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: CRITICAL MULTI-STRANDED APPROACH FOR DETERMINING THE ECOLOGICAL VALUES OF DIATOMS IN UNIQUE AQUATIC ECOSYSTEMS OF ANTHROPOGENIC ORIGIN
Review round: 1
Reviewer: 2

Output:
Basic reporting: 	Comment on language and grammar issues
In general the manuscript (ms) is well written, although it would benefit from a general revision of the English language.
I enclose 2 examples of required improvement of the English language:
-	sentence of lines 71 to 73
-	sentence lines 102-103

	Intro & background to show context
Introduction and background include the main issues that are reported along the manuscript.

	Literature well referenced & relevant
All references cited in the text are listed in the final reference list.
All references in the final reference list are cited in the text.
I didn’t verify the reference formatting for this journal, nevertheless, I found some differences in formatting between references in the final list (i.e.:  separation between authors’ names with a comma, and no type of separation see Bak et al. 2006 and Campeau et al 1999).

	Figures are relevant, high quality, well labelled & described
All figures and tables are useful and of good quality.
A few suggestions were added in the captions of the pdf of the ms.
	Raw data supplied
I didn´t find a caption for the excel table containing the raw data. It would be useful to have a caption explaining what the numbers in the species list mean (probably number of valves counted?). The + sign should also be explained.  I would recommend the insertion of the authorities of the diatom taxa in the excel table, so that at least once along the manuscript it will be possible to read this information.
Why are some environmental parameters missing? In the ms it was only said that 44 samples were taken and that diatoms and water for chemical analysis were performed.
The comma (,) separating the numbers of the environmental parameters should be replaced by and end stop (.) as decimal places should be represented by end stop and not a comma.
Experimental design: 	Original primary research
The research on diatom ecology is not new.  As the authors refer many diatom indices are based on the knowledge about diatom ecology, so in fact the research presented in the ms refers to the improvement/definition of ecological values for some environmental parameters and for 3 extreme environments which were previously mining areas. This primary research did not exist before for these areas, and therefore it is useful new data for scientific community working on diatoms.
	Research question well defined, relevant & meaningful. It is stated how the research fills an identified knowledge gap
Yes
	Rigorous investigation performed to a high technical & ethical standard
The authors performed good field sampling design based on previous knowledge about mining activities, field sampling and laboratorial work seems to have been rigorously performed as well, at least at the diatom level as the raw data provided allow to .confirm that authors were careful with identification and counting of samples. The photomicrographs of diatoms presented are of good quality and diatoms have been correctly identified. Nevertheless, the results extracted from the obtained field and lab data were not extensively explored as I refer several times along the ms in the comments in the pdf. For example: the redefinition or proposal of ecological values for some diatoms are not fully explained or justified through mathematical analysis (objective) but are rather more or less attributed in a subjective way with only verbal definition. The inclusion of available information in the literature on these taxa ecological values is interesting and important and helps to summarize  existing autecological data.
	Methods described with sufficient detail & information to replicate
Yes, although I included in the ms the need to clarify some issues about sampling diatoms, i.e.: what kind of sampling was done (type of substrate for benthic diatoms, phytoplankton net for planktonic diatoms, any other habitat?). The list of diatoms presented show that benthic habitats must have been sampled as the majority of taxa are benthic and not planktonic. Another aspect to clarify and related to the previous comment is the clarification about the type of waterbodies sampled i.e.: are they all lentic in nature (reservoirs) or are there some lotic ones as well (streams).
The methods for chemical analysis were not included. This may be replaced by the reference of the manual with the methods used (for example APHA).
Validity of the findings: 	Impact and novelty not assessed. Negative/inconclusive results accepted. Meaningful replication encouraged where rationale & benefit to literature is clearly stated.
The authors were careful in explaining the novelty and benefits of their research, with which I agree.
	All underlying data have been provided; they are robust, statistically sound, & controlled.
Yes
	Speculation is welcome, but should be identified as such.
I don’t consider that the redefinition and proposal of ecological values, despite the lack of full explanation, are not speculative.
	Conclusions are well stated, linked to original research question & limited to supporting results.
Yes
Additional comments: Authors use the expression “ecological indicator values” throughout the ms, and in fact it is the main focus of the research. I understand that authors use this expression as it is referred in the OMNIDIA software under Van Dam’s  ecological results. Nevertheless, I would like to point out that most autoecological diatom indices are based on 3 parameters: average relative abundance of taxa, the sensibility value (provides information about the response of taxa to degradation and usually varies between 1 and 5) and indicator value (provides information about the degree of stenoicity for each taxon, i.e. measures the reliability of each taxon for a certain environment). So, what I think authors are referring to are not “ecological indicator values” but the sensibility values. I therefore recommend authors to remove the word “indicator” from the expression they use throughout the entire manuscript maintaining as “ecological values” only.
The establishment of new or modified ecological values for the diatom species in this study should be better explained and related to abiotic values and ranges of values. Authors should justify why a certain taxon is changed from 1 class to another in Van Dam’s classification based on the range of chemical parameters. What does eutrophic condition mean? To be eutrophic what concentration of nitrogen and phosphorus must be present in the water?
In order to make the attribution/modification of ecological values more objective and understandable authors should correlate taxa abundances with environmental parameters. It seems that these changes are proposed but in a somewhat subjective way as it is mainly based on the PCA analysis and literature.
The environmental  range reported in the manuscript  for the set of sampling sites is not very wide and includes sites with very specific and particular characteristics, therefore I advise caution in the attribution  of ecological values as these only refer to a small range of environmental conditions.
This manuscript contains interesting information and I recommend it for publication, but after the changes suggested.


