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: STATIC AND DYNAMIC ALTERATIONS IN THE AMPLITUDE OF LOW-FREQUENCY FLUCTUATION IN PATIENTS WITH AMYOTROPHIC LATERAL SCLEROSIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The authors investigated ALFF and d-ALFF alterations in ALS patients, and found some interesting results. The ALS patients can be more accurately classified from HC by combining ALFF and d-ALFF features compared with any single feature.
1.	There are many grammar errors and confusing sentences in the whole manuscript. E.g. what does the subtitles (comparison of ALFF, comparison of d-ALFF, comparison of ALFF and d-ALFF, comparison of brain regions) mean? What does the “unusual areas” mean? They should check the whole manuscript, revise those sentences carefully and make it be understood more easily. I suggest the authors ask a native English speaker or company with English editing for help.
2.	According to the title, it seems that the d-ALFF is more appropriate than ALFF in understanding mechanism of ALS. But the article told us that combination of ALFF and d-ALFF is better. So the authors should discuss what the advantage of d-ALFF is for understanding mechanism of ALS, or maybe the title “Characterizing stationary and dynamic amplitude of low-frequency fluctuation in ALS” is more appropriate.
3.	In section of Data acquisition, the voxel size is 3x3x4, matrix size is 64x64, and the data was scanned axially. So the thickness would be 4 mm instead of 3 mm. The authors should check the information more carefully.
4.	What is the resampled voxel size in the normalized images?
5.	In Table 1. There should be p-value for the comparison of gender. Also it’s better to include GM information in Table 1.
6.	Since there was no difference in gender and age, why the authors regressed out them in statistical analysis
7.	There are some errors in the sentence “As shown in Table 2 and Figure 1, the ALFF in ALS patients increased in right inferior temporal gyrus, right medial superior frontal gyrus and right medial superior frontal gyrus”
8.	In table 4, the authors included brain areas to movement, visual, memory, emotion or cognition network. How did they do the work? Is there any atlas used?
9.	 “We have investigated whether d-ALFF is able to discriminate ALS patients from healthy control” and “In addition, the classification performance with ALFF, d-ALFF and combined ALFF and d ALFF were compared”. The two sentences were overlapped and should be revised.
10.	In discussion, the word “hyperactivity” is not correct because this is a resting-state study instead of task study.
11.	Discussing the result of significant correlation between ALFF and ALSFRS-R score, disease progression rate


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: STATIC AND DYNAMIC ALTERATIONS IN THE AMPLITUDE OF LOW-FREQUENCY FLUCTUATION IN PATIENTS WITH AMYOTROPHIC LATERAL SCLEROSIS
Review round: 1
Reviewer: 2

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


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

Input:
Title: STATIC AND DYNAMIC ALTERATIONS IN THE AMPLITUDE OF LOW-FREQUENCY FLUCTUATION IN PATIENTS WITH AMYOTROPHIC LATERAL SCLEROSIS
Review round: 1
Reviewer: 3

Output:
Basic reporting: 1.     The English language should be improved to ensure that an international audience can clearly understand your text. Some examples where the language could be improved include lines 44-46 – ALS is a neurodegenerative disorder manifested as cognitive and motor impairments, typically resulting in death from…...
2.	The introduction is weak. A lot of details have been given as for the application of d-ALFF while no efforts have been spent to provide a clear hypothesis and rationale of the study. A paragraph reporting study hypothesis should be included.
Experimental design: 3.	Both DPARSF and DPABI appeared in the Methods, have you used both of them or only DPABI?
4.	AlphaSim correction is not suggested. After AlphaSim was questioned by a previous study, it’s correction of multiple comparisons updated with a statistical threshold of P < .050 (an uncorrected height threshold of P < .001, in which the z statistical image was used to estimate smoothness) based on the modified version of DPABI.
5.	Why EPI template and not DARTEL?
6.     In the correlation analysis, the correction of multiple comparisons is needed.
7.	In Classification analysis, how many features were used and what are they? Please clarify. No rigorous validation (e.g., cross validation of K-fold cross validation) is employed. And strictly, there must be one-fold data that is never used in any training process. Is it true in the present work? More details are deeded to address those questions.
Validity of the findings: no comment
Additional comments: 9.	The discussion section is very weak and lacks the explanation/justification and or discussion of the obtained results.


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: STATIC AND DYNAMIC ALTERATIONS IN THE AMPLITUDE OF LOW-FREQUENCY FLUCTUATION IN PATIENTS WITH AMYOTROPHIC LATERAL SCLEROSIS
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: I am satisfied with the revision , and suggest acceptance of the paper


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

Input:
Title: STATIC AND DYNAMIC ALTERATIONS IN THE AMPLITUDE OF LOW-FREQUENCY FLUCTUATION IN PATIENTS WITH AMYOTROPHIC LATERAL SCLEROSIS
Review round: 2
Reviewer: 2

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: All my concerns have been addressed. I am satisfied with the revision and this manuscript can be accepted.


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: STATIC AND DYNAMIC ALTERATIONS IN THE AMPLITUDE OF LOW-FREQUENCY FLUCTUATION IN PATIENTS WITH AMYOTROPHIC LATERAL SCLEROSIS
Review round: 2
Reviewer: 3

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: This article is innovative and I hope the author will have better papers to publish in the future.


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: IN VITRO ANTI-DIABETIC EFFECTS AND PHYTOCHEMICAL PROFILING OF NOVEL VARIETIES OF CINNAMOMUM ZEYLANICUM (L.) EXTRACTS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Dear editor-in-chief,
Thank you for sending the paper for peer review.
Paper  referred to the diabetes and phytochemical profiling of novel varieties of Cinnamomum zeylanicum (L.) extracts. They conducted chemical analysis. They found  the potential antidiabetic properties of the Sri Wijaya Cinnamon accessions, which exhibited significant α-amylase and α-glucosidase inhibitory activities.

Paper, is suitable for publication. I checked all pages. However, a plagiarism check is also necessary. They talked on the anti-diabetic impact of cinnamon, however,  this medicinal plant had anti-hypertensive effect too. There, I would like the authors to read and cite and use in the text, the following article;
Mahmoodnia L, Aghadavod E, Rafieian-Kopaei M. Ameliorative impact of cinnamon against high blood pressure; an updated review. J Renal Inj Prev. 2017;6(3):171-176.
Experimental design: N/A
Validity of the findings: Good
Additional comments: No additional comments


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

Input:
Title: IN VITRO ANTI-DIABETIC EFFECTS AND PHYTOCHEMICAL PROFILING OF NOVEL VARIETIES OF CINNAMOMUM ZEYLANICUM (L.) EXTRACTS
Review round: 1
Reviewer: 2

Output:
Basic reporting: See general comments below
Experimental design: See general comments below
Validity of the findings: See general comments below
Additional comments: Review of manuscript title: In vitro anti-diabetic effects and phytochemical profiling of novel varieties of Cinnamomum zeylanicum (L.) extracts by Wariyapperuma Appuhamillage Niroshani M Wariyapperuma et al.

General comment
The study attempts to identify the potential antidiabetic properties of the Sri Wijaya Cinnamon varieties with significant α-amylase and α-glucosidase inhibitory activities. Three different varieties of cinnamon submitted to distint extraction methods were prepared and tested for their enzyme inhibitory activity. The results showed that extracts prepared using pressured water and as a decoctions were more potent in inhibiting α-amylase and α-glucosidase activity, and had the potential to extract higher content of proanthocyanidin than other methods of extraction. The hypothesis is quite attractive, experimental design is sound and methods seem adequate, in particular the exhaustive chemical characterization of the extract; results are clearly presented and discussed and look promissing for a future utilization in humans. Search for plant foods and their specific components which might be relevant in a nutritionnal prevention and coadjuvant treatment of type 2 diabetes is a high demanded topic in current nutritional biochemistry and studies dealing with this subject should be welcome. Some specific comments are detailed below:

Specific comments
1)	Lines 110-130, line 156 and lines 249-250; it should be mL, with capital L, as in the rest of the manuscript.

2)	Figure 1 is missing statistical symbols; although in line 182 is reported that there were significant differences among extract yields and extraction methods, there is no indication of such statistical differences in the figure or figure legend.

3)	Line 252; it should say Metformin.

4)	Line 256; proanthocyanidins are considered as a subgroup or subfamily of polyphenols, perhaps it would be more correct to state that polyphenols, in particular proanthocyanidin polimers…


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: IN VITRO ANTI-DIABETIC EFFECTS AND PHYTOCHEMICAL PROFILING OF NOVEL VARIETIES OF CINNAMOMUM ZEYLANICUM (L.) EXTRACTS
Review round: 1
Reviewer: 3

Output:
Basic reporting: The article is clearly written and structured in the corresponding sections.
Only minor grammar mistakes have been marked in the text.
Please, keep consistency when alfa-amylase and alfa-glucosidase are written throughout the text: use always the same criteria: capital letters for the first "a" and "g", or not.
Literature references are correct and updated.
Figures and tables are correct and justified.
Results are considered relevant but they don't support the hypotheses or the conclusions of the work: authors stated that benzoic acid, cinnamyl alcohol, banzyl alcohol and the derivative from dimethoxyphenol, which are identified as the major compounds in the extracts, are believed to be responsible of the inhibitory activity. Nonetheless, these compounds are part of the essential oil composition and authors do no discuss any research work proving the cited  activity in any research model. What about the polyphenols composition? Only the total amount has been calculated, not the kind of specific polyphenols: flavonoids, phenolic acids… which are widely known as strong active compounds
Experimental design: The research is well designed and fulfills the aims and scope of the journal.
The research question is well defined and the results are relevant, but do not support the conclusions held by the authors: phytochemical profile of the essential oil by GC is really interesting but does not prove any relationship with the in vitro activity.
What about the polyphenols?

