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: COMPARATIVE GENOMIC ANALYSIS OF THE PRINCIPAL CRYPTOSPORIDIUM SPECIES THAT INFECT HUMANS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The premise for this work was that trees based on a few genes can lead to conflicts and the authors proposed to overcome this by conducting a phylogenetic analysis based on whole-genome seq variation. The authors downloaded 23 high-throughput short read datasets from SRA, from 3 Cryptosporidium species (10 for C. hominis, 9 for C. parvum and 4 for C. meleagridis), reconstructed assemblies and identified seq variants from the assembly alignments. A max likelihood phylogenetic analysis was conducted on the concatenated variant positions, which the authors say agrees with the accepted phylogenetic history for the three species and supported the monophyly of alleles from a locus, gp60, often used to type Cryptosporidium samples. This manuscript reflects a good amount of work, but there are serious flaws with the study and some opportunities missed as well, which maybe can be used as a basis for a solid comparative genomics work.

1.	Basic reporting

The premise of the work --the need to use genome-wide data to resolve potentially conflicting gene-level phylogenetic incongruency (mentioned in the abstract only)—is flawed in this study: a) there is no mention in the intro to studies that have shown inconsistency between gene and species tree involving this three taxa, (b) two of the species used are very closely related and the third is a reasonably distant relative of theirs, so with this phylogenetic pattern incongruencies between gene-species tree will be exceedingly rare, (3) the authors use only three species (no outgroup) so there is an unrooted network of three taxa not a phylogeny, and finally (d) no reference is made to the premise in the introduction despite an extensive literature on the topic of gene tree-species tree conflicts. That is prob because this premise is a bit of a strawman, in that gene-species tree issues are not a problem for three taxa with this pattern of relationships.

The phylogenetic relationship between these three taxa should be discussed in the intro, as well as the extent of nucl sequence divergence between them (all of which are well established).

English is acceptable for the most part, accession numbers for the raw data are provided and the assemblies have been deposited with CryptoDB, the std, NIH-supported, public repository for these taxa. Figures need to use larger font (especially bootstrap values).
Experimental design: I applaud the authors for the effort of building genome assemblies and calling SNVs based on assembly alignment. Especially for the analysis of rapidly evolving genes, this approach to calling seq variants can be vastly more fruitful than read mapping, where reads may not map at all when there is extensive sequence divergence between the taxa being compared. This is where I thought they were going with this study, ie, determining if the comparison of assemblies countenances them to probe the evolution of genes (or segments of genes, or intergenic regions) that are not amenable to study from read mapping due to extensive seq divergence (or the presence to read mapping inconsistencies).

Another interesting topic that can be explored from assemblies (namely with several assemblies per species) that cannot be addressed through read mapping is whether there are genes conserved and specific to C. hominis or C. meleagridis that are absent in C. parvum (the source of the reference genome to which all data was aligned).

There are several problems with the phylogenetic analysis:
-	expand on the phylogenetic methods. Namely, the name of the evolution model should be  spelled out entirely the first time around and the abbreviation used after that. Same thing for the phylogenetic method and the programs used to assess support for internal branches. Models (Transversion Model, TVM) and methods should ALL be cited (some are but not all).
-	Present table with BIC values that support the selection of TVM;
-	The analysis uses no outgroup, so the phylogeny is unrooted (if rooting on longest branch, make sure to state);
-	Most importantly: Fig 2 (phylogeny) is depicted with a scale, suggesting that branch lengths are proportional to change (also stated in the figure legend) but this not the case. What is depicted is an unscaled cladogram, with branches lengths proportional to number of branches. The (scaled) phylogram will show VERY long branches between species and nearly non-existent branches within species, since the intraspecies variation is miniscule (~2000 SNPs) compared to between species (200K – 600K variants).
-	The authors state that some of the branching patters within species are not well supported. This is expected given that these are recombining eukaryotes, ie, no single tree is expected to represent the evolutionary history of the entire genome. This should be addressed.
Validity of the findings: The only aspect of the study that is novel is the generation of the assemblies. The raw data was generated by others, the relationship between species is well established, and no incongruency is expected between gene trees and species tree for this triad of taxa, so no phylogeny is required to use the complete genome. There is the potential that the gp60-based phylogeny is incongruent with the history of the whole genome (which, in a recombining organism, is an average of the history across all positions) but recombination can easily explain that. Again , the authors did not observe such incongrency, possibly because they only have 4-10 genomes from each species, with only 1 or 2 sequenced per subtype which limits the possibility of finding such inconsistencies…
Additional comments: There are interesting questions that can be addressed with the whole genome assemblies and it would be interesting to see what the authors come up with.


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 GUT MICROBIOTA IN THE COMMON KESTREL (FALCO TINNUNCULUS): A REPORT FROM THE BEIJING RAPTOR RESCUE CENTER
Review round: 1
Reviewer: 1

Output:
Basic reporting: Guan et al. conducted an interesting and important study of Common Kestrel gut microbiome. This study will be useful for the species conservation. I recommend this paper is accepted after the revision of my comments. Moreover, I strongly suggest the author should find a native English speaker and an expert in gut microbiota to improve the language.
Experimental design: Materials & Methods
The limitation is the samples from just 1 bird.
L 91. How the fecal could reflect the healthy condition? I am confused.
L134-136. PCA, PCOA and NMDS are redundant. You just need one approach.
L137-138. Reworte.
Validity of the findings: No comment.
Additional comments: General: The abstract should be rewrote.
L 35. Change composition and structure to community and delete microbiota.
L40. Move“A total of 28 phyla, 70 classes, 183 orders, 329 families and 681 genera were detected among the gastrointestinal bacterial communities.”to the first sentence of results.
L42. Remove this sentence “Further research....”.
L43. Remove this sentence “In addition....”
Introduction.
Reworte this sentence in lines 80-81.
Discussion
1.This section should be rewrote and language should be further improved.
2. The author mainly discussed the bacterial composition of each phylum and speculated the potential function. I think the author should also compare the results with the recently published paper of birds on mBio.
3. The author collected 9 samples from one animal in different days. And of course, the author observed the bacterial difference, so why this happen? I guess this may be linked to the healthy and treatment. This should be discussed.


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 GUT MICROBIOTA IN THE COMMON KESTREL (FALCO TINNUNCULUS): A REPORT FROM THE BEIJING RAPTOR RESCUE CENTER
Review round: 1
Reviewer: 2

Output:
Basic reporting: This manuscript uses clear, unambiguous, English language, but uses certain terms quite casually without support. For example, in line 76 you reference the birds to have “special” attributes, but then do not follow up with what they are or how they are unique and thus having a different importance for microbiome studies.

Your introduction needs to start more broadly and the first paragraph should flow better instead of jumping from fact to fact when talking about kestrel natural history and biology. You should consider starting more broadly about the microbiome and then honing in on the species specific information. Expand upon the knowledge gap being filled.

In lines 72 through 76 you suggest that studies on these species have increased, however, after a preliminary literature review it appears that this is not the case. Please rephrase to state that in recent years, these species have been studied in regards to their microbiome, but that there isn’t a positive trend for each of these species.

For lines 72 through 76 consider adding Cockerham et al. 2019 and Fuirst et al. 2018 as an additional recent microbiome studies on Western and herring gulls. Especially for referencing microbial associations with urban and non-urban environments.

Please consider italicizing “Falconidae” in line 51

Where is your ICUN citation for lines 53-55

In line 80 do not use the word “elementary”

I would suggest replacing Figure 1 with a plot that shows the alpha diversity values for all metrics so that we can see the variation among samples. Rarefaction curves should supplemental material.

The text in Figure 4 appears stretched and blurry.
Experimental design: Molecular operational taxonomic units (OTUs) are often used for microbiome studies, but have been quickly shifting to using ASVs. OTUs use a 97% threshold, but you could avoid this and its ecological limitations, by using amplicon sequence variants (ASVs). Especially for a more descriptive microbiome study, using the most recent analytical methods is important. I would recommend following the protocol from Callahan et al. 2017. ASVs are a threshold-free metric and can be selected against databases using the DADA2 pipeline for example.

I do not see any mention of removing contaminants from samples or running negative controls. Was this done? If so, please state this explicitly.

In line 129 please explicitly state your rarefication depth.
Validity of the findings: The validity of these findings is quite limited due to small sample size. Please consider increasing the sample size for final publication.

The overall impact of this research is not mentioned in detail. The results are detailed and acceptable, but, there needs to be more emphasis on the significance of this study other than it being another avian microbiome study.

There should be more detail on where each of the nine birds were retrieved from and how that geographic and environmental origin could influence variation in the microbiome diversity and composition.

Analysis of this data is statistically sound, but I would highly encourage revising the analysis to use ASVs instead of OTUs. OTUs are not replicable between studies, and as a descriptive study, using ASVs would provide more detail and replicable methods for future research. See Callahan et al. 2017 as previously mentioned.
Additional comments: I commend the authors for their thorough research and expansive analyses from these samples. The manuscript is clearly outlined and written well. However, given that this is a more surface-level microbiome study, there should be much more emphasis on the importance for this. There is an overabundance of comparisons of taxonomy to other studies, but barely any link to why we need this information in the first place. Additionally, please consider revising the analysis to follow DADA2 ASVs rather than OTUs. Lastly, a descriptive microbiome study would definitely be strengthened by a larger sample size.