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

Input:
Title: CRITICAL MULTI-STRANDED APPROACH FOR DETERMINING THE ECOLOGICAL VALUES OF DIATOMS IN UNIQUE AQUATIC ECOSYSTEMS OF ANTHROPOGENIC ORIGIN
Review round: 1
Reviewer: 3

Output:
Basic reporting: See Attached PDF
Experimental design: See Attached PDF
Validity of the findings: See Attached PDF
Additional comments: No additional comments


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

Input:
Title: CRITICAL MULTI-STRANDED APPROACH FOR DETERMINING THE ECOLOGICAL VALUES OF DIATOMS IN UNIQUE AQUATIC ECOSYSTEMS OF ANTHROPOGENIC ORIGIN
Review round: 2
Reviewer: 1

Output:
Basic reporting: I recommend an acceptance of the manuscript for publication in your journal. The comments on the improvements in the first review have been made to the best possible satisfaction. In the resubmission of the revised manuscript I see no need for further improvement. The authors have revised the manuscript in an acceptable manner and thus contributed to the increase in quality.
Experimental design: The improvements have been made to the best possible satisfaction.
Validity of the findings: All underlying data have been provided.
Additional comments: I recommend an acceptance of the manuscript for publication in your journal. The comments on the improvements in the first review have been made to the best possible satisfaction. In the resubmission of the revised manuscript I see no need for further improvement. The authors have revised the manuscript in an acceptable manner and thus contributed to the increase in quality.

 For improvement:
- at the beginning of the sentence, please enter the whole genus name for C. invisitatus - line 503, S. binatus lines 535-536, S. parvus – line 557, D. moniliformis – line 569, E. subminuta – line 593, M. smithii – lines 601-602.


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: TEFFECTR: AN R PACKAGE FOR STUDYING THE POTENTIAL EFFECTS OF TRANSPOSABLE ELEMENTS ON GENE EXPRESSION WITH LINEAR REGRESSION MODEL
Review round: 1
Reviewer: 1

Output:
Basic reporting: The article is well written, with the standard sections.

The code to use their algorithms is provided through github, which makes it easy to use, although there are shortcomings explained in "Experimental design".

Table 1 is missing some text in the caption in the pdf, it is fine in its own document.

A great job has been done with the bibliography, with a good search of genes related to breast cancer.
Experimental design: The expression "nearby genes" is used in several locations to decide which genes are affected by a TE. but it is only defined on line 234, and in several captions.
It can be modified by the user in the algorithm, but the maximum distance is a basic parameter in this article, so it should merit some reasoning as to why use 5kb by default, or why use other distances.

No justification is provided for discarding the multimapped reads (line 158), it should be motivated. Also the minimum amount of reads to provide a reasonable result should be indicated, because due to the discarding of the multimapped reads it seems to me there is going to be few reads counted as TE.

The use of covariants dilutes the effect of the TE in the lm, using other variables to fine tune the lm. The only justification I have found to use covariants is that the results are better, (line 277).
Validity of the findings: Only the effect on known genes is looked up, so it seems the algorithm is to first look for important genes in the literature, then analyze nearby TEs, instead of first looking at the TEs and then providing the genes that matches the best.