Methods are correctly described
Validity of the findings: The findings are correct and relevant for the field of research, but they are limited to support the conclusions of the authors.
The performed in vitro studies are not enough to support the treatment of diabetes mellitus, as stated on lines 320-321. Previous in depth studies in cell cultures and experimental animals should be conducted to confirm the observed tendency and to identify the compounds which are responsible.
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: IN VITRO ANTI-DIABETIC EFFECTS AND PHYTOCHEMICAL PROFILING OF NOVEL VARIETIES OF CINNAMOMUM ZEYLANICUM (L.) EXTRACTS
Review round: 1
Reviewer: 4

Output:
Basic reporting: Though Cinnamomum has been extensively researched, the topic was very interesting and curiosity pulling to know in depth. Here some of my points of view:
- The English  writing is clear and good enough to understand the content.
- The obtaining plants accession needs more botanical or genetical background justification. You must be clearly classified what do you mean by Cinnamon accession, is it variety or subculture category. The business source information https://www.lankabusinessonline.com/sri-lanka-develops-75 new-Cinnamon-varieties which could not be opened yet, should not be your main information according to your material source.
- so did with the reason why have to take so many varieties on the extraction.
Experimental design: - The research question is not well defined. There was steam distillation followed by oleoresin n-hexane extraction which not equally compare to many kinds of water extraction method.
- critical important variables were not be controlled (the material weight for extraction, the temperature and the quantity of solvent used)… SD (40 g) while others 10 g – line 93-97
- no standard/ guidance on the extraction method (SE line 98, DWE, IWE line 102-103)
- The extractive method produced non-volatile compounds most. The GC-MS is not suitable for analysis components, except for SD.
Validity of the findings: -	The SE and PWE of SW & SG yields seem wide SD, so did with SE on α-glucosidase and seem need be refined further.
Additional comments: To improve your finding, please describe the method precisely, including the limitation of the research
here some references related:
-Marongiu B, Piras A, Porcedd S, Tuveri E, Sanjust E, Meli M, Sollai F, Zucca P, and Rescigno A. Supercritical CO2 extract of Cinnamomum zeylanicum: chemical characterization and antityrosinase activity. J. Agric. Food Chem. 2007 2007, 55, 10022–10027
-Ervina M, Lie HS, Diva J, Caroline, Tewfik S, Tewfik I. Optimization of water extract of Cinnamomum burmannii bark to ascertain its in vitro antidiabetic and antioxidant activities. Biocatalysis and Agricultural Biotechnology 19 (2019) 101152
-Kasim NN, Ismail SNAS, Masdar ND, Ab Hamid F, Nawawi WI. Extraction and Potential of Cinnamon Essential Oil towards Repellency and Insecticidal Activity. International Journal of Scientific and Research Publications, 2014 (4.7)
Wardatun S, Rustiani E, Alfiani N, Rissani D. Study Effect Type of Extraction Method And Type of Solvent To Cinnamaldehyde and Trans-Cinnamic Acid Dry Extract Cinnamon (Cinnamomum burmanii [Nees & T, Nees]Blume). J Young Pharm, 2017;9(1)Suppl: s49-s51


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: NON-SARS-COV-2 GENOME SEQUENCES IDENTIFIED IN CLINICAL SAMPLES FROM COVID-19 INFECTED PATIENTS: EVIDENCE FOR CO-INFECTIONS
Review round: 1
Reviewer: 1

Output:
Basic reporting: In this study, the author examined infection of COVID-19 and other viruses in publicly available 68 SRA data using Fastv program, which is published in bioRxiv:2020.2005.2012.092163. 10.1101/2020.05.12.092163. Author identified infection of COVID-19 in 65 SRA data. In addition, several viruses were identified.
Experimental design: In fact, this manuscript should be rejected due to several reasons. The first is that there is no novelty of this study. Similar study is already done by Chen et al. who developed Fastv program. The difference is just the number of SRA data for virus identification. Although the author increased the number of SRA data for virus identification, I cannot find any valuable results. Title should be also changed. Most SRA data were derived from transcriptome data not metagenome data. Maybe co-infection might be common in COVID-19 infected samples. I cannot find any results which support the identification of other viruses in the examined SRA data.
Validity of the findings: There is no result associated with validation of the findings in this study.
Additional comments: The manuscript is really poorly written. There are lots of errors in English. Many sentences and paragraphs were poorly written. The materials and methods were not sufficiently written. I only see the detailed information of Macbook pro which was used for data analysis. The results were really badly written. Without proper results, how the author can write the discussion part?

There were lots of errors. These are some examples.
In abstract
L32: sixty-eight public next-generation sequencing libraries -> sixty-eight public next-generation sequencing data
L34: Screen the raw reads for viral genome sequences, including SARS-COV-2, using an alignment-free method based on K-mer mapping and extension. -> The sentence is not complete.

L65 (Viruses 2020) -> The reference is not correct.
L66 98 percent -> 98%
L104 A reference for Fastv is required.
L109 libraries -> data

Materials and methods
L114-118 The hardware for data analysis may be not necessary.
L120-126 Authors selected SRA data from six projects. Which thresholds were used to select SRA data?
L126 Table. 1 -> Table 1
L 128-138 It is necessary how authors conducted data analysis using Fastv program in detail.

L142 We conducted -> I conducted (there is only one author in this manuscript)
The Table 1 is not informative. Author should describe sample information for individual SRA data.
L149-150 No information associated with PRJNA631042 is available.
Results and discussion are terribly written. Author should write results in detail.
Tables and figures were poorly prepared.


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: NON-SARS-COV-2 GENOME SEQUENCES IDENTIFIED IN CLINICAL SAMPLES FROM COVID-19 INFECTED PATIENTS: EVIDENCE FOR CO-INFECTIONS
Review round: 1
Reviewer: 2

Output:
Basic reporting: This paper reported identification of possible viral co-infections in samples from SARS-CoV-2 infected patients. This article is well written and easy to follow. However, Figures and Tables need a major improvement. In general, the results section was quite vague where random outputs from Fastv software are shown.
Experimental design: The author simply used Fastv to analyse 68 publicly available SARS-CoV-2 samples from SRA database. Fastv outputs for all analysed samples are not provided. The author should make the Fastv output for all samples publicly available.
Validity of the findings: Whilst the authors discovered possible co-infections in a number of samples, the significance of these identification as well as their relevance for the accurate estimates of infectious disease burden was not explored. The authors should either attempt to describe how these co-infections could be driving disease progression or outcome, or outline that the consequence of these co-infections are still unclear.
Additional comments: Table 1 should be placed in supplementary. Can also add more information about each sample such as read length, total sequences, total bases, etc.

Figure 1's resolution is too low. Impossible to read.

Remove "Figure.a:" from Figure 1 text.

Which sample does Figure 1 represent?.

Line 158: Figure.2, a: should be Figure 1

Line 161: Figure.2, b: there is no description of this Figure in the manuscript.

Line 166-168: The author should provide sampleIDs for those possible co-infected samples.

Line 168: Figure 3. Why use Krona to visualise these 3 samples? Are they the only samples that contain possible co-infection?

For all co-infected samples, what are their coverage and depth? Can they be false positive?


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: NON-SARS-COV-2 GENOME SEQUENCES IDENTIFIED IN CLINICAL SAMPLES FROM COVID-19 INFECTED PATIENTS: EVIDENCE FOR CO-INFECTIONS
Review round: 2
Reviewer: 1

Output:
Basic reporting: In this study, the author examined infection of COVID-19 and other viruses in publicly available 68 SRA data using Fastv program and Kranken 2.
Experimental design: As compared to the previous version of the manuscript, the revised manuscript is very much improved. However, author did not provide many results derived from the data analysis such as bacterial taxonomy, bacteria phages, and non-SARS-CoV-2.
Validity of the findings: Author used two different programs for the virus identification. Comparative analysis might be useful.
Additional comments: Authors used two different programs for the virus identification. Comparative analysis might be useful.

L95 identification experiments -> Not clear to understand. Please revise it.
L96 using fastv, along with -> by fastv using the pre-computed ~

Supplementary data
Fastv results such as peerj-50170-SRR10971381.html file should be summarized. Please do not show the raw results as it is.

L133 We validated -> I validated (There is only one author in this manuscript. Check it thoroughly in the manuscript.)
L183 Author stated that coverages ranged from 47.55% to 53.22% indicating that the obtained sequences for the rhabdovirus is not complete genome. Please revise it.
L208 Please revise the sentence as follows.
A rank code, indicating unclassified (U), domain (D), kingdom (K),
In the results, I would like to suggest generating phylogenetic trees using obtained viral sequences.

L228 Please revise the sentence. It is not easy to understand.
The value of identifying underlying co-infection(s) is gaining greater appreciation
L232 urgently required -> delete urgently
L253-260 It might be deleted.
In discussion, it is necessary to discuss about identified bacteria.
Authors used two different programs for the virus identification. Please compared the difference between two different methods in the results and discussion.