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 GUT MICROBIOTA IN THE COMMON KESTREL (FALCO TINNUNCULUS): A REPORT FROM THE BEIJING RAPTOR RESCUE CENTER
Review round: 1
Reviewer: 3

Output:
Basic reporting: Although there are phrasing issues to improve clarity, the manuscript is clearly written, unambiguous, and professional English without grammatical errors is present throughout. The authors appear to have written the paper carefully.

The literature cited is robust and relevant. However, the introduction and background do not show the proper context, especially for a journal with a wide-ranging audience such as PeerJ. The introduction begins by introducing the study organism, the kestrel. However, the methods of the paper are much more far reaching- the kestrel here serves to show the importance of describing gut microbiota for general conservation and husbandry purposes, as well as to illustrate effects of surgery and drugs on an animal. The authors do nothing to place the unique value of their data in this context. This results in a paper that is less novel.

The structure of the paper is fine and follows PeerJ standard format. Figures are clear and concise, although I am not sure if the authors utilize colorblind friendly palettes.
Experimental design: No experiments are performed; the paper serves to report on gut microbiota in the kestrel. The research question is defined, and a knowledge gap, notably the lack of gut microbiota data in birds despite its potential importance, is defined. However, once again I think the authors miss the context of their own study- this isn’t just reporting on a bird that was captured and sampled. It is reporting on a bird that was injured, recovered, performed surgery on, and changed its regiment from life in the wild to captivity. But none of these aspects of the bird are highlighted with a context or relevant citations. I think that the unique background of the kestrel is what makes the study special- in addition to what the authors already provide- that birds are unique animals with an “extremely special body structure.” So while a knowledge gap is identified, I think another one is out there that is very beneficial to address.

The methods on the molecular and bioinformatic work are provided with sufficient detail to replicate. However, more detail on the sample collection would improve the manuscript. The kestrel’s unique history is not clear. The reader knows that the common kestrel was “carefully treated with several surgeries and drug therapies.” The details of these events could have a major impact on the microbiome, but we don’t know anything about what happened. For example, were antibiotics a part of the treatments?

We also need to be provided information about the samples. We don’t know anything about when they were collected relative to the life history of the kestrel. Information is vague- lines 90-92: “nine fecal samples that may reflect the real health condition were collected from the common kestrel after relevant treatments on different days.” This makes it sound to the reader as if all treatments were collected after different types of treatment, with the treatments taking place during different days. However, in the discussion, a few small details about sample collection are provided, lines 209-210 say “observed mainly in several samples that were collected during surgeries or drug treatments, such as E1 and E4.” This changes things, and implies that certain samples occur during treatments, but that others did not. If so, what was the duration of time between the last treatments? What treatments were received? Were any sampled before treatments? Any after treatment on the bird was complete?
Validity of the findings: A lot of data are provided in Tables, and data are available via FigShare. The speculation and claims in the discussion are appropriate. However, as with the introduction, I think the discussion should be framed within the context the authors provide- but also- with the link to learning about the microbiota of a bird with a unique life history. This will require the use of different citations to give context to this different point.
Additional comments: Introduction:

General comment: I think that the introduction’s format should be completely redone. Instead of starting general, and narrowing down to the study at hand, this introduction starts specific, and the context of the study is only given at the very end. The power of this study is that it characterized the gut microbiota as a way to potentially inform conservation efforts and husbandry of kestrels. It shows that gut microbiota analyses are a tool, that is often overlooked, during conservation. However, the introduction doesn’t get this point across- instead it starts with kestrels. The scope and impact of this study could reach far beyond kestrels to the general application of gut microbiota analyses coupled with conservation, however this more far-reaching point is lost in the introduction. I would flip, or reverse, the section.

Line 51: PeerJ is a journal with a general audience, may wanted to specify Aves or at least Falconiformes.

Line 54: Readers are likely not familiar with China’s scheme for classifying protected species. It is worth defining what second-class protected means. I do not have a context for this definition.

Line 55: I don’t think the kestrel is a typical opportunistic forager, I’m not sure what this means. Its hovering ability is exceptional.

Line 59: avoid the use of the term “wintering” as it is difficult to understand for readers based in more tropical latitudes or the southern hemisphere. “Non-breeding” is more appropriate. Also, the distinct predatory strategies at different times of the year here are mentioned, but not described. Would be good to describe the difference between the strategies.

Line 61: urban and rural kestrels have differences- but what are they? It’s better for the reader to know the characteristics of the differences (which has higher reproductive success, hunting success?) than to simply know that the differences exist in an undefined direction.

Line 65: probably don’t need this many citations. What does “comparatively comprehensive” mean? I don’t understand what was compared here. Are all these many studies comparing rural and urban?

Line 68: what is a research hotspot? May consider rephrasing.

Line 70: can be phrased better. “With the rapid progress of sequencing techniques, reports on gut microbiota have suggested important roles in…”

Line 73: probably don’t need to pluralize these species. Also, the species could be organized phylogenetically. Are there more raptors to add to this list (could remove non-raptors so that there are not too many citations)?

Line 76: “specialized body structure” could remove the phrase “extremely special.” Also, what about birds are extremely special? This is a general audience that is being addressed and may need more concrete detail.

Line 77: Additional used twice in this sentence. Overall this sentence’s phrasing could be revamped.

Methods:

Line 87-93: Definitely needs to be addressed: for sample collection, I think it is incredibly important to know what the kestrel’s diet was in captivity. Were any samples acquired before the kestrel was fed in captivity (and so would reflect the diet of a wild bird?). What subspecies of kestrel was used in this study?

Line 93: this is more of a descriptive study so I am not sure “experiments” is the correct word.

Line 130: I don’t think this is the correct way (“Team”) to cite R.

Results:

Line 141-147: Better to not have a single sentence paragraph, all one paragraph.

Line 150: Again, better to not have a single sentence paragraph. Can merge here.

Line 154: I don’t understand why this is remarkable.

Line 163: In which samples did these genera account for the largest proportion? I think it is sensible to compare these here.

Line 169-172: I think you mean “the hierarchical clustering trees showed”

Discussion:

General: I think you should start your discussion by interpreting the results, then zoom outwards to explain the context. Here, the discussion starts with the context. It would be better to save this until the end. So, I would invert the way that both the introduction and discussion are written.

Line 187-189: Very general sentences that don’t say much. Could be supported by citations.

Line 193: limited, or unknown? If limited, need a citation here.

Line 202: first mention of cockatiels, requires Latin binomial

Line 224: better phrasing “and an ancient symbiosis may be linked to the common ancestor of amniotes” or something of that nature

Line 234: I’m not sure what this sentence means- a hospital like environment contributed to the detection of this genus?

Line 237: The word “moreover” implies a connection to the next sentence. But here, you’re switching topics to a new genus.

Line 241: First mention of turkey, storm-petrel, requires binomial

Line 245: Do you mean the “presence of Bifidobacterium is closely related to”?


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 GUT MICROBIOTA IN THE COMMON KESTREL (FALCO TINNUNCULUS): A REPORT FROM THE BEIJING RAPTOR RESCUE CENTER
Review round: 2
Reviewer: 1

Output:
Basic reporting: In my initial review, I discussed that the original manuscript contained clear, unambiguous, and professional English throughout. However, the new edited portions contain English that is written with many more issues than the original paper itself. In this way, the paper has actually regressed from its original submission. Basic mistakes (such as the use of "23th" for example) show that the edits likely did not receive the same amount of scrutiny as the original paper.

As I mentioned in my original review, the literature cited is robust.

Figures are shared properly. The paper is self-contained. It does not pose direct hypotheses as it is more of an exploratory study.
Experimental design: While the authors did incorporate my suggestion of mentioning the history of the kestrel, I do not believe that the changes are sufficient, and perhaps I was not clear about what I was asking for. The kestrel has a strange history, of being found injured and emaciated, as well as undergoing multiple treatments with different types of chemicals and surgeries. While the authors provide some anecdotally framed information about relevant samplings in the discussion, this is too challenging for the reader to piece together. I suggest mentioning these treatments clearly in the Methods, and mentioning all treatments corresponding to all sampling periods. This information would be greatly enhanced by a supplementary table detailing the sampling period, date, and relevant surgery, chemicals, or other notes (kestrel ate a mouse, for example) for that day. This would allow the reader to come to their own conclusions about the findings, rather than relying on the author’s selective interpretations in the discussion.

Additionally, the authors continue to use what I consider to be a comparatively weaker knowledge gap in the lack of knowledge about bird microbiota. I think the more interesting angle here is a medical one, regarding the kestrel's health and surgeries. However, if the editor and other peer reviewers do not find this to be a problem it could be published in the current context.
Validity of the findings: This is all fine.
Additional comments: General:

The authors may have misinterpreted my question about subspecies. Could the authors please identify the subspecies of kestrel they are observing? This is essential. I am not asking for a comparison of subspecies- just labellng the current kestrel that has been worked on.

Overall my main comment is that the care that went into the original paper is not reflected in the edits. The edited content is plagued by poor English and poor formatting (see literature cited).

Also very little context is given to the findings as a whole. The authors do a good job of explaining where specific findings fit into the field (this bacteria that we found also does this, and this, and so on) but not where their findings, in summation, fit into the field. Placing emphasis on this in the beginning of the discussion would be good. Again, I think that this study being performed on a captive bird that underwent surgery is a good place to start.

