein type they used, and from where these sequences come from so is very hard to accept that a global vaccine could be developed using these findings. Furthermore, the results and consequently discussion sections lack some information about best epitope selection, as well as docking results and validation. I suggest some changes below.

Lines 256 – 257: what criteria the authors used to put a 250 nM IC50 cutoff? Please, explain this in methods or in results/discussion sections.
Line 260: What is this score of 65%? I didn’t find this in methods. Include for what reason you used this.
Lines 329 – 354: Docking results section needs to be re-written with more details. There is no validation for docking results, and there is no 2D interaction maps peptide-protein. Please include the 2D interaction maps for each docking calculation. I recommend including an epitope re-docking for validation: 3C9N, 5IEK and 5VGD crystals are protein-peptide complexes. I should use some of them for re-docking and validation of your docking methods.
Lines 434 – 435: in Physicochemical properties analysis what you consider as stable structures?
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: COMPUTATIONAL PERSPECTIVES REVEALED PROSPECTIVE VACCINE CANDIDATES FROM FIVE STRUCTURAL PROTEINS OF NOVEL SARS CORONA VIRUS 2019 (SARS-COV-2)
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript has a clear and professional use of English.
Although the COVID-19 literature increases by the day, and there are many preprints, the references used are adequate for the methodology applied.
The paper has good structure, the protein models are properly drawn and colored, although figure 4 is impossible to read. Maybe they can be included at full size as supplemental material and just make a reference to the important alleles in results and discussion.
In figure 5, it would be nice if some sequence numbering is included. Also, it is not clear if the images correspond to different proteins or are views of the same protein. Please explain in more detail.

In line 225. and 227, it should be “physicochemical” instead of “Physiological”, probably a typo.
In the same section please also include the prediction of stability if expressed on E. coli

Maybe design the figure using a “surface glycoprotein” label, and then include figures a-d. Then a label “ORF3a protein” and the corresponding g-h images and “membrane glycoprotein”, and a fourth label “nucleocapsid phosphoprotein” for i-j images.
Experimental design: The methods are described in detail and the parameters used in each algorithm are included.
I could not find details of the program used to display the structures, please include the reference and the PDB access for each structure (if taken from there) or if these are your predictions, how they were modeled.
Validity of the findings: Novel work that predicts potential targets towards a COVID-19 vaccine. The pipeline is clear and it helps the non-specialist to understand the procedures. Table 2, although detailed, is great because it resumes all the scores that point out towards the target peptides that are worth further investigation.

I consider important, since physicochemical properties are reported, that the author proposes the challenges of expressing these peptides as vaccines, Which ones would be more suitable for bacterial expression, pros, and contras, and also if there are issues with glycosylation. What are the more adequate systems for expressing and purifying the peptides?

Labeling in figure 2 could be done with 1-letter amino acid symbols to reduce the crowding of the stick figures. Letters in figure 2c are too small, in 2F hard to read, in 2i too big. In all of them, a one-letter amino acid code would be better.

Finally, I suggest that the author takes a final review of the current literature since there are new reports by the day, of the current status of vaccine development.
Additional comments: The paper is novel and understandable, please keep it updated so it stays as an important resource in the avalanche of COVID-19 publications.


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: COMPUTATIONAL PERSPECTIVES REVEALED PROSPECTIVE VACCINE CANDIDATES FROM FIVE STRUCTURAL PROTEINS OF NOVEL SARS CORONA VIRUS 2019 (SARS-COV-2)
Review round: 1
Reviewer: 3

Output:
Basic reporting: Comments for the author on English.
Figure 4 - text in figure is far too small, impossible to read, it might need a key.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: The introduction is highly informative and the literature is well referenced and appropriate. It provides good background and context to the reporting of the study. There are numerous minor grammatical errors, in addition to those I have identified in my comments, so I would recommend careful and thorough proofreading. The research question is well defined and very timely. The knowledge gap is identified clearly, as is the approach to the intended filling of the gap.

The study addresses the important question of identifying effective epitopes for SARS-CoV-2 by the  coherent application of a number of well established bioinformatics methods that predict antigenicity, immune protein epitopes, immunogenicity, allergenicity and toxicity, in this case applied to viral protein sequences, which when taken together allow cross-referencing of their results to address the specific question concerning epitope-based vaccine design. The methods are appropriate to the problem and the authors should be commended on a thorough and well-directed study. However, some clarifications of the core analytical approach and extensive grammatical corrections are required.