Figure 2-4 Author identified many reads associated with bacteria phages. Please write results and discussion about the identified bacterial phage.
I can not find any tables or figures addressing the identified non-SARS-CoV-2. Please include them.
In addition, author should provide a table or a figure showing identified bacteria by Kranken2.


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: NON-SARS-COV-2 GENOME SEQUENCES IDENTIFIED IN CLINICAL SAMPLES FROM COVID-19 INFECTED PATIENTS: EVIDENCE FOR CO-INFECTIONS
Review round: 2
Reviewer: 2

Output:
Basic reporting: The author has addressed some of the concerns I previously raised.  However, more information is still needed at various places in the manuscript.
Experimental design: Download links and references for all the tools and databases used in this study need be added to the manuscript. All parameters also to be shown. This is to ensure reproducibility.
Validity of the findings: All analysis outputs have to be provided to show the validity of the findings described in this manuscript
Additional comments: LINE 241: "SRA-tools package (version 2.9.1), Kraken 2, and fastv (version 0.9.0)"
- Please add download links and references for these tools.

LINE 247-248: "The sequence data were downloaded as .sra files using the prefetch tool, then extracted to. fastq files using the NCBI fastq-dump tool. "
- Please add download links and references for these tools.

LINE: 285 "a k-mer-based program "
- Please elaborate. A k-mer-based program for?

LINE 292-294 "A standard database containing RefSeq complete bacterial, archaeal, and viral genomes, along with the human genome and a collection of known vectors (UniVec_Core) was downloaded."
- Please add download links for these datasets.

LINE 294: "Then the database was built using"
- Please provide database building parameters (e.g. default parameters?)

LINE 311-312: "We converted the Kraken 2 output in HTML format using the program ktImportTaxonomy"
- Please add the download link for this tool.

LINE 326-328: "Figure 1: SARS-CoV-2 detection by fastv. SARS-CoV-2 was detected in WIV07 patient, where twelve SARS-CoV-2 hits were included in the genome list and ordered by k-mer coverage (99.4972% to 99.2308%). Mismatches were highlighted in red. "
- Provide more description. What are x and y axes representing?

LINE 330: "SARS-CoV-2 was detected in all of the other 68-sequence date"
- date -> data
- all of the other 59 sequence data

LINE 330-331: "SARS-CoV-2 was detected in all of the other 68-sequence date except three samples (Bioproject PRJNA631042) were negative for SARS-CoV-2. "
- Which sample/run ID were negative?

LINE 430-432: "Influenza type A (8%), rather than, SARS-CoV-2 (2%) was found to be dominant in the WIV07 patient (Figure 2)."
- Is this consistent with Fastv result?

SECTION "Kraken Taxonomic classification"
- Provide results for all samples

LINE 472-474 "A previous study (Lai et al. 2020) showed that reverse transcription polymerase chain reaction (rRT -PCR) could not detect SARS-CoV-2 in two patients co-infected with influenza A virus."
- Why cannot detect?
- How often does this happen?

Sample SRR11772648 that is provided as supplementary data is not found in Table S1.

Only 5 Fastv outputs are provided as supplementary data. The author should make all 68 Fastv outputs available.

From the 5 Fastv outputs that are provided as supplementary data, each output appears to be generated using slightly different Fastv parameters. The author should be consistent with how Fastv is applied to all of the 68 datasets. This is to ensure that all of the results are comparable and without biases.

Here are the commands that were used to generate the 5 outputs provided.

./fastv -i SRR10971381.fastq -c viral.kc.fasta.gz
./fastv -i SRR11092059.fastq -c viral.kc.fasta.gz -g SARS-CoV-2.genomes.fa -k SARS-CoV-2.kmer.fa
./fastv -i SRR11092062.fastq -c Downloads/microbial.kc.fasta.gz
./fastv -i SRR11092062.fastq -c viral.kc.fasta.gz
./fastv -i ncbi/public/sra/SRR11772648.fastq -c Downloads/microbial.kc.fasta.gz -w 10


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

Input:
Title: NON-SARS-COV-2 GENOME SEQUENCES IDENTIFIED IN CLINICAL SAMPLES FROM COVID-19 INFECTED PATIENTS: EVIDENCE FOR CO-INFECTIONS
Review round: 3
Reviewer: 1

Output:
Basic reporting: The author properly revised the manuscript according to reviewers' comments.
Experimental design: The author properly revised the manuscript according to reviewers' comments.
Validity of the findings: The author properly revised the manuscript according to reviewers' comments.
Additional comments: The author revised the manuscript according to reviewers' comments.
I suggest the manuscript for publication.


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: NON-SARS-COV-2 GENOME SEQUENCES IDENTIFIED IN CLINICAL SAMPLES FROM COVID-19 INFECTED PATIENTS: EVIDENCE FOR CO-INFECTIONS
Review round: 3
Reviewer: 2

Output:
Basic reporting: I would like to thank the authors for carefully revising the manuscript. I believe all my concerns have been properly addressed.
Experimental design: All my concerns regarding Experimental design have been addressed.
Validity of the findings: All my concerns regarding findings of this study have been addressed.
Additional comments: Here are a few more errors I came across in the manuscript

Line 129 "extracted to. fastq files"
- Remove . next to 'to'

Line 381 "detected in SRR11092062 (Figure 7) and SRR11092063 sequence date."
- Change 'date' to 'data'.


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: OPTIMIZING AND PURIFYING EXTRACELLULAR AMYLASE FROM SOIL BACTERIA TO INHIBIT CLINICAL BIOFILM-FORMING BACTERIA
Review round: 1
Reviewer: 1

Output:
Basic reporting: English use needs to be greatly improved. As it stands now, the manuscript is very hard to follow. I recommend extensive revision by a native speaker or the use of professional editing services.

The Introduction and Discussion sections need significant improvement. The Introduction should include relevant previous work related to strategies for biofilm removal and the potential of amylase enzymes in antimicrobial treatments. Since the focus of this paper is this type of enzymes, previous works where they are used and the rationale of their interest should be better explained. Right now the Introduction is a series of scarcely connected sentences. By the way, biofilms are considered a health threat, but not really a "threat for nature"; they are the usual way in which bacteria (pathogenic or not) are found in nature. As for the Discussion, it should not be repetitive with the Results section. In this particular case it may be even better to combine both.

Figure 1 is of poor quality and not informative, it can be removed. Alternatively, cropped images of the assays with all the isolates and the different target bacteria can be included, with their corresponding labels, but I deem it unnecessary since the quantitative data are shown.
Experimental design: The data from the biofilm inhibition experiments need to be normalized with respect to culture growth. Since antimicrobial activity (that is, growth inhibition) has been observed for some of the isolates on agar plates, it is more than likely that an inhibitory effect also takes place in liquid cultures. This would give the false impression of limited biofilm formation, but of course if bacteria cannot grow they cannot form a biofilm.
I also think biofilm assays should be done in a more exhaustive way in terms of analyzing at different timepoints. 48h seems too long for at least some of the bacteria assayed. Finally, why are the medians presented instead of averages and standard deviations (or standard errors)? This is the most usual way of representing this type of data.

Given the differences in enzyme activity between the purified amylases, I think it is not correct to use the same volume (100 µL, according to the Methods section) of each amylase solution for the plate growth inhibition and biofilm assays. At least, additional assays should be done adjusting the amount used, in order to have approximately the same activity units in all cases. That way one can discern between effects due to higher or lower enzyme activity obtained from each Bacillus strain and those effects related to enzyme specificity towards different bacteria.
Validity of the findings: Without the modifications and additional information indicated above, the validity of some of the findings is questionable.

The Conclusions should reflect (and be limited to) the main findings of the study. In my view these correspond to the antimicrobial potential of several Bacillus isolates, the fact that part of it (but not all) can be due to amylase activity, and the characteristics of the different amylases purified.
Additional comments: The overall concept of the work is scientifically valid and some results are potentially interesting. However, all the aspects indicated above need to be addessed.


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: OPTIMIZING AND PURIFYING EXTRACELLULAR AMYLASE FROM SOIL BACTERIA TO INHIBIT CLINICAL BIOFILM-FORMING BACTERIA
Review round: 1
Reviewer: 2

Output:
Basic reporting: The article presents a report of valuable pharmacological activity that can contribute to the development of new therapies against the problem reported in the study.

However, I had some doubts and would like to make some suggestions.

1. Abstract: Something about purification could be mentioned in the methodology, at least the resin you used.

2. Figure 1 "Antagonistic efficacy of isolated Bacillus sp. From soil against some human pathogenic bacteria" is related to some direct result or only indicates the representation of antagonistic efficacy? This figure is mentioned a few times in the results, but it was not clear. In line 230, for example, it was said that figure 1C represents the activity of B. mycoides and M. roseus against E. coli, but figure 1C shows only one plate, where, in the legend, it does not say which bacteria is being represented, moreover, figure 1 does not have a scale and the images are not standardized.

3. Figure 2 "Optimization and purification conditions of amylase enzyme from selected Bacillus sp." could present more information in the legend. There is a point that was not clear: what are the purification conditions in this image?

4. Some minor corrections and suggestions in writing: a) line 61, correct the citation; b) line 80, correct "soli"; line 104: the sentence "The medium inoculated with the tested isolates" is not adequate; line 145, suggest removing the sentence "The used pathogenic strains in the study work".
Experimental design: Materials and methods:

1. About the "Effect of pH", describe the ranges of pH variations used.

2. One of the most important points to ensure that the information can be replicated is to provide the concentration used, especially in antibacterial assays. In line 153, the volume of 100 μl was mentioned, but the concentration in that volume was not mentioned. What concentrations of antibiotics were used? Wouldn't it be interesting to perform a serial dilution with antibiotics / isolates? Inform what is the concentration of "crude enzyme preparations" to use in chromatography.