Line edits corresponding to the numbering system in the "track changes" version of the document.

Line 33:
Replace “extremely special” to “specialized”

Line 38:
Eliminate “in this study”

Line 41:
Eliminate “could also”

Line 48:
“Research” not “researches”

Line 48:
First sentence of new intro is a run on sentence, could break into two sentences

Line 53:
“Animals” plural. “has gradually become a” This sentence is not using proper English.

Line 70-72:
Re-read reviewer 2’s comment regarding this sentence. It still insinuates that studies on the species themselves have increased, not on birds as a whole. It still needs to be rephrased.

Line 72:
“With regard.”

Line 73:
Avoid casual abbreviations in scientific writing: “isn’t.” What does “positive trend” mean? Confusing.

Line 74:
You changed this sentence here, now change it in the Abstract.

Line 75:
This sentence is also not using proper English and needs to be edited. Also, not all birds are warmer than ambient temperature, for example in extremely hot desert environments.

Line 75-81:
I appreciate the additional information but this section is using poor English. I can’t put in the time to correct all of the English, the authors need to consult someone.

Line 86:
Should “BirdLife International” citation include a period in it?

Line 108:
Eliminate “the” in “the common raptors”

Line 109:
Eliminate “the” in “the food chains.” Eliminate the in “the common kestrels.” Is the kestrel a top predator? Presumably other larger raptors can prey on it.

Line 118:
“the injured common kestrel could not fly and was found…” Eliminate “first.” First is implied with “found”

Line 228:
Should “R Core Team” citation include a period in it?

Line 239-241:
Probably a mistake that could be eliminated.

Line 247:
“Are shown” as opposed to “were shown”

Line 282:
May want to relist these results with the smallest percentage first- it does the best job of illustrating your point, so may want to put it up front.

Line 304:
Eliminate “and depicted in”

Line 307:
“the variation” Eliminate “the”

Line 308:
Eliminate “in this case” - it is implied.

Line 312:
Eliminate “the”

Line 314:
“continues to be” Eliminate “was”

Line 318:
“were” past tense, because you have now characterized it.

Line 324:
Can probably just say “birds” here

Line 349:
“23rd” “June, the day after”

Line 350:
“22nd” June. This document needs to be reviewed by a native English speaker.
Line 351:
Unclear, revise English

Line 355:
“combined with”

Line 369:
Was “only” slightly lower. Otherwise using “although” in this sentence does not makes sense.

Line 374:
“Carbohydrates, sugar, and even by digesting fiber in some species”

Line 379:
Eliminate “special” ?

Line 382-390:
Again, information is appreciated but I don’t have time (or think it is my job) to make every English correction. Someone else needs to review the manuscript generally, but especially these new edited sections.

Line 393:
Don’t need to specify that Clostridium difficile belongs to Clostridium

Line 393-394:
Again, English issues.

Line 397:
“Remarkably.” Sentence is vague, need to back up with some numbers

Line 398:
Eliminate “the” in “the surgery” “While E5 was collected”

Line 414:
This is indicating relative abundance correct?

Line 419:
Do you mean “notably”?

Line 430-432:
Again, poor English. “Although the kestrel underwent surgery on 4th July, the reason for the high proportion…”

Literature cited:

There is inconsistent italicization and species capitalization of Falco tinnunculus in the literature cited.

I am not sure that the BirdLife International citation is formatted correctly, as I do not think a reader could retrieve information based on this citation.

The new citations are added very sloppily, without proper page numbers, often including URL links, and are inconsistent.


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 AND IN VIVO HYPOLIPIDEMIC PROPERTIES OF THE AQUEOUS EXTRACT OF SPIRULINA PLATENSIS, CULTIVATED IN COLORED FLASKS UNDER ARTIFICIAL ILLUMINATION
Review round: 1
Reviewer: 1

Output:
Basic reporting: The objective of the work may clearly be stated at the end of the introduction.
The detailed methodology of the work may be presented in a Flowchart form
The literature review may include previous works on similar topic .The following may be useful .
“Spirulina – From growth to nutritional product: A review,” Trends Food Sci. Technol., vol. 69, 2017, doi: 10.1016/j.tifs.2017.09.010.
Comparative study on the growth performance of Spirulina platensis on modifying culture media,” Energy Reports, vol. 5, 2019, doi: 10.1016/j.egyr.2019.02.009.
Experimental design: Line 122 justify the absorbance measurements at three different wavelengths 663, 645 and 480 nm.and its physical significance.
Indicate unit g /L instead of mg/mL
Provide the equation for the growth rate of spirulina.
Page 35: Indicate results discussed either in tabulated form on graphically to understand effect of each process variable. A comparison of the data with literature should also be done.Comment on mechanism of Spirulina cultivation with time.
What is the unit of SP growth rate, explain and check units.
Author should provide quantitative data for growth rate and biomass content at different cultivation conditions.
Compare the results obtained with published literature and discuss.
There is more variation in SP growth rate (Table 3) with colorless than color . Explain
Page 97: What information can be derived from GC-MS. This has to be discussed. What is typical chemical composition of algae and what could be the possible application other than the work reported in this study .
Validity of the findings: What are the contribution of work towards global community and audience future deployment of spirulina cultivation
The conclusions need to be re-written in point by point form with key numbers obtained from the study. Conclusions seems to be weak.It should be extended .
No reports are available on cultivation of blue-green algae Spirulina platensis in
399 Erlenmeyer flasks of different color under artificial illumination.Justify.
Similarly future scope should be focused.
Additional comments: The paper titled “In vitro and in vivo hypolipidemic properties of the aqueous extract of Spirulina platensis, cultivated in colored flasks under artificial illumination” is very interesting and within the scope of the journal.The above suggestions are provided to improve the quality of the manuscript.


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

Input:
Title: IN VITRO AND IN VIVO HYPOLIPIDEMIC PROPERTIES OF THE AQUEOUS EXTRACT OF SPIRULINA PLATENSIS, CULTIVATED IN COLORED FLASKS UNDER ARTIFICIAL ILLUMINATION
Review round: 1
Reviewer: 2

Output:
Basic reporting: Lack of literature findings
Experimental design: Investigation is to be improved by varying other parameters inaddition to the coloured flask variation
Validity of the findings: Conclusion is not well defined and key findings are not represented. Sprirulina is one of the richest protein sources in microalgae. How much protein content in dry weight it would contain.
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 AND IN VIVO HYPOLIPIDEMIC PROPERTIES OF THE AQUEOUS EXTRACT OF SPIRULINA PLATENSIS, CULTIVATED IN COLORED FLASKS UNDER ARTIFICIAL ILLUMINATION
Review round: 1
Reviewer: 3

Output:
Basic reporting: The work of Al-Saman et al., entitled “In vitro and in vivo hypolipidemic properties of the aqueous extract of Spirulina platensis, cultivated in colored flasks under artificial illumination” examined the effect of hot water extract of S. platensis grown in colored glass bottles on cholesterol levels reduction in vitro and in vivo. In general the manuscript is well prepared and outlined. However, some points must be revised for a better understanding of the study. The considerations follow below:

Abstract
1.	Add the aim of the study to the summary.
2.	There is a lot of information missing from the methods section. For example, what was the biological model used? What are is the characteristics of the model (rats, mice..)? What route of administration was used? What dose administered and for how long?
3.	It is missing a conclusion in the summary.
Introduction
1.	In the first paragraph of the introduction (line 56-57), please remove the following sentence "Cholesterol can be both good and bad."
2.	The references used to support spirulina are very old, especially those related to composition, such as Bujard et al., 1970; Habib et al., 2008. Are there no more current references?
3.	The purpose of the work should be made clearer.
Experimental design: Materials and methods
1.	What was the final concentration used for the in vitro anti-cholesterol assay test?
2.	Why did they use two-week-old young mice, not adult mice?
3.	The concentration of 15 mg / mL was based on what?
4.	Please reference the protocol methodology in vivo.
5.	Why, when combined, atorvastatin and spirulina had their dose reduced?
6.	How is the glucose level of the animals?
Validity of the findings: Results and discussion
1.	Have any in vitro cytotoxicity before been performed for further in vivo studies?
2.	In the "In vitro cholesterol-lowering activity of Spirulina platensis cultivated in colored flasks" section, in the last paragraph. Please explain how the antioxidant capacity decreases serum lipid levels.
3.	The discussion about the influence of the extract on the lipid profile must be deepened.
4.	In the description of body weight results, please add the value of p.
5.	In order to better understand the cardioprotective role of the extract, if possible, it would be extremely important to investigate the activity of paraoxonase-1, since it plays an important antioxidant role associated with HDL. Otherwise, the addition of a possible mechanism of action could be added in the discussion.
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: IN VITRO AND IN VIVO HYPOLIPIDEMIC PROPERTIES OF THE AQUEOUS EXTRACT OF SPIRULINA PLATENSIS, CULTIVATED IN COLORED FLASKS UNDER ARTIFICIAL ILLUMINATION
Review round: 1
Reviewer: 4