Several times it says "health vs tumor" tissues, but it seems it isn't covered in the results, it only uses the "health vs tumor" as another covariant to provide a better lm.
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: TEFFECTR: AN R PACKAGE FOR STUDYING THE POTENTIAL EFFECTS OF TRANSPOSABLE ELEMENTS ON GENE EXPRESSION WITH LINEAR REGRESSION MODEL
Review round: 1
Reviewer: 2

Output:
Basic reporting: The authors investigated the effect of transposable elements on gene expression with regression model, which is a very relevant topic to the field at the moment. They have based theirs results on the correlation between the expression of TEs and a gene in a give genomic location developing a tools to calculate the parameters of correlation. However,  this correlation not show  the effect the expression of TEs relating to his adjacent genes.
Experimental design: The authors use a mapping bam file to evaluate the expression TEs, nevertheless they describe perfunctorily which are the parameters of the alignment. The method should be described with sufficient information. On the other hand, even though the multi mapping not be as precise  as single mapping reads, you can get a better determination of which loci and types of elements are unregulated in your samples whether you evaluate the multimapping reads. I think that the multi-mapping reads could to be analyzed with Squire https://doi.org/10.1093/nar/gky1301 or with TEtranscripts DOI: 10.1093/bioinformatics/btv422-

The authors use a Repeat Masker TEs to localitation TEs on genome, however this database contain both activa a inactive TEs so it is very difficult to ensure  that  the expression TE has effect on the expression genes.

On this sense, in the title, the autor focus us on the effect the expression TE on near genes, however in the text they say:  ‘Still, it should be …… proximed genes’, so the title do not reflect the result of this work', a title overambitious.
Validity of the findings: If the author used a dataset normal and tumor tissue, why they focus only a few genes? Why they only investigated genes at 5 kb upstream? why not other distances? In the heathy tissue, which is the expression this TEs?

It would be helpful if the authors could something comment on methylation in these tumors and whether or not it is likely contributing to the expression patterns, this would add some rigor to the study and further support the results without needing to perform additional experiments. It would further be helpful if the authors would state some how that if the methylation differences were the reason, one would expect global differences for different classes of elements or theirs near genes.
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: TEFFECTR: AN R PACKAGE FOR STUDYING THE POTENTIAL EFFECTS OF TRANSPOSABLE ELEMENTS ON GENE EXPRESSION WITH LINEAR REGRESSION MODEL
Review round: 2
Reviewer: 1

Output:
Basic reporting: It continues to be well written, with the standard sections.
Experimental design: All questions presented in the first review have been answered correctly.
Validity of the findings: All questions have been answered correctly. The article introduces an improvement of existing algorithms and programs.
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: TEFFECTR: AN R PACKAGE FOR STUDYING THE POTENTIAL EFFECTS OF TRANSPOSABLE ELEMENTS ON GENE EXPRESSION WITH LINEAR REGRESSION MODEL
Review round: 2
Reviewer: 2

Output:
Basic reporting: Overall, the submitted paper has been improved regarding to the older version,  is well-written with sufficient intro about the background of the TE and its potential relationship with cancer. Also results and discussion has been improved, just as the tittle.
The authors has answered all my concerns, so, I recommend this paper to be published in PeerJ .
Experimental design: No comment
Validity of the findings: No comment
Additional comments: No comment


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

Input:
Title: MANAGEMENT OF RECURRENT URETERAL STRICTURE: A RETROSPECTIVELY COMPARATIVE STUDY WITH ROBOT-ASSISTED LAPAROSCOPIC SURGERY VERSUS OPEN APPROACH
Review round: 1
Reviewer: 1

Output:
Basic reporting: The standard reporting is fine.
Experimental design: -
Validity of the findings: See my general comments.
Additional comments: Authors performed a retrospective study upon their experience in treating recurrent benign ureteral stricture. The tested the hypothesis that redo ureteral reconstruction is as effective as open procedure. They considered 41 patients comparing 22 robot-assisted surgeries with 19 open surgeries. The study was not a RCT. They found no significant intergroup difference in terms of age, gender composition and ASA scores. Robot-assisted group showed shorter operative time, less estimated blood loss, shorter hospital stays and higher cost. Similar success rates: in the robot-assisted (85.71%) and the open group (82.35%). Authors concluded that robot-assisted procedure for recurrent stricture after previous ureteral reconstruction is as effective as open procedure and is associated with shorter operative time, less estimated blood loss and
shorter hospital stays, but higher cost.
This is an interesting study, but there are some flaws: the sample is not homogeneous (Initial etiologies for ureteral stricture were clarified into congenital, stone disease, vascular anomalies and polyp), the study is retrospective and not RCT and no clear indication to open vs. robotic was cleared in the manuscript. We would like to see in the follow-up the assessment of renal function. Is it known preoperatively?