3. Line 186: what was the temperature used in the incubation?

4. In the methodology, DEAE-Sephadex A-25 resin (line 137) is mentioned, but in table 4 it is Sephadex G-200.
Validity of the findings: Results:

1. Bradford's methodology (1976) was cited, but concentrations were not mentioned.

2. Why hasn't the purification graph (chromatogram) been added? My suggestion at this point is to add the graph and make a polyacrylamide gel (SDS-PAGE) monitoring the purification steps.

3. The topic of discussion can be much more interesting if the effects of dilution are added to the results.
Additional comments: In general, the absence of concentration was what most caught my attention, as it was not mentioned in the methodology, making it difficult to reproduce the experiments.


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: OPTIMIZING AND PURIFYING EXTRACELLULAR AMYLASE FROM SOIL BACTERIA TO INHIBIT CLINICAL BIOFILM-FORMING BACTERIA
Review round: 2
Reviewer: 1

Output:
Basic reporting: The manuscript has been somewhat improved, but in my opinion, the main problems have not been corrected, namely presenting biofilm data normalized with respect to culture growth and adjusting the amount of amylase used to compare differences in specificity. The responses to these issues are not satisfactory enough. The manuscript needs another round of revision in terms of English use and typographic errors.
Experimental design: Same comments as in the first version. I appreciate the addition of an extra timepoint in the biofilm assays, but other aspects have not been satisfactorily responded to. Normalization with respect to growth is required in this case to support the conclusions. The question of amylase specificity has not been properly adressed, but this may be less relevant, as long as the authors clearly state the growth differences between strains and the fact they are not trying to compare efficiency of the different amylases but of each amylase vs. total supernatant of the same strain.
Validity of the findings: no comment
Additional comments: no comment


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

Input:
Title: OPTIMIZING AND PURIFYING EXTRACELLULAR AMYLASE FROM SOIL BACTERIA TO INHIBIT CLINICAL BIOFILM-FORMING BACTERIA
Review round: 2
Reviewer: 2

Output:
Basic reporting: Standardize the space between the sections in the methodology (lines 215, 221 and 227);

Standardize the left side alignment (lines 363-392).
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: With the latest changes, the paper makes important contributions to the study of the development of new antibiotics.


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: CHANGING HEALTH COMPLIANCE THROUGH MESSAGE REPETITION BASED ON THE EXTENDED PARALLEL PROCESS MODEL IN THE COVID-19 PANDEMIC
Review round: 1
Reviewer: 1

Output:
Basic reporting: •	Line 2 - 4: Your abstract needs modification. How you relate fear with health messages is not clear. Health messages are not necessarily be fearful I suggest that you improve the description at lines 2-4 to provide more description for rationale.
•	Line 5 - 6: The title doesn't tell about the first aim (“EPPM in naturalistic context…”)...rather it focuses only on the second objective.
•	You described the association between health proposal on Susceptibility. However, it is not clear what kind of susceptibility you referring for, physiological or perceived. I think you mean perceived susceptibility. Be explicit in description.
•	Your description of the rationale in Line 35-36, you mentioned as there are inconsistent findings. It needs more elaboration on the inconsistent findings by providing necessary studies.
•	In line 84-85 of the introduction, you mentioned that “Even though the degree to which one is influenced by the pandemic may vary, the cognition of its perceived threat can be developed spontaneously.” However, the cognition of perceived threat also vary to a large extent as the influence varies.
•	The way you write the hypothesis needs modification. It looks, in any way the hypothesis is true regardless of the data.
I suggest to formulate it like;
H1: Perceived efficacy has a positive/negative/ (depending on whether one- or two-sided test) effect on self-efficacy.
H0: Perceived efficacy has no effect on self-efficacy.
•	Table: you frequently mentioned “Based on a preliminary experiment….”. Could you cite the results of the preliminary experiment or upload as supplementary material?
•	The interpretation column of the table need to be revised. The one written here is a general rule not specific to your study and context. It is also a redundancy from the hypothesis section.
•	The table in the analysis plan is not relevant. All are redundancy from the hypothesis section and sampling. You can summarize it using a single paragraph or simple table provided that there is no specific analysis for each hypothesis.
Experimental design: •	In your research question, line 28-29 you described “to examine how people health compliance intention is influenced by various factors…….”. This research question is not reflected in the title. Your title needs modification to accommodate most of the research aims.
•	Line 12-13: The study aimed to compare one vs two-time messages. However, the description in line 12-13 (‘the times of exposure’) and also in the main manuscript looks as you have multiple exposure times. It is not possible to assess the association of the ‘times’ of exposure with response efficacy. The difference between one vs two messages might not be necessarily equal to the difference in two vs three messages. Your interpretation seems continuous exposure variable but it is binary.
•	In this study, a number of hypotheses are being tested. Multiple comparison is still an issue. As you are evaluating the statistical significance of multiple parameters in a model, the type I error is expected to inflate. You should describe how to control the multiplicity issues. What type of statistical adjustment will you use?
•	In line 197-199 you described how you determine sample size. However, the sample size calculation is not clear. The ‘findRMSEAsamplesize’ function of R requires a degree of freedom to calculate sample size and the sample size varies accordingly. What degree of freedom did you used? How much is the expected dropout rate and you need to adjust for it as well.
•	In you inclusion criteria, in line 222-224: Will you use the nationality as screening or they will fill out the questionnaire regardless of their nationality?
•	The data exclusion and sample size adjustment is not clear. Needs more elaboration.
•	In line 246-247 of the data exclusion section you mentioned that participants with SD of 0 will be excluded. I didn’t get the reason to exclude the participants with SD of 0. The measurement is only twice, it is highly possible to have same score i.e. SD of 0. Why you exclude them?
•	The quality check and control needs descriptions in detail what quality checks will be performed during the design, data collection, and management.
Analysis plan
•	It is recommended to use Tucker–Lewis index in addition to RMSEA and CFI to assess the model fit.
•	Page 24: The items looks similar. Any validity test for these items? How is the inter-item correlation of the questionnaire? In general, the psychometric validity of all the tools to be used need to be described in detail.
Validity of the findings: •	As a protocol, I couldn’t assess the validity of the findings comparing with the data.
•	You mentioned about the preliminary experiment you conducted, however, neither the data nor the results are provided.
Additional comments: •	Line 11-12: It says “SEM is used to....” As it is a protocol, it needs to be corrected to “SEM will be used….”
•	You repeatedly used the phrase “we predicted” for instance line 12, 15… You mean hypothesized? These two terms are different.
•	Line 108-111: Put the direct translation of the message in English.
•	Line 254-256: This sentence should be part of the exclusion not the quality control
•	The description written in the “Sampling plan” column of the Table is repetition. You should remove it.
•	RMSEA, CFI – Put the long form at first instance - root mean square error of approximation - RMSEA, comparative fit index - CFI.


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: CHANGING HEALTH COMPLIANCE THROUGH MESSAGE REPETITION BASED ON THE EXTENDED PARALLEL PROCESS MODEL IN THE COVID-19 PANDEMIC
Review round: 1
Reviewer: 2

Output:
Basic reporting: Yang and colleagues seek to probe factors relating to the EPPM model of fear-motivated health behaviors as well as frequency of measure exposure that may affect behavior intention in the context of the Covid-19 pandemic. Such a study may identify features that have led to relative success in managing the pandemic in some countries relative to others. The proposed study would certainly be beneficial in our attempt to understand and combat the pandemic. Further, the proposed study benefits from a clean and elegant experiment, that can be easily understood by respondents and then further operationalized for analysis. One omission is of preliminary analyses of the pilot data. The study also does not appear to involve any neuroscience and therefore should not be listed as such .
Experimental design: •  2. Experimental design

1.	I recommend changing the ACQ to something less subjective. You risk biasing your sample towards those that already formulate their beliefs based on evidence, which does not comprise the general population, or at the very least, lose participants who find it humorous to answer > 1. Perhaps a simple request to select a number on the scale could serve as an ACQ.
2.	There is also a concern regarding the nationality restriction, given that the question regards a global pandemic. There may be IRB limits to your participant pool but it may be advantageous to expand the survey population, if possible, to other nationalities.
3.	1. In line 23, the message to be repeated is first assessed according to favorability but it is unclear what is meant by this measure. Is it assessing how much respondents: 1) like the message; 2) agree with the message?
Validity of the findings: N/A
Additional comments: Major comments

It is a bit unclear what the specific aim /question of the study is. In the abstract, I can identify 4: response efficacy ~ frequency of exposure; susceptibility ~ frequency of exposure; behavior intention ~ perceived efficacy; behavior intention ~ perceived threat.
The stated aims in the rest of the report are fuzzier. There are some attempts, in lines 77-78; 96-98; 101-103, but they are vague. This part of the report needs to be tightened.
With regards to the specific hypotheses listed, there are too many and some may not be pertinent, notably hypotheses 1-4.

Perhaps providing a real-world translation in the interpretation column could help. But it is not clear why finding a relationship between perceived and self-efficacy is of interest or utility in the context of the proposed study. The same criticism underlies hypothesis 2. With hypotheses 3 and 4, one can argue that it is highly likely that perceived threat (i.e. some average value of susceptibility and severity) is related to susceptibility and severity.