Output:
Basic reporting: •	Does Spirulina platensis grow in the country of the authors ? is it harvested in some farms ?
•	The english should be improved
•	Why are the references Ishikawa and Jeffrey not in bold ?
•	We did not understand well the use of letter a, b, c,d representing the statistical difference. It should be well explain.
Experimental design: •	Why the authors did the culture in flask ? we think that it is important to tell us what are the challenge with the culture of Spirulina platensis in their country. It is harvested elsewhere.
•	They did not test  the presence of the major pigment of Spirulina platensis, phycocyanin
•	Line 129 : please do not begin the sentence with a number. See how it is done in line 132
•	Line 142 : powder or power
•	Line 156 : what is EI mass spectra ?
•	Line 171 : and each group containing 8 mice
•	Line 179 : ‘’of 1 g cholesterol for each 1 kg diet plus from S. platensis’’ . It seems to be a non sense
•	Line 180 : why not the same concentration of spirulina and atorvastatine  like in other group. The concentration of Spirulina and atorvastatine are divides by 2
•	Line 187 : Where the test tube dry ? or with any anticoagulant ?
Validity of the findings: •	Lines 365 to 367 : what does that means ? toxic renal effects are only revealed by the dosage of urea ?
•	Line 392 : is it spirulina 7.5 mg/ml ? or 75 mg/kg ?
•	Does the presence of proeminent Kupffer celles, edema in the liver means inflammation ?
•	In the heart, what the congestion of coronary vessels suggest ?
Additional comments: •	We propose to harmonise the spelling of chlorophyll a : See line 38 and line 42
•	Line 56 : cholesterol plays an essentiel role. Not the essential role
•	Is cholesterol only essential for heart ? We suggest to put 2 or 3 roles of cholesterol
•	Line 60 : major cause not chief cause
•	The english will have to be improved . Ex : lines 66, 67, 71,72,73,74. The traduction is not well done
•	Line 76 : this allowed… not led
•	Line 82 : teh reference are too old. There are new references in Africa
•	Line 90 : there a in vivo and in vitro studies done in Africa on the hypolipidemic effects of Spirulina platensis


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 AND IN VIVO HYPOLIPIDEMIC PROPERTIES OF THE AQUEOUS EXTRACT OF SPIRULINA PLATENSIS, CULTIVATED IN COLORED FLASKS UNDER ARTIFICIAL ILLUMINATION
Review round: 2
Reviewer: 1

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


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

Input:
Title: IN VITRO AND IN VIVO HYPOLIPIDEMIC PROPERTIES OF THE AQUEOUS EXTRACT OF SPIRULINA PLATENSIS, CULTIVATED IN COLORED FLASKS UNDER ARTIFICIAL ILLUMINATION
Review round: 2
Reviewer: 2

Output:
Basic reporting: Nothing to say
Experimental design: Nothing to say
Validity of the findings: Line 368 : what does that means ? toxic renal effects are only revealed by the dosage of urea ?
Here is the response of the authors : Mice were not treated with urea; but the toxic renal effects associated with increased serum urea and creatinine levels

Please add creatinine in the sentence . We want to let them know that evaluation kidney function is explored by the two, urea and creatinine, and mainly by the glomerular filtration rate as recommended by kidney societies
Additional comments: Nothing to say


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: EFFECTS OF PLASTIC MULCHING ON THE ACCUMULATION AND DISTRIBUTION OF MACRO AND MICRO PLASTICS IN SOILS OF TWO FARMING SYSTEMS IN NORTHWEST CHINA
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: The method of MiPs separation is not correct at all. The flotation separation method used in this paper comes from "zhang, S., Yang, X., Gertsen, H., Peters, P., Salanki, T., Geissen, V., 2018. A simple method for the extraction and identification of light density microplastics from soil. Sci Total Environ 616-617, 1056-1065". In fact, the method given in this paper is not to separate plastic film, but plastic particles or fibres. The tiny plastic film in the soil is always wrapped with soil particles, and it is impossible for distilled water to float them out，and at least, saturated salt solution must be used for flotation agent. It is possible that the use of incorrect flotation solution resulted in large deviation in MIPS data and no MiPs  found in most of those samples. So, based on the uncertainty of experimental method, MiPs data is not credible.
Validity of the findings: this is a great topic
Additional comments: The manuscript "Effect plastic mulching on the accumulation and distribution of macro and micro plastics in soil - A case study of two farming systems in Northwest China" investigated the accumulation of plastics in the agricultural soils. For now, most of the studies have estimated the occurrence of microplastics in aquatic environments. Very few studies have talked about the terrestrial environments. From this point, this manuscript has some novelty. However, the modification of the writing English is highly needed. The logic of the results was not clear. Why the accumulation and distribution were shown separately? It will be better to organize one section for macroplastics, and one section for microplastics. Additionally, this no information about the amount of microplastics in abstract. Also, there is no need to tell the categorization of macroplastics in the abstract. Overall, this is a great topic, and the authors should organize this manuscript better before it can be accepted.

There was no line numbers in this manuscript.

Title
There is no need to emphasize that this work is a case study.

Abstract—it will be better to re-write the abstract
The grammar of the first sentence was quite strange, please modify this sentence.

From the first 0-30 cm? from the top

Introduction
Modify the first sentence.

Modify "Plastic mulch proves" to previous studies proved or it has been proved that

Using plastic mulch ----plastic mulching

There is a growing concern

Not only these studies have reported the accumulation of plastic debris in the farming lands. For instance, Huang et al. 2020, Environmental Pollution "Agricultural plastic mulching as a source of microplastics in the terrestrial environment"

There is no clear hypothesis in the introduction. That may be the potential reason that this manuscript looks confusion. The author should talk about the farming practices such as the level of mechanization, mulching pattern, significantly influence the profiles of plastic residues in the soil.

It is very impressive that the authors showed the details of study area.

For the identification of microplastics, any references? If the authors developed this method, the limit of detection and the recovery should be added.

The author said it was the wind dispersed agricultural plastic debris to other environments. This reason seems not very robust. More discussion is needed.

It will be better if the author can give the FTIR 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: EFFECTS OF PLASTIC MULCHING ON THE ACCUMULATION AND DISTRIBUTION OF MACRO AND MICRO PLASTICS IN SOILS OF TWO FARMING SYSTEMS IN NORTHWEST CHINA
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript is well written and suitable for their publication. It covers an interesting topic about macro- and microplastics in agricultural soils, one of the primary sources of plastics in terrestrial ecosystems. However, in my opinion, the manuscript needs several improvements (main attention for question 12, 12.1 and 13).

1. I like the supplementary data file. It's very good. However, in my opinion, Table 1 should be as Table S1 (Supplementary data), and Tables S2, 4, 6 and 8 should be moved to the manuscript and not as Supplementary data. Besides, I saw that Table S2 and Table S6 is the same information that is written in Figures 3 and 5. Is it right? I suggest to avoid duplicate information. In my opinion, it should be more useful to left the tables for comparison studies, and delete the figures. Besides, Figure S1 should be added to Figure 2 (merged in the same figure).
Experimental design: 2. L96-101 I don't understand the differentiation between <2 and > 0.25 cm2, because Microplastics are also called to particles less than 5 mm, and since some years ago, MiPs are called to particles less than 1 mm and not 2 mm. I understand that < 2 mm was chosen due to particle soil size, but in my opinion, these questions should be clarified and improved. Also, a density of less than 1 g cm3 was chosen by authors, why? Which criteria was followed?

3. L105. Which farming system? Greenhouses? Open cultures? Which cultivation (Strawberry?)? These sentences (and also 109-110 lines) should be improved, or even give some pictures in the manuscript or Supplementary data. This information is given in lines 121... and for this reason, I suggest to improve this section. For example, lines 121- should be moved to 108-. It should be more readable, give first general information and after give information for each soil.

4. L124-127. I don't understand why plastic mulch is used for maize crop. Is Early sweet corn that needs plastic mulch to improve their grown? https://doi.org/10.21273/HORTSCI14667-19 https://lib.dr.iastate.edu/farms_reports/521/ Besides, how is removed the plastic mulch? Mechanically? Handmade? By wind?

5. L150. "MaPs were manually collected using a quadrat sampling method. For each sampling field, we randomly selected three quadrats 100 cm long, 50 cm wide and 30 cm deep which covered two crop rows (Fig. S1)" Which area was sampled? 1 Ha? How many samples by each study area? Improve this section. I suggest add a figure for the manuscript or to the Supplementary Information.

6. L153. I think that bag showed in Fig S2 is a PP mesh bag with probable plastic contamination to samples. Besides, why the samples were not sieved directly in the field?

7. L162-163. This method is new, or authors are following a published methodology? I suggest add also a citation as indicated by Line 199

8. L195. "polypropylene (PP) plastic bags." Figure S2b, 2c?

9. "plastic-free room" How? Please, explain this.

10. "A control field where no plastic mulch had ever been used was selected from each study site." Microplastics can be carried by wind, car tyres (agricultural machinery as a source of soil contamination), or even by fertilizers. I suggest improving this explanation.

11. Keywords: I suggest to add the plastic polymer (low-density polyethene) and not macro- and micro-plastics because they are in the title.
Validity of the findings: 12. I like the proposed method for microplastics extraction due to it's simple and very easy to use for non-resources laboratories. However, some authors also proposed to add Fenton Reagent or KOH for organic matter digestion. Considering that these are agricultural soils, and the organic matter content must be higher than 2%, why was no further digestion of organic matter added? See https://doi.org/10.1021/acs.est.9b04618 or https://doi.org/10.1021/acs.est.8b01517 Unnecessary? I suggest to improve this information. Besides; some authors also make a plastic identification by FTIR. Why was not used in this plastic identification and only by optical microscopy? Unnecessary?