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: MANAGEMENT OF RECURRENT URETERAL STRICTURE: A RETROSPECTIVELY COMPARATIVE STUDY WITH ROBOT-ASSISTED LAPAROSCOPIC SURGERY VERSUS OPEN APPROACH
Review round: 1
Reviewer: 2

Output:
Basic reporting: Clear and unambiguous, professional English used throughout.
The article must be written in English and must use clear, unambiguous, technically correct text. The article must conform to professional standards of courtesy and expression.

Reviewer considerations:
In the PDF document of the authors themselves, there are notes throughout the document where, in my opinion, the writing needs to be improved to make it more understandable.

Title of the manuscript is redundant in the text, it can be improved:
Current:
Management of recurrent stricture after previous ureteral reconstruction: Robot-assisted laparoscopic surgery versus open surgery

Suggestion:
Management of recurrent ureteral stricture: "Comparative study with robot-assisted laparoscopic surgery versus open approach"

Abstract:
Background:
To report our experience in treating recurrent benign ureteral stricture and demonstrate whether robot-assisted procedure for redo ureteral reconstruction is as effective as open procedure while remaining less invasive.

They wrote it down here it is only the purpose of the study and prior to this lack a short but important text as background.

Methods and Results:  Both sections of the Abstract should reflect the type of patients the study is about: most recurrent UP obstruction and less recurrent ureteral stenosis.

Conclusions:  According to the instructions for the authors, Conclusions are not included in the ABSTRACT, only until after the Discussion.

Subjets: Lack.
Keywords: Lack.

The english language check of:
Introduction to References: In the same PDF of the authors' manuscript.

Literature references, sufficient field background/context provided.
The article should include sufficient introduction and background to demonstrate how the work fits into the broader field of knowledge. Relevant prior literature should be appropriately referenced.

Reviewer considerations:
In the introduction I suggest including relevant information from more publications in the adult population, since most of their references in children and mainly about pyeloplasty.

Professional article structure, figures, tables. Raw data shared.

Reviewer considerations:
The instructions for authors according to the suggested format, some aspects are missing, that in the authors' own PDF register notes clarifying and sometimes deleting sections.

Figures should be relevant to the content of the article, of sufficient resolution, and appropriately described and labeled.

Reviewer considerations:
Figure 1. Improve the description of their images:
(A)	Modified 45 ° lateral decubitus position for upper ureter stricture repair.This image shows above all the location of the trocars.

(B-D) Dissection and transection of the ureter.  There are 3 transoperative images, where they should highlight the characteristics of the surgical anatomy and details of the surgical technique they used.
Further:
Images D, E and F are not good images, since they do not adequately show the transoperative details of anatomy and surgical technique. I suggest changing them for other better images.

Table 1. The title is incomplete, it must describe the information contained in the table.
I suggest to better organize the presentation of the data in the table and if necessary use table footers in "Items" that are extensive.
For example: Symptoms, n (%), n (%) should be placed on the column of cases and percentage, also median (range), and "n".
It is also convenient to adjust the percentages to 100% in both columns of Symptoms.
Item "History",  I suggest changing it to: Prior or Primary Procedures.

Tables 2 and 3: Also their titles are incomplete and it is also necessary to better organize the presentation of the data.
Table 2. In Item “Postoperative complications”, at the table footer, inform that the classification of the degrees of complications are based on the Clavien and Dindo Classification.
Further:
Table 2. Indicate that the Items: age, operative time, blood loss and hospital stays, in their results include the standard deviation

All appropriate raw data have been made available in accordance with our Data Sharing policy.

Reviewer considerations:

The raw data (or code) be opened, and is well described in English., Only the abbreviation VAS, which is located in column "P" of the spreadsheet does not guess the meaning.

Self-contained with relevant results to hypotheses.

Reviewer considerations:
The variables analyzed and the results are adequate to respond to the hypothesis raised, and in the same PDF of the authors there are notes where its writing needs to be improved.
Experimental design: Original primary research within Aims and Scope of the journal.
Research question well defined, relevant & meaningful. It is stated how research fills an identified knowledge gap.