Minor comments:

1. In the opening paragraph of the introduction, you use colloquial and vague language (“It has been a while…”). Please be more specific, so as to allow the reader to contextualize the problem (e.g. “As of writing, it has been x months since the outbreak (…)”. Second, the impact on life and financial well-being are not incalculable, as both life and economic loss can be counted.

2. Some language throughout the report is a bit colloquial (e.g. “sum up” instead of “in summary) and there are a few typos (Hypothesis 4 is listed twice in the table). A revision of the writing would benefit the report.

3. Please change “times of exposure” to “frequency of exposure” to avoid confusion.


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: CHANGING HEALTH COMPLIANCE THROUGH MESSAGE REPETITION BASED ON THE EXTENDED PARALLEL PROCESS MODEL IN THE COVID-19 PANDEMIC
Review round: 1
Reviewer: 3

Output:
Basic reporting: The project meets basic report standards. The table is somewhat large and difficult to read, however, as the cells aren't always aligned well. (eg., sampling plan doesn't track all the way down with the other cells)

There are a few minor English errors in the manuscript and it should be thoroughly proofread.
Experimental design: The experimental design is poor. The primary manipulation isn't very strong (1 vs 2 repetitions) and there is no control group. Most of the hypotheses are regarding structural relationships that are well-established and unlikely to be impacted by repetition very extensively. There aren't safe guards to ensure that any changes aren't due to demand characteristics.
Validity of the findings: I doubt the current study will lead to robust repetition effects, and it is lacking evidence that it should.
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: CHANGING HEALTH COMPLIANCE THROUGH MESSAGE REPETITION BASED ON THE EXTENDED PARALLEL PROCESS MODEL IN THE COVID-19 PANDEMIC
Review round: 2
Reviewer: 1

Output:
Basic reporting: •	I still have some concerns on the description in line 96-103. The declaration of WHO as pandemic alone could not be considered as the health treat by all the people to the same extent. The way people react to the this health treat (COVID-19) considerably vary according to the type of health messages communicated by each country and even specific contexts. For instance, the health messages in Belgium are different from the messages in Uganda or Ethiopia, which in turn leads variation in the influence on peoples. I agree that the government  measures could be the same as WHO standard. However, the health messages and respective reactions significantly varies within and across countries.
•	The formulation of the hypothesis is improved. I still have minor issues. The H1 is one sided while the Ho is two sided. For instance, if perceived efficacy has a negative effect on self-efficacy, what conclusions would be made? This is the same for some of the hypotheses.
Experimental design: •	Good to add the degree of freedom. However, not clear why select 70 as df? Since it is a protocol, it is better if you add a description to justify it.
•	I suggest to put age as relevant characteristics as inclusion criteria.
Validity of the findings: Not applicable
Additional comments: Thank you for the opportunity to review this manuscript once again. Overall, the manuscript has significantly improved. The preliminary experiment substantiates the need for the present study. I have a few minor 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: CHANGING HEALTH COMPLIANCE THROUGH MESSAGE REPETITION BASED ON THE EXTENDED PARALLEL PROCESS MODEL IN THE COVID-19 PANDEMIC
Review round: 2
Reviewer: 2

Output:
Basic reporting: n/a
Experimental design: Overview: Thank you for revising your report. I still maintain that H1-4 are not true hypotheses, as the outcome variables are constructed from the dependent variables. Related coefficients are not included in supplementary 1, Figure 1’. I leave it to the authors to consider when submitting their results.

Some examples of this relationship:
Abstract: Here, you claim that “perceived threat of COVID-19, which underlies perceived susceptibility”, so why test this in H4?

Lines 64-65: If perceived efficacy is composed of self-efficacy and response efficacy, I still don’t understand why their relationships is being tested in Hypotheses 1 & 2. You could use these hypotheses as positive controls however.
Validity of the findings: n/a
Additional comments: Line 187: While the term “ social distancing” has gained ground, it is at base incorrect.  Please change the term to “physical distancing”.


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

Input:
Title: THE EFFICACY OF IL-6 INHIBITOR TOCILIZUMAB IN REDUCING SEVERE COVID-19 MORTALITY: A SYSTEMATIC REVIEW
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1.	The language used is error-free and unambiguous, conveying the necessary scientific details.
2.	Reference list is clear, correct and sufficient. Some comments listed below would need more references to be added, but the current ones are ideal for the written content.
3.	The authors have mentioned how 3/4th of the severe cases present with IL6 mediated respiratory failure. Alluding to the remaining 1/4th cases (with appropriate references) would be helpful.
4.	A schematic showing the mechanism of action of TCZ and its downstream effects would be useful.
5.	Please clarify what “potentially repeating patient data” indicates.
Experimental design: 6.	This being a review article, it addresses all the relevant clinical trials and primary research articles to arrive to scientific conclusions about efficacy of TCZ in COVID-19 patients.
7.	Are there any trials of TCZ on younger patients suffering from COVID-19? If so, since they weren’t in this review analysis, what were the results and maybe mentioning those in the discussion section would be useful.
8.	Why did the one mentioned study derive results showing increased mortality with TCZ treatment compared to SOC? Please elaborate.
Validity of the findings: COVID-19 affected individuals and results/conclusions arising from the studies are still in nascent phases and ever-changing based on recent research.
Hence even though this review suggests that TCZ could be a potentially beneficial treatment for patients, the authors have highlighted the caveats of the research.
Data is statistically analysed and speculations have been clearly outlined.
Additional comments: The manuscript is a great attempt at bringing together all the relevant research for IL6 inhibitors, especially TCZ in COVID treatment. It would be of importance to the field, as we slowly know more about this virus and its deleterious effects.


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

Input:
Title: THE EFFICACY OF IL-6 INHIBITOR TOCILIZUMAB IN REDUCING SEVERE COVID-19 MORTALITY: A SYSTEMATIC REVIEW
Review round: 1
Reviewer: 2

Output:
Basic reporting: No comments
Experimental design: No comments
Validity of the findings: No comments
Additional comments: Detailed systematic review!


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

Input:
Title: THE EFFICACY OF IL-6 INHIBITOR TOCILIZUMAB IN REDUCING SEVERE COVID-19 MORTALITY: A SYSTEMATIC REVIEW
Review round: 1
Reviewer: 3

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: This systematic review is based on efficacy of IL-6 inhibitor Tocilizumab in Covid-19 patients. The authors presented the review based on 16-case control studies gathered from PubMed search. Considering the ongoing pandemic COVID-19 and lack of effective therapies to treat patients affected with severe disease, careful evaluation of clinical data on existing/new anti-viral drugs is important and much needed. However, currently couple of studies are published on efficacy of Tocilizumab in COVID-19 patients as systematic review and meta-analysis (PMID: 32712333, 32713784 & Aziz M et al 2020). The authors can improve the article with following…
1)	The introduction part of the review is somewhat lengthy and covered topics which doesn’t have scope in the present review.  Authors can present a short and informative introduction that largely talks about IL-6 role in physiology, dysregulation of immune pathways, IL-6 role in COVID-19 pathology, mechanism of Tocilizumab action and known adverse effects (other IL-6 inhibitors). Instead of presenting the topics on other anti-viral drugs, authors can cite relevant existing literature.
2)	Authors did not talk about IL-6 levels and other biomarkers (post-treatment levels, if any)?
3)	Discussion can be improved by adding points that can help ongoing/future clinical trials to better assess drug efficacy.


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

Input:
Title: THE EFFICACY OF IL-6 INHIBITOR TOCILIZUMAB IN REDUCING SEVERE COVID-19 MORTALITY: A SYSTEMATIC REVIEW
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: Authors have addressed all the concerns.


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

Input:
Title: INTEGRATED NETWORK PHARMACOLOGY AND MOLECULAR DOCKING APPROACHES TO REVEAL THE SYNERGISTIC MECHANISM OF MULTIPLE COMPONENTS IN VENENUM BUFONIS FOR AMELIORATING HEART FAILURE
Review round: 1
Reviewer: 1

Output:
Basic reporting: NA
Experimental design: NA
Validity of the findings: NA
Additional comments: In this manuscript  “Integrated network pharmacology and molecular docking approaches to reveal the synergistic mechanism of multiple components in Venenum Bufonis for ameliorating heart failure” Ren et al. have performed network and molecular docking analysis of VB and heart failure associated targets and revealed the role of adrenergic signaling. Although authors have done multiple bioinformatics analysis but manuscript in its current form lacks key methodological details and biological insights gained from the current analysis.  Following are the comments that need to be addressed to strengthen the manuscript:

Major comments:

1. Section 3.1: What are 260 potential targets of components of VB. Authors should discuss them in detail.  Similarly authors should also discuss the heart failure related targets they have identified. Is there any overlap between VB and heart failure related targets?

2. Why enrichment analysis was not done using GSEA instead of DAVID? GSEA is more powerful method to identify enriched pathways and GO terms. What does x axis and percentage implies in Figure2? It is not clear if these are FDR corrected p-values? Use full names of  MF, BP and CC  instead of abbreviations.

3. Network diagram in Figure 3 is not informative at all. What does three circles represent?  Instead of mapping interactions using STRING authors can use other experimentally validated datasets (eg. iRefWeb) to make this figure interpretable.  Figure text is unreadable. How many hubs were identified? Please discuss biological role of these hub genes.