12.1. I understand (lines 387-401) that authors do not have the facilities for organic matter digestion or high-density plastic extraction (only plastic with density less than 1 g cm3 were extracted). I suggest to add this information in the material and methods to show the limitations of the present study because this is a significant limitation of the present study. No more source of plastics in these sources.

I suggest to improve this question in the material and methods, or even in the introduction (e.g. line 100), to justify why high-density plastics were not extracted.

13. I did not see in the results and discussion information about the shapes (or colour) of Macro and microplastics. All plastic items were found as fibres or undefined shapes? If possible, please add this information.
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: EFFECTS OF PLASTIC MULCHING ON THE ACCUMULATION AND DISTRIBUTION OF MACRO AND MICRO PLASTICS IN SOILS OF TWO FARMING SYSTEMS IN NORTHWEST CHINA
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have responsed all the comments.
Experimental design: The authors have responsed all the comments.
Validity of the findings: The authors have responsed all the comments.
Additional comments: This manuscript can be published now.


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

Input:
Title: EFFECTS OF PLASTIC MULCHING ON THE ACCUMULATION AND DISTRIBUTION OF MACRO AND MICRO PLASTICS IN SOILS OF TWO FARMING SYSTEMS IN NORTHWEST CHINA
Review round: 2
Reviewer: 2

Output:
Basic reporting: The submitted paper has been improved since last submission. Now, several sections are more clear and easy to read. However, i have my doubts regarding to extraction method, although this question was clarified and improved.

I think that all questions were addressed.
Experimental design: No comment.
Validity of the findings: I have my doubts regarding to extraction method, although this question was clarified and improved. However, I think that all questions were addressed. The paper is interesting.
Additional comments: The submitted paper has been improved since last submission. Now, several sections are more clear and easy to read. However, i have my doubts regarding to extraction method, although this question was clarified and improved.

I think that all questions were addressed.


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: EXPRESSION AND PHYLOGENY OF MULTIDRUG RESISTANCE PROTEIN 2 AND 4 IN AFRICAN WHITE BACKED VULTURE (GYPS AFRICANUS)
Review round: 1
Reviewer: 1

Output:
Basic reporting: This manuscript is interesting and well prepared.
Experimental design: In this study, only 2 vulture samples were collected. One was used for NGS analysis, and another was used for PCR and Sanger sequencing. Could the other add more samples for both NGS and Sanger sequencing to make the data more valid? Alignment of the sequences from both analyses will give the consensus information of the MRPs’ genetic background in vultures.
Validity of the findings: To predict the molecular structure and function of MRPs, the in silico analysis was used in this study. Could the authors give discussion on the molecular functional study of MRPs using other approaches (for example the molecular cloning study, and the gene or protein manipulation)? Advantages and disadvantages of the analyses should be discussed.
Additional comments: No additional comments


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

Input:
Title: EXPRESSION AND PHYLOGENY OF MULTIDRUG RESISTANCE PROTEIN 2 AND 4 IN AFRICAN WHITE BACKED VULTURE (GYPS AFRICANUS)
Review round: 1
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The manuscript entitled “Expression and phylogeny of multidrug resistance protein 2 and 4 in African white backed vulture (Gyps africanus)” is quite well structured and written. The authors have addressed each of my concerns and I am happy to recommend acceptance of this manuscript in its present form with just some corrections required.


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: EXPRESSION AND PHYLOGENY OF MULTIDRUG RESISTANCE PROTEIN 2 AND 4 IN AFRICAN WHITE BACKED VULTURE (GYPS AFRICANUS)
Review round: 2
Reviewer: 1

Output:
Basic reporting: n/a
Experimental design: n/a
Validity of the findings: Since the prediction of the molecular structure and function of MRPs in this study is based on only 2 vulture samples and relied on the in silico analysis, the reviewer suggests that the authors should give discussion on the molecular functional study of MRPs using other approaches (for example the molecular cloning study, and the gene or protein manipulation). Advantages and disadvantages of the analyses should be discussed comparatively.
The discussion detail will clarify the reliability and the bias issue of the results in this study.
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: EXPRESSION AND PHYLOGENY OF MULTIDRUG RESISTANCE PROTEIN 2 AND 4 IN AFRICAN WHITE BACKED VULTURE (GYPS AFRICANUS)
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The Authors have successfully addressed all reviewer suggestions.


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: COMPREHENSIVE PATHWAY-RELATED GENES SIGNATURE FOR PROGNOSIS AND RECURRENCE OF OVARIAN CANCER
Review round: 1
Reviewer: 1

Output:
Basic reporting: NA
Experimental design: NA
Validity of the findings: NA
Additional comments: In this manuscript “Comprehensive pathway-related genes signature for prognosis and recurrence of ovarian cancer “ Zhao et al. have  analyzed the gene expression profiles of ovarian cancer patients to develop a gene signature of overall survival and recurrence free survival using regression analysis. Although manuscript is written well but authors need to perform more rigorous data analysis to support their findings. Following are some of the comments that need to be addressed:

1.	Figure 1: What does size on x- axis indicates? Are these p-values FDR corrected? How these barplots sorted ? Do authors find similar enriched pathways in TCGA data also?

2.	What is the significance of using LASSO over ELasticNet which can also handle coexpression patterns of genes more efficiently.

3.	Are risk scores significantly different in alive and dead patients? A boxplot representation of risk score in these two categories and statistically calculating the pvalue of significance can clearly show this instead of a scatter plot representation, which could be misleading.

4.	What does authors want to represent in heatmap of Figure 3? Expression levels of genes shown in figure are not different in low and high risk groups. For a better representation gene expression should be normalized across the patient data.

5.	How does cutoff between low and high risk identified? What is the overlap of genes in OS and RFS.

6.	Figure 4C –D lacks legend. What does these color represent? It is not clear why red lines (risk scores) have so much variations in FPR and TPR values?


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: COMPREHENSIVE PATHWAY-RELATED GENES SIGNATURE FOR PROGNOSIS AND RECURRENCE OF OVARIAN CANCER
Review round: 1
Reviewer: 2

Output:
Basic reporting: The article is written in a professional article structure. However, it contains unclear sentences due to grammatical errors, lack of formal language and precision, and concepts could be further explained. I would suggest that the authors review the manuscripts to provide precise examples and professional language.

Line 42 is missing a space before "(": 2020 cancer statistics (Siegel & Miller 2020)
Line 42-44: the sentence structure is unclear
Line 46 OC is still a highly malignant tumor which threat women's lives: should be rewritten in a professional manner, OC is a highly malignant tumor, "which threatens women's lives" should either be replaced with mortality statistics
Line 51: it is unclear why the current methods are unsatisfactory, no performance values arre provided
Line 54 identified some genes: "some" should be removed, either state the number of genes or just use examples
Line 57 "And": replace with a better connector such as furthermore
Line 65 "In this study, we were committed to explore": we explored (or investigated)
Line 66 "and attempted to identify the potential prognostic": say what you identified, remove "attempted".
Line 69, 70, and 72 say "both of the two": say "both"
Line 71 "Besides": use "In addition"
Line 120-122: fix the grammar and sentence structure as it is unclear
Line 122 "there are a lot of": state how many
Line 124-125 "and so on": either state them all or say "such as"
Line 128: it is not stated how many genes were tested
Line 151 "9-gene signature was a helpful overall survival's indicator of OV": this is not professional language and OV has not been defined
Line 164 "age, grade and so on": use professional language
Line 197 "studies has": studies have
Line colors used in the plots of figure 2 and 4 are not explained in the figure legend.
Figure 3 should show the color legend in risk score plots for A and B.
Figure 4 and 5 should show the hazard ratio and its 95% confidence interval
Experimental design: In this manuscript the authors use public available datasets to identify genes with prognostic value and tested their model. The research question is well presented. However, there are sections that should require other analysis.

Section 2.1: detailed clinical and demographic information is only provided for the TCGA samples, this information should be provided for all samples in order to asses the data that has been used in the study.

Section 2.3 and 2.6: it is not specified where the models were performed, was it R or other software?

Section 3.1: Why were only hallmark genes used for this study? Is it expected that only hallmark genes will have prognostic value? Further, the author should correct their p-values by multiple hypothesis testing.

Section 3.2: The correlation method and correlation threshold are not specified. Therefore, it is not possible to asses the quality of the genes for OS and RFS.
Line 132-133, it is not defined how this gene sets were defined.
Mean or median values should be provided for OS and RFS
Hazard ratios and their 95% confidence intervals should be shown in line 145.

Section 3.3
The authors use only one external dataset as validation for their prognostic markers. If other datasets are used to create the gene sets, are the same gene sets found? The authors should perform cross validation for their analysis to prove the consistency of their signal. Therefore the suggested experiments is to use a cross-validation using each of the datasets as validation while the others to identify the gene sets.

Section 3.5
Clinical and demographics data was only provided for one dataset. Was this the only dataset used for this analysis? In addition, identified race as a cofounder of the results should concern the authors. As race could imply socioeconomic differences in the patients and ultimately the difference in OS could be driven by these factors instead of the gene signatures.