Reviewer considerations:

This is the aim:
In the current study, we aim to report our experience in treating recurrent UPJO or ureteral strictures and demonstrate whether robot-assisted repair in redo ureteral reconstruction is as safe and effective as open repair.
I consider that the research question is correct.

Rigorous investigation performed to a high technical & ethical standard.

Reviewer considerations:
This study is retrospective, observational and comparative, it was approved by the ethics committee of hospital institution. Therefore, it does not violate ethical principles.

Methods described with sufficient detail & information to replicate.

Reviewer considerations:

The methods are described enough to reproduce the study.
Validity of the findings: Impact and novelty not assessed. Negative/inconclusive results accepted. Meaningful replication encouraged where rationale & benefit to literature is clearly stated.

Reviewer considerations:
The results obtained by the researchers support the hypothesis raised in the objective of the investigation, I only consider that the days of postoperative stay, for the reason stated by the researchers themselves, depending on the institutional care policy, I suggest they should eliminate it, to avoid invalid conclusions.

All underlying data have been provided; they are robust, statistically sound, & controlled.

Reviewer considerations:
The limitations of the study are recognized by the researchers, the most important being: limited number of cases of both study groups and the significant difference in follow-up time between both study groups.

Conclusions are well stated, linked to original research question & limited to supporting results.

Reviewer considerations:

The conclusions are appropriate and consistent with the research question and are supported by the results, except for:the statement of:shorter hospital stays.That it contrasts with the results obtained in the investigation and that in the discussion they comment on it, so, I suggest you should not take this data into account (consider it not valuable).
Additional comments: As a general recommendation, researchers should avoid issuing personal opinions.

In the PDF document of the same researchers, notes with corrections and suggestions to improve the manuscript are indicated.

Attachment of the Reviewer:
The PDF of the researchers themselves.

https://www.ncbi.nlm.nih.gov/pubmed/?term=recurrent+UPJO+%2B+Robotic+surgery/ recurrent UPJO + Robotic surgery
Publications 20  (adults and children).

https://www.ncbi.nlm.nih.gov/pubmed/?term=recurrent+UPJO+%2B+Robotic+surgery/ recurrent ureteral obstruction + robotic surgery
Publications 30  (adults and children)… Several duplicate publications.

https://www.ncbi.nlm.nih.gov/pubmed/?term=recurrent+UPJO+%2B+Robotic+surgery/recurrent ureteral obstruction + open surgery
Publications 112  (adults and children). Several of these useful and interesting.

Annex:
Abstract of a comparative study of robot versus open surgery in children.

J Pediatr Urol. 2015 Apr;11(2):69.e1-6. doi: 10.1016/j.jpurol.2014.10.009. Epub 2015 Feb 24.
Failed pyeloplasty in children: Is robot-assisted laparoscopic reoperative repair feasible?
Asensio M1, Gander R2, Royo GF1, Lloret J3.

Abstract
OBJECTIVE:
In this study we aim to demonstrate that robot-assisted laparoscopic (RAL) reoperative repair is safe and effective and even less technically demanding than open repair for recurrent ureteropelvic-junction obstruction (UPJO).
STUDY DESIGN:
A retrospective study was conducted of all cases of failed open pyeloplasties who underwent RAL reoperative repair at our institution between January 2010 and December 2013. The general surgical procedure was the same we previously described for robot-assisted laparoscopic pyeloplasty. Success was defined as: improvement in the degree of hydronephrosis at ultrasound, improvement of diuretic washout time at postoperative diuretic renogram (<15 min), improvement or at least stable differential renal function and absence of symptoms. These radiographic and symptomatic criteria of success were considered the primary outcomes. Secondary outcomes included complications and length of hospital stay.
RESULTS:
Between 2000 and 2013 a total of 153 patients underwent open Anderson-Hynes dismembered pyeloplasty. Of these 9 (6%) had recurrent UPJO. Four patients underwent open redo pyeloplasty. As a result, our study population comprised 5 children who underwent reoperative RALP repair. Patient characteristics and outcomes are summarized in the table below. Our success rate was 100%.
DISCUSSION:
Due to the low failure rate of open dismembered pyeloplasty there is no consensus on the best surgical approach for recurrent obstruction. While endoscopic approaches have been favored in adults, children have shown better success rates with repeat pyeloplasty. Laparoscopic salvage pyeloplasty for failed open procedures has become more popular and has been shown to result in excellent outcomes while providing the advantages of minimally invasive surgery. To date, the literature regarding the use of RALP for failed open procedures in the pediatric population is scarce. Only 2 pediatric series of robotic reoperative pyeloplasty have been reported by Helmal et al. (9 patients) and Lindgren et al. (16 patients) with a success rate of 100 and 88%, respectively. Although this is one of the first published studies about robot-assisted laparoscopic reoperative repair for failed open pyeloplasty in pediatric patients, we acknowledge the limitations of our study due to the small number of patients, its retrospective nature and limited follow-up time.
CONCLUSIONS:
The incidence of failed open pyeloplasty is as low as 5% and management remains controversial. As reported by other authors, we believe that crossing vessels play a particularly important role in secondary obstruction and adversely impact the outcome. Redo pyeloplasty, open or minimally invasive, is associated with high success rates (80-100%) and therefore considered the treatment of choice by the majority of authors nowadays. Additionally, RALP for secondary procedures has demonstrated to be safe and even less technically demanding when compared to the open approach, providing the advantages of minimally invasive surgery.