4. Authors have not provided the technical details of docking experiment? Is this a rigid or flexible docking method? How many docking runs were carried out? Discuss the biological implications of the results. What are the unit’s of binding energy?

Minor comments:

5. Line 103: What does the confidence score implies to identify VB compound –drug pairs? Is it a drug and Chinese medicine similarity score?

6. Discussion section of the manuscript is verbose and should only focus on the biological insights gained from this analysis and its implication for novel drug usage for heart failure.

7. Figure quality is very poor and text font is unreadable.


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: INTEGRATED NETWORK PHARMACOLOGY AND MOLECULAR DOCKING APPROACHES TO REVEAL THE SYNERGISTIC MECHANISM OF MULTIPLE COMPONENTS IN VENENUM BUFONIS FOR AMELIORATING HEART FAILURE
Review round: 1
Reviewer: 2

Output:
Basic reporting: Overall speaking, the writing was acceptable.
More details should be added to the docking part.
Experimental design: The paper structure is really complicated, emerging 4 databases (FDA drug-target, drugbank, OMIM, String-db), 4 different bioinformatics tools (MedChem Studio: drug-drug similarity through chemical components; cytoscape network analyzer; David for enrichment analysis; docking in Discovery Studio), and other efforts such as collecting VB components, etc.
Despite these efforts, it looks like the stacking of all these fancy methods.
Validity of the findings: When the authors merged VB-related targets and HF-associated targets and analyzed the network features of the merged dataset. The authors were assuming that all VB-related targets were effective. This means that instead of analyzing how each of VB-related targets might affect heart function, the authors were treating these two sets of genes equally and analyzing the overall function of the merged PPI network. It’s not surprising that a lot of HF-related pathways were extracted out.
Additional comments: Review of  Manuscript 49255v1, "Integrated network pharmacology and molecular docking approaches to reveal the synergistic mechanism of multiple components in Venenum Bufonis for ameliorating heart failure "

Venenum Bufonis (VB, or Chan Su in China) has been extensively used as traditional Chinese medicine (TCM) for treating heart failure (HF) since ancient times. Similar to a lot of other TCM, the detailed molecular mechanisms of how VB might work are largely unknown.
In the current paper, the authors applied integrated computational biological methods to address how various components of VB might work. Firstly, 42 components of VB were collected from previous studies. Then, the VB-related targets were inferred by the relationship of VB components----Known FDA drug similarities ---- Drug targets. Meanwhile, the HF-associated targets were inferred from two sources: Drugbank HF targets(109 targets) and OMIM human genetics(199 targets). The linkage between VB-related targets and the HF-associated targets was addressed through PPI network from String-db. The network structure was analyzed using Cytoscape Network Analyzer plugin to find the hubs. 158 hubs, including 93 VB-related and 65 HF-associated targets, were found important based on the network topology. The functions of these hub genes were addressed through gene enrichment analysis using GO/KEGG genesets. Potential pathways were discussed and interpreted as the potential functions of VB. Finally, molecular docking between VB components and their potential targets were performed using the Discovery Studio platform.
The paper structure is really complicated, emerging 4 databases (FDA drug-target, drug bank, OMIM, String-db), 4 different bioinformatics tools (MedChem Studio: drug-drug similarity through chemical components; Cytoscape network analyzer; David for enrichment analysis; docking in Discovery Studio), and other efforts such as collecting VB components, etc. However, despite all these work, the hierarchical nature of the architecture of this paper makes it not suitable to publish at the current stage, and scientific research is not a stacking of what you could do using your currently available data/methods.
When the authors merged VB-related targets and HF-associated targets and analyzed the network features of the merged dataset. The authors were assuming that all VB-related targets were effective. This means that instead of analyzing how each of VB-related targets might affect heart function, the authors were treating these two sets of genes equally and analyzing the overall function of the merged PPI network. It’s not surprising that a lot of HF-related pathways were extracted out.
Other comments:
1. Why not do the gene enrichment analysis of the VB-related targets directly.
2.From line170-180, the author could list them in a table
3.Figure 3, larger fond size,
4.Figure 4, color is too complicated. Because of the nature of PPI network, it’s almost impossible to tell whether it’s activation or inhibition. Still, the authors should try their best to note each pathway whether it’s activation or inhibition.
5. In the docking part, the author should write it clearly how they perform the docking in detail. Such as, what are the targets? Is it all the hub genes or only the VB-related targets? If it was the hub genes, which part of the genes was selected for docking? Regulatory domain? enzyme domain? Or structure domain? What are the small molecules in the docking? Is it the VB-related components? For the CIE, for each of the targets, is there any gold standard of what value makes sense? Or what’s the CIE for a known target-small molecule for each target?


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: INTEGRATED NETWORK PHARMACOLOGY AND MOLECULAR DOCKING APPROACHES TO REVEAL THE SYNERGISTIC MECHANISM OF MULTIPLE COMPONENTS IN VENENUM BUFONIS FOR AMELIORATING HEART FAILURE
Review round: 1
Reviewer: 3

Output:
Basic reporting: - There contains many grammatical errors and typos in this manuscript. The authors should re-check and revise carefully. The use of language should be improved significantly.
- Quality of figures needs to be improved. Now there are some parts that cannot be seen clearly in the manuscript.
Experimental design: - Methodology hasnot been explained well, it makes difficult for other people in reproducing their results. The authors should explain their methods clear and transparent, not only the use of some packages and software.
- What is the confidence rate of retrieving data from literature?
- GO database and analysis had been used in biomedical works such as PMID: 31921391 and PMID: 31277574. Therefore, the authors should refer more works in this description.
- Why did section 3 is "results and discussion", but section 4 is "discussion" again?
- Why was the confidence score of greater than 0.6 considered as the target proteins of the known drugs?
Validity of the findings: - There are many metrics related to molecular docking analysis. Why did the authors only use "-CIE"?
- The application of this study is too narrow. It is important that the authors discuss and compare their works with some general studies on molecular docking-based or network pharmacology-based Venenum Bufonis.
- The authors should compare their performance results with the previously published works on the same dataset.
- What are orange and green chars in Fig. 2?
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: INTEGRATED NETWORK PHARMACOLOGY AND MOLECULAR DOCKING APPROACHES TO REVEAL THE SYNERGISTIC MECHANISM OF MULTIPLE COMPONENTS IN VENENUM BUFONIS FOR AMELIORATING HEART FAILURE
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: My previous comments have been addressed satisfactorily.


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: BIASES IN GENOME RECONSTRUCTION FROM METAGENOMIC DATA
Review round: 1
Reviewer: 1

Output:
Basic reporting: This manuscript described the biases in the genome reconstruction process from metagenomic data. The authors explored several factors that might affect the genome reconstruction by comparing their unicyanobacterial consortia paralleled genomic and metagenomic datasets. They reported that repeated sequences and genomic regions with variant nucleotide compositions are more likely to be missing, and that missing regions are strong biased toward rRNA, tRNA, and mobile elements.

Although most of the conclusions drawn from this study are not very surprising, the study itself is plausible in that it dig through the elements that might affect the genome reconstruction process. There are however still room to improve this study. I give my review as follows.

1. The dataset that the authors used for probing the problem is the unicyanobacterial consortia that they developed as model systems, which however represents just one system. This makes their conclusions a bit weaker since their results were only drawn from that very system. I would appreciate if the authors can extend their study to one or two more systems, be them simulated or real ones with genuine answers.

2. The microbial population complexity may also affect the genome reconstruction results. Looks like the unicyanobacterial consortia is a simple system since not too many genomes can be recovered and reconstructed. Perhaps the study can be extended to a more complex microbial community. The authors may also be able to say something about the relationships between microbial community complexity and the reconstruction process.

3. I wonder when the authors term MDR (missed detection region), do they distinguish contigs that were not binned correctly between contigs that were filtered out due to short contig sizes? For example, 16S rRNA genes cannot usually be assembled very well and were usually split into pieces. Hence these fragmented were usually filtered out in the very first step of binning due to the contig length requirements of most binning tools. This also applies to most repetitive elements since they confuse assemblers. I guess the analysis of MDR is focused on those long enough to be included in the binning process and would very much appreciate if the short contigs can also be included in the analysis as well.

4. (line 109) how come tetranucleotide frequencies need to be extracted across all six reading frames? Tetranucleotide need to be considered for only forward and reverse-complement strains. Please explain or revise this part.

5. In table 3 there is a “mean tetranucleotide frequency,” however I do not know what does that mean. Are all tetranuclotide frequencies for the CDR or MDR regions of organisms be calculated and averaged? Perhaps the authors can explain more in both table 3 and the text.

6. Two words are used interchangeably in this manuscript: repeat and redundancy. I however feel that the word “redundancy” is a bit misleading and was not used very frequently throughout genomic analysis. Alternatively the term “per-base coverage” may be more informative. Please consider using terms that are more widely used and comprehensible.

7. Figure 2 is quite clear in delivering the message to readers. I wonder if the values such as TNF, GC%, or “redundancy” is derived by a sliding window?
Experimental design: no comment
Validity of the findings: no comment
Additional comments: no comment


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

Input:
Title: BIASES IN GENOME RECONSTRUCTION FROM METAGENOMIC DATA
Review round: 1
Reviewer: 2

Output:
Basic reporting: In the manuscript entitled ‘Biases in genome reconstruction from metagenomic data’, authors describe genomic regions associated with ten microbial populations that were missing in metagenome-assembled genomes, yet present in culture representatives. Authors found that the missing regions exhibit distinct traits compared to the rest of the genomes studies, and conclude that high-completion metagenome-assembled genomes are generally good representatives of the metabolic potential of the microbial populations they represent.