In addition, the prognostic model in this study was not compared to other prognostics models on the same validation dataset. There we can't asses the quality, validity and improvement of this new prognostic method.
Validity of the findings: The conclusions of this paper are not supported by the results provided in the manuscript. The authors should use the cross-validation to test the robustness of their gene set and validation results. In addition, the correlation of race with OS and RFS is concerning, which may explain the difference in this parameters instead of the gene sets used. Further, the authors can't conclude that their model may be a superior prognostic indicator for OS and RFS if it is not compared to the other methods.
Additional comments: No further 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: COMPREHENSIVE PATHWAY-RELATED GENES SIGNATURE FOR PROGNOSIS AND RECURRENCE OF OVARIAN CANCER
Review round: 1
Reviewer: 3

Output:
Basic reporting: The authors present a bioinformatic analysis for identifying genes signature for prognosis and recurrence of ovarian cancer. This is a well-known study and the authors proposed a new gene signature on it. Since the idea is of interest, there are major points that need to be addressed.

The authors should improve the English language usage in the paper, there contain many grammatical errors or typos such as:
- ... prognostic value in an independent GEO datasets.
- ... was the fifth major causes of mortality of women ...
- Despite the moderately improvement has been made in diagnosis and treatment of ...
- ...
They should re-check the errors and consider rewriting the content of the article in concise language.
Experimental design: The authors should provide detail on the data that they used (number of data, how many data for training, validation, ...).

Did the authors consider the inspection and removal of batch effects when merging different datasets from GEO?

Source codes should be provided for reproducing the results.

ROC curve & AUC were used in previous works in biomedical such as PMID: 31921391, PMID: 32613242, and PMID: 31362508. Therefore, the authors should refer more works in this description to attract broader readership.

Why did the authors use different datasets for different analyses, i.e., GEO in signaling pathways and TCGA in gene signature? In my opinion, the authors could use one set for generating the signatures and the other for validation.
Validity of the findings: Why was the performance from ROC curve analysis chaotic and inconsistent?

The authors should compare their gene signature with the previous works on ovarian cancer.

For this kind of problem, the authors should perform more analyses related to GO enrichment analysis and transcription factor analysis.
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: COMPREHENSIVE PATHWAY-RELATED GENES SIGNATURE FOR PROGNOSIS AND RECURRENCE OF OVARIAN CANCER
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors responded to my previous comments properly.
Experimental design: The authors responded to my previous comments properly.
Validity of the findings: Section 3.4 name is confusing. Also, include some metric value in the text to show how much better this new signature is
Section 3.6: is it independent considering the risk score association with other characteristics? The authors should add a socioeconomic part in their discussion. It is important to show the possible effect of race given that the study is retrospective and race could bias/cofound effects
Additional comments: No comments


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

Input:
Title: COMPREHENSIVE PATHWAY-RELATED GENES SIGNATURE FOR PROGNOSIS AND RECURRENCE OF OVARIAN CANCER
Review round: 2
Reviewer: 2

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


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: SALIVARY MICROBIAL CHANGES DURING THE FIRST 6 MONTHS OF ORTHODONTIC TREATMENT
Review round: 1
Reviewer: 1

Output:
Basic reporting: A.	Text
The English language should be improved, with more professional terms. Some examples include:
“Many studies revealed that placement of orthodontic appliances influenced cleansing of the oral cavity, thus inducing the accumulation of dental plaque (Lee et al. 2005; van Gastel et al. 2008).”
“The etiology and progression of caries or periodontitis are closely related to the oral microbiome rather than a single or several species. “(What does it mean?)
“Our results indicated that microbial richness (gender, species or levels?)

B.	Intro & background
i. Text: “Other evidence also showed that improper orthodontic force could alter the oral microbial ecosystem and increase the potential for pathogenicity (Ong & Wang 2002).”
My question: Is this topic actually reported by literature in this way? Please check the reference.
ii. In concern about the importance of saliva in diagnosis of microbial environment, the reference citations are not adequate.
Text: “Saliva, which is regarded as a microbial repository and transport medium, is also widely used in dental research in addition to dental plaque. Various studies indicated that saliva was affected by oral health status as well as the quantity and types of microbes during orthodontic treatment”.
My question: In the references, there are only HolgersonL et al. 2020. Jing analysed the salivary levels of S. mutans and Lactobacillus, and Zhao et al 2019 analysed in aligner appliance, what should not be compared to brackets. I think it is necessary comparison with researches that was analysed interaction among microorganisms in the saliva.
iii. The aim is not clear:
Text: “Salivary microbial communities were characterized and compared to better understand microbial changes during the early stage of orthodontic treatment.”
My question: what does “microbial communities” mean? Phyla, gender, species? Or group or complex.
Experimental design: i.    The research is within scope of the journal.
ii. The question is relevant, but it is not clear: “Salivary microbial communities were characterized and compared to better understand microbial changes during the early stage of orthodontic treatment”
My question: Did they compare phyla, genders, species? Did the sequencing also evaluate species? The results did not show this clearly. And it is important to support the topics addressed in discussion.
iii. Description of methodology is based, clearly, in the literature and, then, is correct. But some aspects of the malocclusion sample are also very relevant, like crowing, crossbite.
Saliva sample: Time that saliva was collected. The aliquot utilized in the analysis. What kind of buffer solution was utilized?
I think it is necessary to describe this aspect.
Validity of the findings: i. The results are inconclusive.
ii. The statistic is adequate.
iii. The figure 4 is inconclusive.
iv. The results showed data about phyla and genera. The discussion addresses specific concerns about dental caries and periodontal disease, in which some species of bacteria are not investigated. And attention: the salivary levels and in situ levels are different, and the comparison with the literature should be considered this aspect.
Therefore, the discussion could not compare the specific results of this research with those literature results pointed by the authors, specially about aligners appliance.
Additional comments: My conclusion: within the points considered, I think this study is relevant and necessary. The orthodontic research should be pointing out more information about microbial interaction in orthodontic treatment. The study was well planned and done. Some adjustments are required to be coherent with the proposal, results and discussion. The major adjustments are necessary to be published.


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: SALIVARY MICROBIAL CHANGES DURING THE FIRST 6 MONTHS OF ORTHODONTIC TREATMENT
Review round: 1
Reviewer: 2

Output:
Basic reporting: In this study, the authors conducted to better understand on orthodontic patients’ salivary microbial changes over a 6-month period orthodontic treatment.
Experimental design: 1. Could authors supple the reasons why 6-month period was chosen for observation?

2. The number of participant patients involved in this study was 19 which was not big enough.

3. In method part, hypervariable region V4 of the bacterial 16S rRNA gene was PCR amplified with the specific premiers. Could authors explain how these specific premiers could be applied for various bacterial in salivary samples?

4. In method part, “Rarefaction curves, α diversity (Observed species, Chao1, ACE), β diversity (Bray-Curtis distance), and OTU relative abundance were calculated by QIIME”. Could authors add some brief introductions on each index?
Validity of the findings: 1. In discussion part, authors analyzed and compared the alternation of the community structure in different term. However little reasons on how contributing those changes was indicated. Please list some reasonable information.

2. From you results, what clinical significance could be inferred? Please add this information on your discussion part.
Additional comments: In this study, the authors conducted to better understand on orthodontic patients’ salivary microbial changes over a 6-month period orthodontic treatment.

1. Could authors supple the reasons why 6-month period was chosen for observation?

2. The number of participant patients involved in this study was 19 which was not big enough.

3. In method part, hypervariable region V4 of the bacterial 16S rRNA gene was PCR amplified with the specific premiers. Could authors explain how these specific premiers could be applied for various bacterial in salivary samples?

4. In method part, “Rarefaction curves, α diversity (Observed species, Chao1, ACE), β diversity (Bray-Curtis distance), and OTU relative abundance were calculated by QIIME”. Could authors add some brief introductions on each index?

5. In discussion part, authors analyzed and compared the alternation of the community structure in different term. However little reasons on how contributing those changes was indicated. Please list some reasonable information.

6. From you results, what clinical significance could be inferred? Please add this information on your discussion part.


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: SALIVARY MICROBIAL CHANGES DURING THE FIRST 6 MONTHS OF ORTHODONTIC TREATMENT
Review round: 2
Reviewer: 1

Output:
Basic reporting: The English language was improved.

i. "Other evidence also showed that improper orthodontic force could alter the oral microbial ecosystem".
This sentence was deleted.

ii. Concern about the importance of saliva in diagnosis of microbial environment the reference citations are not adequate. “Saliva, which is regarded as a microbial repository and transport medium, is also widely used in dental research in addition to dental plaque. Various studies indicated that saliva was affected by oral health status as well as the quantity and types of microbes during orthodontic treatment”. In the references there are only HolgersonL et al. 2020. Jing analysed the salivary levels in orthodontic patients, and Zhao et al 2019 in aligner appliance that should not comparing with brackets.
They modified this section and added more references focusing on studies of salivary microbial changes following fixed appliance installation.

iii. The aim is not clear: “Salivary microbial communities were characterized and compared to better understand microbial changes during the early stage of orthodontic treatment.” What means “microbial communities “
This question was clarified.
Experimental design: The questions were answered and the text was adapted.
Validity of the findings: The findings were validated.
Additional comments: The requests were met, and the limitations discussed. At that moment, I think it should be accepted 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: ASSESSING ANXIETY, DEPRESSION AND QUALITY OF LIFE IN PATIENTS WITH PERIPHERAL FACIAL PALSY: A SYSTEMATIC REVIEW
Review round: 1
Reviewer: 1

Output:
Basic reporting: English: needs some minor revision, to involve a native speaker would be helpful.
Literature: In the Introduction, old/outtaken literature is cited, there is newer epidemiological data. Reading the introduction, I get the impression, that the authors themselves do not treat PFP patients. Help from a physician doing so, would help to improve the introduction.