Others References:

Springerplus. 2014 Oct 3;3:580. doi: 10.1186/2193-1801-3-580. eCollection 2014.
Passing from open to robotic surgery for dismembered pyeloplasty: a single centre experience.
Di Gregorio M1, Botnaru A1, Bairy L2, Lorge F1.

FINDINGS:
From 17 patients who underwent Da Vinci Robot procedure, 15 followed the complete treatment (2 were converted to laparotomy). Two patients had post-operative urine leakage; the stent was changed under sedation without further sequelae. The mean operative time was 189 minutes. The average hospital stay was 4 days. The average follow-up was 25 months. There was only one patient with UPJ stenosis at 6 months and he was treated by balloon dilation. All patients were followed with MAG 3 lasix renal scan, CT or urography. Except the patient with recurrent stenosis, all patients were asymptomatic without objective evidence of obstruction at the present time.
CONCLUSIONS:
Robotic pyeloplasty technique is feasible and gives good results without previous laparoscopic experience.

J Urol. 2011 Apr;185(4):1455-60. doi: 10.1016/j.juro.2010.11.056. Epub 2011 Feb 19.
Long-term experience and outcomes of robotic assisted laparoscopic pyeloplasty in children and young adults.
Minnillo BJ1, Cruz JA, Sayao RH, Passerotti CC, Houck CS, Meier PM, Borer JG, Diamond DA, Retik AB, Nguyen HT.

Abstract
PURPOSE:
Laparoscopic pyeloplasty is one of the more common robotic assisted procedures performed in children. However, data regarding long-term experience and clinical outcomes for this procedure are limited. We evaluated the long-term outcomes in a large series of patients undergoing robotic assisted laparoscopic pyeloplasty at a teaching institution, and the effect of a collaborative program between the robotic surgeons, surgical nurses and anesthesiologists on overall operative time.
MATERIALS AND METHODS:
We retrospectively reviewed 155 patients who underwent robotic assisted laparoscopic pyeloplasty between 2002 and 2009. Operative data, including surgical approach, type of procedure, total and specific operative times and placement of ureteral stents, were determined. Postoperative outcome measurements, including duration of hospital stay, duration of Foley catheter drainage, radiological findings and any subsequent complications, were assessed.
RESULTS:
Mean operative time and length of hospitalization decreased significantly by the end of the study. At a mean followup of 31.7 months the primary success rate was 96% (hydronephrosis was improved in 85% of patients and stable in 11%). The complication rate was 11%, and recurrent obstruction requiring redo robotic assisted laparoscopic pyeloplasty or open pyeloplasty developed in 3% of patients. Success rate and complication rate were unchanged during the study period.
CONCLUSIONS:
This study confirms that even within the confines of a pediatric urology training program successful collaboration between robotic surgeons, surgical nurses and anesthesiologists can lead to shorter operative times and hospital stays. Long-term surgical success and complication rates were comparable to open surgery.