I note that links to the genomic and metagenomic raw reads used for mapping are not available and need to be added in the material and methods section.
Experimental design: The experimental design used in this manuscript is valid. Authors applied the same bioinformatics methodology to genomic regions present and missing in metagenome-assembled genomes (GC%, sequence composition, coverage, and functional potential). Methods are described with enough details.

I would like to mention one limit of the experimental design. There are many ways to characterize metagenome-assembled genomes, and it would have strengthened to study to test the effect of assembly (using different software) and binning (using both manual and automatic tools) on downstream results. Are the missing regions due to fundamental limitations of assembly and/or binning, or specific to the tools used to characterize the metagenome-assembled genomes? To me, this is the main limitation of the study. However, it does not impact the described observations. In my opinion, it merely limits the extent of the conclusions and does not prevent publication in its current form.
Validity of the findings: The findings of this study are valid, and in line with the field’s understanding of assembly-based metagenomics.
Additional comments: The overall aim of the study is to investigate the genomic regions of microbial populations systematically missing when using assembly-based metagenomics, as compared to cultivation. This is an important topic, as metagenome-assembled genomes largely contribute to our understanding of the microbial tree of life. Authors demonstrated that in the studied microbial community, nucleotide composition of genomic regions missing in reconstructed “genomes from metagenomes” frequently differ from the genome average. This contributes to our understanding of the limits of metagenomic assemblies, and/or binning. The manuscript is well written, and general trends appear to emerge from the analysis of 10 microbial populations.

The manuscript is of interest, and I could not see any flaw in the methodology. However, as far as I could see authors used only one metagenomic assembly software, and only one metagenomic binning workflow. It is unclear how variations in the bioinformatics workflow for assembly and binning impacts the genomic regions determined as missing. As a result, authors can only compare culture representatives with one single metagenomic workflow. I would appreciate an extensive answer of the authors regarding this matter. Would they consider expending their experimental design for a more comprehensive study?

Specific notes:

Introduction:

Ln 42-43: Key references of the pioneer publications supporting the sentence are missing. Please consider introducing a more relevant history of high-throughput sequencing in the context of culture-independent surveys.
Ln 58-59: Please consider reformulating the sentence, as single copy core genes are used to determine the completion, as well as the redundancy, of bins without the need for any reference genomes.
Ln 81-82: what workflow did the authors used, specifically? Many automatic, and a few manual-binning tools are available. They all provide different results.

M&M:

Please provide links to the genomic and metagenomic raw reads used for mapping. The study uses this data to assess coverage values and as a result it is important to make it easily available to the reader.

Results and discussion:

Ln 197: does “save” corresponds to “except”? Please consider using a more commonly used term for an optimal reading experience.
Ln 207: please consider using a different term. Genome reconstruction could refer to the assembly, or the binning. A more specific term would be appreciated.
Ln 221-223: This is incorrect. Duplicated regions will exhibit 2-fold coverage increase across all samples, and thus be clustered with the associated genome when binned using differential coverage. On the other hand, multi-copy plasmids for instance can create problematic situations, if the regulation of copy-numbers changes across samples. Please reformulate the sentence.
Ln 229-230: sequence coverage and genome coverage of what? I understand the metagenomic coverage variations described later on, but not this one. Is it based on reads recruitment from the pure culture? Please explain.

Figure 3: legend does not match the figure. Was the wrong figure uploaded?

Ln 296-298: Unclear. How adding controls not related to the studied environment (generally a black box) will help understand assembly accuracy?
Ln 304: Why 2000nt?
Ln 306: The “enhanced” binning results of this reference are contaminated due to a lack of proper curation step. Please consider using a better reference.

Conclusion:

Ln 325: Please fix typo.


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

Input:
Title: BIASES IN GENOME RECONSTRUCTION FROM METAGENOMIC DATA
Review round: 1
Reviewer: 3

Output:
Basic reporting: ## Minor

* the central figures (Fig. 2, Fig. 3) need better annotation and a legend, otherwise the reader has to spend quite some to switching between the caption text and figure to understand them

l. 59-63: other comparisons to sequenced isolates and controlled simulation benchmarks with complex communities have been made to assess properties such as quality and completeness of reconstructed genomes. The authors should mention and cite some of them.

l. 72: explain axenic, this is a special term mostly unknown to non-biologist readers

l. 94-95: data set vs. dataset (should be consistent throughout the manuscript)

l. 107: "tetranucleotide frequency distance chi squared analysis": why not TNF chi square analysis/test and where is the distance? It's a little confusing because there is also a term called chi2 distance/divergence.

l. 108: typo in "a custom perl scripts"

l. 113: "absolute distance" == Euclidean distance? If needed, provide formula

l. 119: explain shortly what "per-base redundancy" means

l. 121: what is the "arithmetic distance" here? If needed, provide formula
Experimental design: ## Major

l. 81-82: The genome reconstruction alias binning process is treated as a black box, but there are pronounced difference between different binning procedures (see e.g. http://biorxiv.org/content/early/2017/01/09/099127). The process described as "current standard genome reconstruction techniques" is very nebulous and hides the complexity of genome reconstruction. In general, the details of the metagenome sequencing and in-silico processing (assembly, binning) are nowhere described although they represent the actual subject of the study. The choices made here have direct implications on the validity of the findings. Therefore, the authors must disclose which technology (sequencing platform, read lengths, insert size etc.) and algorithms (assemblers, contigs lengths, manual inspections etc., binning program, binning procedure, binning features) their results will relate to.

* Please provide scripts used in the calculations for the distances and p-values together with a minimal documentation and a usage/license statement.

# Minor

* When the authors generate empirical null distributions for the p-value calculation using random draws (for instance l. 112,129, ), it is not entire clear to me what pool they sample from. Is this the same genome including MDRs and CDRs?
Validity of the findings: ## Major

In general, the overall findings confirm what is generally known for genome binning and add additional facts on the functional level. However, results are presented being universal although this extrapolation cannot be made without looking at different data and algorithms.

* It remains unresolved whether the missing regions are a result of an incomplete assembly or the binning. For both steps, there exists a multitude of different algorithms which lead to different output. In l. 151 the authors write that the assembly contained no errors, but it is not clear whether this also relates to missing regions. In l. 188 they write, that that differing GC content in MDRs and CDRs are due to the assembly but unless verified, it could also be an artifact of the sequencing itself.

* In l. 212 and following the authors elaborate on the effects of differential coverage binning. Basically, if absolute counts are used for binning, collapsed regions are missed because they have higher coverage. However, many binners also group such contigs by using information which is not dependent on absolute counts, such as covariance/correlation distances. For instance, the program MetaBAT recruits shorter contigs to existing binss at the end of the binning procedure. The authors must disclose the details of genome binning, otherwise the results are hard to interpret or validate. The authors should also show that their findings are not an artifact of a specific binning program or setting.
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: REVALIDATION OF MORPHOLOGICAL CHARACTERISTICS AND MULTIPLEX PCR FOR THE IDENTIFICATION OF THREE CONGENER INVASIVE LIRIOMYZA SPECIES (DIPTERA: AGROMYZIDAE) IN CHINA
Review round: 1
Reviewer: 1

Output:
Basic reporting: The work of Dr. Chang and Collaborators focuses on the possibility to discriminate among three Liriomyza species of agricultural importance. They re-evaluate three morphological characters and develop a new test based on a multiplex PCR.

In my view, the manuscript is not acceptable in its present form, but has the potential to become acceptable following revision.
The one and only issue that, in my view, may prevent it from being acceptable even after revision,  if not dealt with convincingly, is intraspecific variability. This is possibly the key reason why such tests are not valid/usable and may mislead other authors into using an invalid test in their research.

Language is not always clear and should be improved before publication. As an example see lines 27-19, 45-49, 57-60, 145-149, 154-157, 192-194.

Background is appropriate, but some recent studies (eg: Chen et al, 2019 Sci.Rep; Carapelli et al, 2018 Genes) may be included. Furthermore it is not always clear form the introduction and discussion sections:
a) which morphological characters are considered as inequivocous and which are considered dubious and re-evaluated. A clear distinction between male genital structures, being inequivocous, and the other three, being equivocous, stems from the results of this study. Does this represent the state of the art before this study? Did other authors comment on the validity of these characters in the past?
b) which are the strength and weaknesses of the molecular tests developed in the past and which is the knowledge gap that the authors are willing to cover. Curiously, the authors suggest that 'using samples from different geographic regions' (line 197) may be a limitation, while I would argue that using samples from one single location is the key limitation of this study (see below).
A paper (Zehnder et al 1986) is referenced to in the text but it is not present in the bibliography. Please cross check text and bibliography throughout.

The structure of the manuscript appears correct.

Figures are generally appropriate.
In Figure 4 please specify what interval the central box represents and add marks (usually asterisks are used) to convey the results of the post-hoc tests.
In Figure 6 please indicate which positions are variable within each species. The image (as is) suggests that sequences are invariable within species, that is certainly not the case.
In Figure 7 it is not clear which species panel B refers to. Please prepare a montage figure where all developmental stages of all species are shown and emend the caption accordingly.
In Table 1 the accession number of the complete mitochondrial genome of the three species may be removed.
Table 2, see below.