The reviews was focused on depression and anxiety, many psychological variables were not analyzed. This should become clearer in title/abstract/text.

Raw data was not shared, but is not imported for such a narrative systematic review.
Experimental design: The results/conclusions are nor surprising.
A reader knowing the field knows that mainly simple cross-sectional studies on the topic are available and actually no relevant comparing group studies are published.
Hence, the result of such a systematic review is clear before analysis:
Quality of the data is very poor and limited.

And see 1: This narrative review is only about anxiety and depression. Many aspects of the psychological impairment of the patients were not included in the search strategy and analysis.
Validity of the findings: Due to my comments in 2, the impact and novelty of this review is very low.

Actually, NO general conclusions can be made, as the data quality and heterogeneity of the  included studies was very low.
Additional comments: This narrative review is about anxiety and depression symptoms in patients with PFP.
The study situation in this field is more than poor. There are NO prospective trial, there are nearly NO comparative group studies. The patients included are so heterogeneous that that this review does not allow to conclude anything.

Abstract: “Peripheral facial palsy (PFP) is a unilateral disorder“ –is not true, PFP can also be bilateral.
Abstract: “Data Extraction: On November 1st.” – the year is missing
Abstract: English style: “The aim of this systematic review was to assess the involvement of various psychological variables associated with PFP.” – a patient can have psychological symptoms but not variables. A study uses variables.
Introduction: “Peripheral facial palsy (PFP) is a unilateral disorder” – see above
Introduction. “and innervate the anterior two-thirds of the tongue” misleading, this is chorda tympani function and only sensory function, no motor fuction.
Introduction: “which can be classified as a) idiopathic, the most prevalent and includes Bell’s palsy and Ramsay Hunt syndrome” – wrong Bell is a synonym for idiopathic, Ramsay-Hunt is NOT an idiopathic form, it is related to herpes virus reactivation. Including a physician dealing with such patients would have been a help.
Introduction: “Involvement of the musculature around the eyes is a key clinical finding for differentiating PFP from central facial palsy”- 1) Imprecise, and in incomplete PFP, orbicularis oculi function can be maintained.
Introduction: “PFP affects 20–32 adults per 56 100,000 per year and 2.7 children per 100,000 per year (Peitersen, 2002; Rowlands et al., 2002) 57 with an annual incidence rate of 11.5–53.3 per 100,000 inhabitants (Martyn & Hughes, 1997).” – if you want present epidemiological data, you should cite epidemiological (!) papers and actual papers. Peitersen 2002, for instance, is cited and cited, but has many methodological flaws and is NOT an epidemiological paper.
Introduction: “control of salivary and tear glands” the patients do not have any loss of control of SALIVARY glands.
Introduction: “as to evaluate the involvement of psychological variables such as anxiety”  see above.
Methods: The search concerning psychological impairments of the patients was limited to depression, and anxiety. Therefore, the conclusion is not surprising: “Limited evidence suggests that patients with PFP might have increased levels of anxiety and depressive symptoms” as other psychological factors were not analyzed. What about self-esteem, self-perception, body disfigurement/figurement, psychological/emotional/social stress/distress? – Hence the title, and description of the focus in not correct: “Assessing psychological variables in patients with peripheral facial palsy: A systematic review” – This review was dealing only with anxiety and depression.
Results/Discussion: One important aspect of the heterogeneity of the studies is completely neglected: The duration of the PFP, or better the interval to onset. In some of the studies, patients in the acute phase = flaccid palsy are investigated, in some other studies, many patients have reached the post-paralytic phase and complain mainly of synkinetic/hyperkinetic movements.These are completely different situations with different psychological impact. Furthermore, the underlying etiology may also have a major impact: A cancer patient or a trauma patient can have several other reasons to be anxious or the be depressed.


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: ASSESSING ANXIETY, DEPRESSION AND QUALITY OF LIFE IN PATIENTS WITH PERIPHERAL FACIAL PALSY: A SYSTEMATIC REVIEW
Review round: 1
Reviewer: 2

Output:
Basic reporting: Lines 60-62: Needs further explanation and specificity regarding eye musculature invovement
Lines 62 - 63: Not completely acurate information. Taste can be altered and post auricular pain sensations can occur
Experimental design: -Medline, PEDro, CINAHL and Google Scholar are a limited band of search engines to perform a systematic review
-The main search term 'facial nerve paralysis' was omitted. Suggest should be updated
-Lines 31-33 Seach terms would have benefitted from including Ramsay Hunt, Herpes Zoster Oticus,
-Line 32 'and not MeSH:'etc unclear sentances

- Although 'bias' is noted in the Limitations section line 248, suggest authors further consider bias as it relates to the articles in the review
Validity of the findings: Lines 250-252: incorrect information presented in the review. Coulson 2004 and Muhammad Kassim N, Abdullahi A, Sammani Usman J. 2019 were not a retrospective studies design and these article stated participants had unilateral lower motor neuron facial nerve paralysis. Please suggest authors thoroughly review and check the accuracy of the ways in which all articles are discussed in their review

The conclusion is somewhat contradictory. The authors report that there is limited evidence and the that patients with PFP can have increased anxiety levels and depressive symptoms and their is limited QoL evidence, and yet they say that results show the clinical relevance of the assessment and correct management of psychological factors.
Please rewite to improve clarity and continuity
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: ASSESSING ANXIETY, DEPRESSION AND QUALITY OF LIFE IN PATIENTS WITH PERIPHERAL FACIAL PALSY: A SYSTEMATIC REVIEW
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors addressed many of the queries but not all.
Experimental design: The authors addressed all queries concerning the experimental design but not all answers are convincing:

Query: "Methods: The search concerning psychological impairments of the patients was limited to depression, and anxiety. Therefore, the conclusion is not surprising: “Limited evidence suggests that patients with PFP might have increased levels of anxiety and depressive symptoms” as other psychological factors were not analyzed. What about self-esteem, self-perception, body disfigurement/figurement, psychological/emotional/social stress/distress? – Hence the title, and description of the focus in not correct: “Assessing psychological variables in patients with peripheral facial palsy: A systematic review” – This review was dealing only with anxiety and depression."

Query: "Results/Discussion: One important aspect of the heterogeneity of the studies is completely neglected: The duration of the PFP, or better the interval to onset. In some of the studies, patients in the acute phase = flaccid palsy are investigated, in some other studies, many patients have reached the post-paralytic phase and complain mainly of synkinetic/hyperkinetic movements. These are completely different situations with different psychological impact. Furthermore, the underlying etiology may also have a major impact: A cancer patient or a trauma patient can have several other reasons to be anxious or the be depressed."

It is not enough the say in the Discussion/Limitation that the data is heterogeneous.
The aspects of the two queries should be addressed with more clarity in the Discussion/Limitations because this is of CLINICAL importance and helps the reader to classify the meaning of the results for clinical routine.
Validity of the findings: See. 2.
The limitations have to be addressed more clearly.

My main comment on the findings remains unchanged:
The novelty of this review is  low. Actually, NO general conclusions can be made, as the data quality and heterogeneity of the included studies was very low.
Additional comments: Some queries were not answered adequately.

Query: "Introduction: “control of salivary and tear glands” the patients do not have any loss of control of SALIVARY glands.
Response: you are talking about very specific details. That's not what the review is about. Besides, I can easily argue with you because I use the scientific articles to extract the information.
Look at this: “Because of the efferent parasympathetic branches to the lacrimal and salivary glands and the motor branch to the stapedius, additional symptoms such as hyperacusis, decreased lacrimation, or decreased taste can help in localization of the lesion along the course of the facial nerve.”

Decreased lacrimation is related to the affected tear function but SALIVARY function is normal. There is no primary original literature on patients with peripheral facial palsy available showing an impaired salivary function. This is the reason why I recommended to show the manuscript to a physician dealing with such patients.

Query: "Introduction: “Involvement of the musculature around the eyes is a key clinical finding for differentiating PFP from central facial palsy”- 1) Imprecise, and in incomplete PFP, orbicularis oculi function can be maintained.
Response: Our argument is drawn from the state of the art. You offer clinical brushstrokes without providing any reference…look at this paragraph taken from a paper ..."

Here again, it would have been helpful to show the manuscript to a physician dealing with such patients. Many patients with peripheral facial patients present at onset with an incomplete palsy, i.e., for instance, eye closure can be unaffected. You find data on patients with incomplete palsy in any large series or trial on peripheral facial palsy:

Two examples, RCTs, incompleteness is also an important prognostic factor:

Sullivan et al. N Engl J Med. 2007 Oct 18;357(16):1598-607. doi: 10.1056/NEJMoa072006.
Engström et al. Lancet Neurol. 2008 Nov;7(11):993-1000. doi: 10.1016/S1474-4422(08)70221-7. Epub 2008 Oct 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: ASSESSING ANXIETY, DEPRESSION AND QUALITY OF LIFE IN PATIENTS WITH PERIPHERAL FACIAL PALSY: A SYSTEMATIC REVIEW
Review round: 3
Reviewer: 1

Output:
Basic reporting: Now, finally, all queries are answered.
Experimental design: Now, finally, all queries are answered.
Validity of the findings: Now, finally, all queries are answered.
Additional comments: Now, finally, all queries are answered.