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: MANAGEMENT OF RECURRENT URETERAL STRICTURE: A RETROSPECTIVELY COMPARATIVE STUDY WITH ROBOT-ASSISTED LAPAROSCOPIC SURGERY VERSUS OPEN APPROACH
Review round: 2
Reviewer: 1

Output:
Basic reporting: Authors did changes accordingly the questions reviewers posed. The quality of manuscript was increased. I have no other questions.
Experimental design: -
Validity of the findings: -
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: MANAGEMENT OF RECURRENT URETERAL STRICTURE: A RETROSPECTIVELY COMPARATIVE STUDY WITH ROBOT-ASSISTED LAPAROSCOPIC SURGERY VERSUS OPEN APPROACH
Review round: 2
Reviewer: 2

Output:
Basic reporting: I have no observations in this section.
Experimental design: I have no observations in this section.
Validity of the findings: I have no observations in this section.
Additional comments: It is where I consider it convenient to insert a paragraph and make corrections or minor modifications in the body of the manuscript, the text has been highlighted and note boxes have been placed in the PDF document (it is attached):

Material and methods:

Line 96 (USA).
Line 104 (add a paragraph): We did not include the postoperative stay to analyze it as a variable, because it was not valuable due to the hospital discharge policy of our institution.

Procedures:

Lines 117 and 125 (breakdown figure 1).

Results:

 Add "also" on line 146.
On line 148 add space (14 laparoscopic).

References:

Correct in the 27 references, publication year order and add the DOI (digital object identification).


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: MIGRAINE AURA, A PREDICTOR OF NEAR-DEATH EXPERIENCES IN A CROWDSOURCED STUDY
Review round: 1
Reviewer: 1

Output:
Basic reporting: The writing is clear and unambiguous.  The references show sufficient familiarity with the background and context.  There are several problems with the reference list, but they are trivial and easily corrected.
The figures are mostly relevant and well labeled,.but Figure 3 ("Map showing places of residency of survey participants") is irrelevant and does not contribute anything to the manuscript and should be deleted.
Experimental design: The experimental design is adequate.  There are substantial limitations to the online survey methodology in terms of biased sampling and inability to capture complex concepts, but the authors acknowledge those limitations on lines 261-266 and 302.
Research question is adequately defined and meaningful.
Methods are adequately described.
Validity of the findings: Adequate data are provided for the most part.  However, the authors never described their criteria for REM intrusion.  They listed in Table 1 the questions on headaches, migraine aura, and NDEs, but they never mentioned the specific questions on REM intrusion, nor did they mention their criteria (i.e., how many of those REM intrusion questions had to be endorsed to fulfill the criteria).  Without that information, readers cannot judge how much the REM questions may have overlapped with the NDE questions, leading to a tautological association.
Speculation is not always identified as such. On line 321, the authors write that “one must assume that there has to be a neurological basis” for conscious awareness when cerebral function was not expected.  Why must one assume that?  This assumption is particularly questionable in light of their citation in the next sentence of the study of restoration of cellular function in a pig brain by Vrselja, et al. (2019), in which the authors explicitly disavowed any implication that this effect had anything to do with consciousness when they wrote: “it is important to distinguish between resuscitation of neurophysiological activity and recovery of integrated brain functions (that is, neurological recovery). The observed restoration of molecular and cellular processes following 4 h of global anoxia or ischaemia should not be extrapolated to signify resurgence of normal brain function. Quite the opposite: at no point did we observe the kind of organized global electrical activity associated with awareness, perception, or other higher-order brain functions.”  The present authors, then, should elaborate on the rationale for their assumption.  As they acknowledge on lines 392-395, we do not understand how changing polarization states of neocortical neurons create consciousness.  And yet, they assume that they do create consciousness somehow.
Additionally, their Discussion should address objections to the hypothesized association between REM intrusion and NDEs, such as the occurrence of NDEs in patients receiving general anesthetics and other drugs that suppress REM activity, and the finding of Britton & Bootzin (2016) of increased REM latency in people who have NDEs.
Additional comments: In addition to the substantive issues mentioned above, the style of references is inconsistent.  In some journal article cited, the article title in entirely in lower case, whereas in others, most words are capitalized.  Likewise, some journal titles are entirely in lower case, whereas others are capitalized.
On lines 597-598, the authors list a reference to Long & Holden, 2007.  On lines 599-600, they list the identical reference, this time citing it as Long & JM, 2007.  The second (incorrect) reference should be deleted. On line 107, the authors cited "Long & JM, 2007" but that should be "Long & Holden, 2007".
On line 625, the authors list the Martial, et al., 2019 paper as “In press”.  It is no longer in press, but was published in Volume 69.