No mention is made to data deposition, while I deem that new cox1 sequences should be deposited in GenBank.
Experimental design: The work presented can be considered as original primary research.

The research question is well defined (i.e. developing an improved molecular test for species discrimination) but the knowledge gap this is filling is not well defined or justified. In fact, the limitations of previous molecular methods similarly based on PCR are not presented and it is not clear how this new test improves over available tests.

Some aspects of the methodology are not clearly presented and it cannot be evaluated if the investigation has been conducted under rigorous standards. More specifically, my main concern regards intraspecific variability, an issue that appears to have been overlooked by the authors but, in my view, needs some consideration.
a) how many individuals, and how many from which species, were sequenced (lines 98-102)? Did the authors sequence all individuals listed in Supplementary Table 1?
b) how many sequences were taken from GenBank (lines 103-104)? How many from each species? Which is the geographical representativity of these sequences? Which is the level of intra and interspecific variability?
c) how many variable sites were observed between species (lines 105-107)? How many different sites were fixed and invariable within species?
d) how many specimens were tested to validate the test? Which developmental stages? Three bands per species (adults) and three band from three different developmental stages (stages and species are not indicated) are shown in figure 7. Is this the full panel of tests conducted to validate the essay? I think that a minimum of few tens of individuals per species (including different developmental stages) may be used here.
e) line 153 says that the reverse primer is 'common only to those three Liriomyza species'. I would say that it 'is common' to the three species, while the authors cannot know if it is common 'only' to these. This is relevant as the primer may amplify in other species, something that has not been tested.

Methods (meaning how experiments were performed) are described with sufficient detail. How many samples, and which samples were included is not always clear (see above).
Validity of the findings: Following guidelines of the journal, impact and novelty are not assessed. Nevertheless I think that the applicability of the test should be declared in full clarity.
As from my comments above, I think that natural intraspecies genetic variability is a crucial aspect here. Considering that a) sequence data used for test development may or may not include a sufficient number of reference sequences from outside of China (see my abovestated request for clarification), b) samples used to validate the test are from China (see also my abovestated comment on their number), and c) the fact that we are talking about three invesive species whose native range is not China and may display a sizeable level of genetic diversity in their native range that is not present in China, I would argue that, strictly speaking, the test is valid for China only.
Obviously, the test developed for China by Dr. Chang can be revalidated by other authors working in different areas, but it is important to clearly state the range of applicability of the test as currently proposed.

Underlying data has generally been provided, but a few pieces of information should be addedd:
a) Gen Bank accessions for new sequences (see my comment above).
b) patterns of setae color in Table 2 are not clearly reported. I suggest the authors list, for each species, the visible phenotypes (including uncertain phenotypes that are descibed in the text) and report their absolute and relative frequencies. Here the 'unit of observation' is the individual insect, not the seta. This has been correctly presented in the text but not, in my view, in the table.
c) statistical analysis on wings is correctly conducted (an overall test followed by multiple post hoc tests), but the results are not clearly presented. The p value of the overall test should be reported as well as p values for each of the three post hoc comparisons (or at least the significant ones). Furthermore, in the methods section it is declared that p=0.05 is used as a significance threshold, while in the result section a p=0.001 is considered not significant.

Speculation is not an issue.

Conclusions are generally well stated, apart from the need (presented above) to specify how the morphological reanalysis and the new test improve over current knowledge.
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: REVALIDATION OF MORPHOLOGICAL CHARACTERISTICS AND MULTIPLEX PCR FOR THE IDENTIFICATION OF THREE CONGENER INVASIVE LIRIOMYZA SPECIES (DIPTERA: AGROMYZIDAE) IN CHINA
Review round: 1
Reviewer: 2

Output:
Basic reporting: The present manuscript of Dr. Chang and co-Authors describes the efficiency of three morphological characters in the identification of three Liriomyza species (i.e., L. trifolii, L. sativae and L. huidobrensis). Moreover, the Authors developed of a multiplex PCR approach to enhance our ability to identify these three important pest insects.

The English is not always clear and many sentences are vague and imprecise (see attachment for further details). In my opinion, it should be improved, before publication.

The literature references should be improved in some points (e.g., lines 168-170). The background and the aims are sufficiently described and clear throughout the text.

The structure of the manuscript is clear, as well as the figures and tables are relevant to the manuscript aim. However, I would suggest to improve the Figure 1 by both increasing the resolution and by adding more annotation to help the reader understanding the morphological description. I think the Figure references in the text should be improved (see attachment for further details).

 The raw data are not shared (e.g., the sequences obtained from their first PCR are not shared and no accession number is given).
Experimental design: The manuscript is an original primary research and the research question is clear and relevant.
However, it does not seem to me that the analyses were conducted rigorously. The way in which the methods and the results are described opens to some important critical issues (also highlighted in the attached pdf). The methods are not sufficiently described to be reproducible. For examples, it is not clear how many sequences they used to design the primers. If the sequences were only one per species, how did the Authors account for intra-specific variability of the cox1 gene? Moreover, no other Liriomyza species was included in the alignment as a control that the primers were actually specific for the three Liriomyza species under study. In the analysis, specimens and sequences of Liriomyza langei were not included and it is known that this latter species cannot be morphologically distinguished from L. huidobrensis (Scheffer et al 2014). Therefore, in my opinion, the Authors should have at least included in the cox1 alignment the sequences of L. langei (available on public databases). In this way, the Authors could confirm that the species-specific selection through multiplex PCR was potentially effective also in other areas where L. huidobrensis is present (e.g., America).
Furthermore, it is not clear from the methods (and the results), whether the Authors sequenced or not the multiplex PCR product to confirm that they actually selected the cox1 of the wanted species.
In my opinion, it is fundamental that these points, just highlighted, are clearly described and satisfied prior further reviewing or publishing the manuscript.
Validity of the findings: In my opinion, the novelty and impact of the present work strongly depends on few aspects. The first one is the inclusion of specimens or at least cox1 sequences of L. langei in the alignment to check the efficiency of the primers designed. The second aspect is the sequencing of the multiplex PCR products to confirm that the primer selection actually works. A third aspect that should be better and clearly described is the application of this method to all the developmental stages of L. sativae and L. huidobrensis. The authors reported that this method worked only for all the developmental stages of L. trifolii. If we cannot distinguish larvae from these three species, then this method is not effective to develop useful control strategies for these pest insects. Finally, the submission of the cox1 raw data on public databases is fundamental for reproducibility.
Without these improvements, the present work is inconclusive and ambiguous, with the results not fully supported in the present form.
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: REVALIDATION OF MORPHOLOGICAL CHARACTERISTICS AND MULTIPLEX PCR FOR THE IDENTIFICATION OF THREE CONGENER INVASIVE LIRIOMYZA SPECIES (DIPTERA: AGROMYZIDAE) IN CHINA
Review round: 2
Reviewer: 1

Output:
Basic reporting: The current version of the ms marks a significant improvement over the first submission, especially in terms of language and clarity in the presentation of the results.
Some of the limitations - i.e. geographical representativeness and reliance on few sequences - are still present, but giving a clear account of these aspects in the manuscript now allows the reader to decide if and when to apply the test proposed.
Experimental design: NA
Validity of the findings: NA
Additional comments: NA


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

Input:
Title: SOCIAL COGNITION AND SOCIAL FUNCTIONING IN PEOPLE WITH BORDERLINE PERSONALITY DISORDER AND THEIR FIRST-DEGREE RELATIVES
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1. In table 3, BPD is spelt wrongly as BDP.

2. It is unclear in Table 2 what the numbers in brackets are e.g. 31 (6). I understand that 31 is the number of people in a group. What does the (6) mean?
Experimental design: 1. Throughout the tables 1 and 2, where there are omnibus p values that are significant when comparing the three groups, it would be important for the authors to analyse which of the paired comparisons between two groups are significant. This can be achieved by post-hoc tests e.g. Bonferroni correction after the chi-squared and ANOVA testing.

For example, for any given comparison where the omnibus p value is <0.05: is the significant difference between controls and relatives? Is the significant difference between relatives and BPD patients? Is the significant difference between controls and BPD patients? This would more clearly answer the study question, about whether relatives are more "similar" to controls or more "similar" to BPD patients.
Validity of the findings: 1. Similarly, the results section of the text should be re-written to always focus on the results of the relatives when compared to controls, and also when compared to BPD patients.

Comparisons focusing on the BPD patients, comparing them with controls, are not novel and not as relevant to the overall aim of the study.

For example, "relatives showed statistically similar scores in the MASC to BPD patients. Both relatives and BPD patients scored more poorly than controls".

For example: "On the family relationships subscale of the SFS, the relatives scored better than BPD patients (p value xxx). However, the relatives scored worse than the controls (p value xxx)."

For example " On the SFS subscales of social engagement/withdrawal, interpersonal behavior, independence-competence, and prosocial
activities, plus the full scale and the categorization, there were no statistically significant differences between relatives and controls. However, relatives scored better than BPD patients".

2. Similarly, it would be important in Table 3 to change the structure: have the relatives scores "set" at 0, and then present the coefficients of controls compared to relatives, then present the coefficients of BPD patients compared to relatives. Currently, looking at Table 3, it is not possible to compare relatives with BPD patients which would be of much more clinical interest than comparing controls with BPD patients.
Additional comments: No additional comments