Line by line comments:

"anticipation" should be replaced by "prediction". This word is frequently misused and should be corrected throughout.
Line 146 - missing "For" the recognition...
Line 147 - was - were. This is also a frequent error
Line 150 - 250? The units need to be included, nM?
Line 151 - "Lower IC50 value signifies higher binding" - this should be qualified or prefaced by the term "In general, for similar ligands," as the maximal bound levels of different ligands can vary considerably even at the same site, meaning that comparison of IC50 values for different ligands in bringing about 50% inhibition only relates to the required concentration of that ligand relative to that for the highest attainable inhibition for that ligand. It doesn't allow for comparison of the level of binding of different ligands, or actual concentration of ligand at a given site.
Line 151 - ingenious - not clear what is meant here.
Line 155 - combined score - how is this derived?
Line 156 - performed - made
Line 183 - created - developed
Line 186 - was - were
Line 187 - The - An
Line 190 - was - were
Line 194 - implied - implicated, or better still, taken forward
Line 207 - Eventually - remove this
Line 217 - generated - developed
Line 222 - demonstrate - provides
Line 226 - upper case F in For, should be lower case
Line 240 - the - not needed
Line 249 - the (antigenicity) - not needed
Line 251 - Surprisingly? Why is this a surprise?
Line 251 - Similarly? - the values are very different
Line 252 - immunogenicity value - immunogenicity values
Line 253/254 - Table S2 details 82 epitopes rather than 83. The combined scores are shown in the table. However, it is not clear how these scores are derived. Further, the selection criteria for the 82 epitopes for further study is not described, this requires clarification, particularly if VaxiJen indicated only 49 epitopes to be antigenic. This contradicts the assertion in line 155/156 - "the final selection for further study was performed after the (prediction) of antigenicity by VaxiJen v2.0 server....", and suggests that a proportion of the negative results obtained using VaxiJen, which represent an important observation, are being overlooked.
Line 257 - was - were; IC50 values
Line 267 - allergic - allergenic; allergen - allergenic
Line 268 - was - were
Line 289 - fetched - gathered
Line 291 - should read - although E protein epitope “LLFLAFVVF” exhibited binding....
Line 298 - non-allergen - non-allergenic
Line 299 - candidates
Line 308 - two other epitopes...
Line 318-320 - ....ORF3a was found to be present.... (the two sentences could be condensed into one)
Line 321 - sentence is not clear at all, should be rephrased
Line 324 - resulted - result
Line 330 - considered to be potential...
Line 347 - As a representative of what?
Line 356 - amid - across
Line 369 - was - were
Line 384 - candidates
Line 385 - adjective missing before "antigenicity"
Line 387 - the spike protein
Line 396 - values
Line 400 - candidates
Line 415 - remove "only"
Line 430 - exhibited - displayed
Line 444 - design a peptide-based
Line 460 - Cells - cells (lower case)
Line 468 - thresholds
Line 470/471 - further ensure the acceptance - further supports the feasibility
Line 472 - incomplete sentence - it looks like it should be part of the previous sentence?
Line 478 - considered as the most important epitopes, no need for "rest of the"
Line 482 - criterion
Line 485-488 - reference should be made to Table-S4
Line 488 - The SARS-CoV-2 outbreak has resulted...
Line 505 - the selected
Line 506 - simplified?
Line 507 - no need for "investigation"
Line 514 - most up-to-date
Line 515 - exhibits notably good performance
Line 519-521 - incomplete sentence
Line 525 - The majority
Line 527 - the ElliPro - "the" not needed
Line 532 - An earlier study
Line 535-536 - we propose the identified potential vaccine candidates should be pursued in clinical trials.
Line 539 - at the earliest - early
Line 542 - vaccines - vaccine
Line 543 - directives - directions / avenues; no need for "the"

Figure 1 legend - proteins
Throughout Figure 2 and Figure 3 legends - odd use of "as"
Figure 4 legend - proteins
Figure 4 - text in figure is far too small, impossible to read, it might need a key.

Otherwise, the figures are very good, relevant and the legends are informative.

The tables are fine. Table 4 should perhaps be moved to the supplementary.

There are no legends provided for the supplementary tables.

In all, this is a good manuscript, in my view very worthy of publication, which successfully applies and integrates a number of bioinformatics tools and resources in a coherent, meaningful way to address the challenge of the day. The basis of some scores and selection or filtering processes requires clarification and the manuscript needs busy checking of the English to ensure the widest possible understanding of the findings.


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: COMPUTATIONAL PERSPECTIVES REVEALED PROSPECTIVE VACCINE CANDIDATES FROM FIVE STRUCTURAL PROTEINS OF NOVEL SARS CORONA VIRUS 2019 (SARS-COV-2)
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The authors adjusted the manuscript as recommended. I would like to thank you for agreeing with my suggestions for improving the work. The manuscript is now suitable for publication.