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: CLINICAL SIGNIFICANCE OF PRETREATMENT PROGNOSTIC NUTRITIONAL INDEX AND LYMPHOCYTE-TO-MONOCYTE RATIO IN PATIENTS WITH ADVANCED P16-NEGATIVE OROPHARYNGEAL CANCER—A RETROSPECTIVE STUDY
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript lacks syntax in English and grammar, such as lines 31, 34, 36, 53, 58 are not understandable. The authors are suggested to improve on it.

There are a few missing references, such as lines 82, 187, 193.

The manuscript carries all the required scientific information and is well structured.

The manuscript contains relevant results.
Experimental design: 1)	Authors are suggested to perform the correlation analysis of PNI and LMR with tumor recurrence & extra-nodal extension and make a graph using the Table 1 data.  This correlation will be helpful in understanding the direct relationship of PNI and LMR with tumor growth and staging.

2)	Similarly, the authors are suggested to make a graphical representation of  PNI and LMR data correlation with other critical parameters such as tobacco use, drinking, alcohol consumption using the data from Table 2.

3)	In Table 7, I see that different head and neck studies have proposed different LMR values which are ranging from 2.8 to 4.97. It would great if authors can comment on this variation, and what could be the factor affecting the LMR score? This information might be helpful in selecting the criteria along with LMR calculation.

4)	Line 141 says that 66.9% of patients were dead at the very end of the study then how the PNI and LMR correlation values were calculated with OPSCC patients DFS and OS.

5)    It would be nice if authors can add the details of “Youden’s Index” calculation and “variance inflation factor” in the materials and method section.
Validity of the findings: no comments
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: CLINICAL SIGNIFICANCE OF PRETREATMENT PROGNOSTIC NUTRITIONAL INDEX AND LYMPHOCYTE-TO-MONOCYTE RATIO IN PATIENTS WITH ADVANCED P16-NEGATIVE OROPHARYNGEAL CANCER—A RETROSPECTIVE STUDY
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript entitled “Clinical significance of pretreatment prognostic nutritional
index and lymphocyte-to-monocyte ratio in patients with advanced p16-negative oropharyngeal cancer” by Tsai et al. describes the effect of PNI and LMR on the survival of patients with advanced p16-negative oropharyngeal cancer. The study included 142 patients for the determination of cut off values of PNI and LMR for improved survival. The results show the higher survival rate in patients with higher levels of PNI and LMR. The manuscript is well written, and interesting. Discussion section is also very informative where authors have explained the possible mechanism for the effect of PNI and LMR on the survival rate. However, it is important to address few questions to further improve the manuscript.
Experimental design: Authors have considered two factors LMR and PNI. Did authors also look at other factors say platelet count or neutrophil or total WBC count? If authors include these values, it will complete the data of blood profiling and also will strengthen the manuscript which will help further research for oropharyngeal cancer treatment.

What are the post-treatment values of PNI and LMR? Authors have not mentioned about post-treatment conditions.
Validity of the findings: Did authors also collect data for the patients if they had any underlying condition which effect patients’ survival?

The importance of current study should be discussed in the discussion section. For example, how this study can be useful for future therapy of p16-negative oropharyngeal cancer.
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: CLINICAL SIGNIFICANCE OF PRETREATMENT PROGNOSTIC NUTRITIONAL INDEX AND LYMPHOCYTE-TO-MONOCYTE RATIO IN PATIENTS WITH ADVANCED P16-NEGATIVE OROPHARYNGEAL CANCER—A RETROSPECTIVE STUDY
Review round: 1
Reviewer: 3

Output:
Basic reporting: The authors have utilized a number of clinical parameters and determined that pre-treatment nutritional index (PNI) and lymphocyte to monocyte ratio (LMR) is a potential prognostic indicator of overall survival in patients with advanced p16 negative oropharyngeal carcinoma. The study is generally well written and concise but can benefit from some additional details within the methods section and discussion.
Experimental design: Experimental design and set up is adequate for the questions the authors have attempted to answer in this study. However, there is lack of clarity on the different factors utilized for multivariate analyses in tables 3-5 (which also are not referenced in the text). The authors  have chosen to focus on PNI and LMR as prognostic factors without discussing much about ENE as the only other significant predictor in univariate analysis. The figures are not as self explanatory and can benefit from modifications as suggested below-

1.	Figure 1 A and B need clear annotations/ legends as to what they are depicting. It is unclear which curve represents PNI and LMR. The figures show an AUC of 61 and 64% respectively, which although not unacceptable, are on the lower side of what would be considered ideal for such analyses.
2.	Figure 2 and 3 would benefit from adding the total number of patients for each curve as well as the hazard ratios in addition to the existing p values in the plot
Validity of the findings: The findings are consistent with previously reported literature and the authors have carefully discussed the possible explanations for their observations.
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: CLINICAL SIGNIFICANCE OF PRETREATMENT PROGNOSTIC NUTRITIONAL INDEX AND LYMPHOCYTE-TO-MONOCYTE RATIO IN PATIENTS WITH ADVANCED P16-NEGATIVE OROPHARYNGEAL CANCER—A RETROSPECTIVE STUDY
Review round: 2
Reviewer: 1

Output:
Basic reporting: The Authors have improved on syntax in English and grammar. The authors have made the necessary improvements and have cited the relevant literature references. Moreover, the manuscript is well structured and has all relevant data and figures to support the observation and investigation.
Experimental design: The experimental design is based on a previously published report which seems to be standard for PNI and LMR investigation in patient samples. Additionally, the data collection and analysis from 142 patient samples make the study more impactful.
Validity of the findings: The findings of this article are in sequence with the previously published report. In the revised manuscript, the authors have made necessary changes that further support the study.
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: CLINICAL SIGNIFICANCE OF PRETREATMENT PROGNOSTIC NUTRITIONAL INDEX AND LYMPHOCYTE-TO-MONOCYTE RATIO IN PATIENTS WITH ADVANCED P16-NEGATIVE OROPHARYNGEAL CANCER—A RETROSPECTIVE STUDY
Review round: 2
Reviewer: 2

Output:
Basic reporting: The revised submission of the manuscript entitled “Clinical significance of pretreatment prognostic nutritional index and lymphocyte-to-monocyte ratio in patients with advanced p16-negative oropharyngeal cancer – a retrospective study (#51116)”, describes the effect of nutritional status and inflammation on the survival of cancer patients. Authors have answered all the comments/concerns point-wise and improved the manuscript accordingly. The abstract, introduction and discussion sections are improved very well.

Line 226, "That’s may be the reason." needs language change.

Line 233-238, “Several limitations should be addressed in current study”. This sentence itself is not clear. The entire paragraph should be changed to strengthen the manuscript. For instance, why the mentioned studies were not done, should be described in an effective way so that it does not undermine the current manuscript.
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: CLINICAL SIGNIFICANCE OF PRETREATMENT PROGNOSTIC NUTRITIONAL INDEX AND LYMPHOCYTE-TO-MONOCYTE RATIO IN PATIENTS WITH ADVANCED P16-NEGATIVE OROPHARYNGEAL CANCER—A RETROSPECTIVE STUDY
Review round: 2
Reviewer: 3

Output:
Basic reporting: Article meets standards
Experimental design: No comment
Validity of the findings: Authors have made necessary changes. No further changes required
Additional comments: No additional comments


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

Input:
Title: USING HIGH-RESOLUTION MELTING TO IDENTIFY CALLIPHORIDAE (BLOWFLIES) SPECIES FROM BRAZIL
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript is written in clear proffessional language. Introduction show advanteges and limitations of existing methods of species identification important for forensic entomology. Structure of the manuscript conforms PeerJ standards. Figures are high quality and relevant and well described. Provided References are well selected and up to date.
Experimental design: Performed research are in scope of the Journal. Research aim - delivering simple and robust method for identification of forensically inportant Calliphorode species in Brasil - is well defined. To reach this aim is necessary to fill the gap - design approptiate primers. The Authors successfully done it.

Description of methods, however is insufficient.
Line 70 and 71. „Mitochondrial COI barcode sequences of those species were downloaded from BOLD systems“. What were the criteria for selecting the sequence to be analyzed?.
For example, in Supplementary Table 2 authors chosed 5 sequences of Chrysomya megacephala for analysis. For this species in BOLD there are 833 public records with sequences, 47 of its from Brazil, but the Authors did not selected any sequence of Chrysomya megacephala from Brazil. Why?

Line 73. The Authors selected two regions containing polymorphic variations for species distinction. It would be uselul include sequences alignment in supplementary material. The Authors do not indicate what the differences were in the amplicon sequences.

Line 75-76. uMelt-DNA Melting software version (available at  https://www.dna.utah.edu/umelt/um.php) - link outdated
Validity of the findings: The Authors provide well supported data. Forensic entomology will benefits from publication of this manuscript.
Additional comments: No additional comments