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: MIGRAINE AURA, A PREDICTOR OF NEAR-DEATH EXPERIENCES IN A CROWDSOURCED STUDY
Review round: 1
Reviewer: 2

Output:
Basic reporting: The paper is well-structured, clear, and well-written, however, I would suggest to the authors to carefully go through their manuscript to correct minor typos and to be consistent along the whole manuscript. For example, they wrote “laypeople” and a bit further “lay people”, line 276 a bracket is missing, etc. Please also double check the references for typos too.

In addition, the numbering of the Figures are not correct (no consistency between the manuscript text and the attached Figures). Please ensure that Tables and Figures are properly referred within the main text.
Experimental design: Kondziella and colleagues conducted an interesting study, as it is the first study to properly assess the potential association between NDEs and migraine. They discuss here a fascinating topic in the science of consciousness, the NDEs, which has not received the scientific and medical attention it deserves. Its strength resides mainly in the number of individuals included, the method used, an interesting discussion and of course its results. I think that this study is important and suitable for this journal.

This is a good paper, albeit some improvements can be made:
- Although it is important to describe the link between REM sleep, migraine aura and NDEs -as they did in the introduction, I think the introduction could be improved by being more concise on this aspect of the introduction. This could leave space for introducing a bit more the literature concerning the presence of hallucinations in migraine patients. In particular, it has been shown that hallucinations are not infrequent in people who have migraine and it would be worthwhile to develop more this literature.
-	In the ‘Near-death experiences and evidence of migraine aura’ section of the Materials & Method, it is not clearly stated that the authors also included the experiencers reporting NDEs in a context where they “just” felt that they will die while there was no true life-threatening context. Please more detailed the inclusion criteria somewhere in the Materials & Method.
-	Do the authors have more details regarding the context in which the participants lived their NDE? To my opinion, a limitation of this study is that they did not get (self-reported) details regarding the precipitating factors/contexts in which experiencers have experienced the NDE. Indeed, the sample of people who claimed that they “just” felt that they will die while there was no true life-threatening context could actually include a very broad variety of precipitating factors/contexts. Indeed, my own expertise gives me the opportunity to attest that some people might have felt an impression of dying while they actually “only” fainted. According to me, this should clearly be stated in the manuscript.
-	Interestingly, the most frequently reported NDE features are ‘abnormal time perception’, ‘extraordinary speed of thoughts’, ‘exceptional vivid senses’, and ‘feeling separated from one’s body’. This frequency pattern might be discussed, as they are not completely consistent with available studies investigating features frequency using standardized scales in NDE reports. Indeed, for example, ‘exceptional vivid senses’ and ‘extraordinary speed of thoughts’ are features reported less often than the feeling of peace/well-being and out-of-body experiences. Moreover, it would be interesting to detail the frequencies of the NDE features reported only by the sample of experiencers who reached 7/32 on the GNDES -and, if the authors wish, maybe have a Figure or Table to illustrate it as well.
- In the first paragraph of their discussion, the authors discussed unpleasant NDEs. That’s interesting and relevant because very little is known on unpleasant NDEs. I would suggest to add the most recent publication studying distressing NDEs, which is Cassol et al. (2019) published in Memory. I think it is very relevant here.
Validity of the findings: The statistics used by the authors are appropriate from what I can tell. The authors' conclusions are well stated and linked to their research question.
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: MIGRAINE AURA, A PREDICTOR OF NEAR-DEATH EXPERIENCES IN A CROWDSOURCED STUDY
Review round: 1
Reviewer: 3

Output:
Basic reporting: By assessing the prevalence of NDEs in people presenting or not migraines with aura, this paper adds an interesting piece to the NDE literature. However, my general feeling about the introduction is that authors focus extensively on how their previous study about REM sleep intrusion addresses the biases of the study by Nelson et al. It would benefit the reader to hear a bit more about the content of NDE and NDE-like memories from previous research.
Experimental design: no comment
Validity of the findings: no comment.
Additional comments: Abstract:
-In the abstract, authors could give a little bit more details in the part dedicated to the methods.
Introduction:

Introduction:
-Line 60: distortion of time perception and speeded thoughts are two distinct/independent features according to the Greyson NDE scale, and speeded thoughts are not considered as a subtype of altered time perception.

- Line 107: there is a typo in the reference placed into brackets, it should read (Long & Holden, 2007). Besides, this reference appears twice in the references at the end of the manuscript.

Discussion:
-Line 238, it should read (Gallup, 1982).

-Line 242, authors write that NDEs are almost always associated with well-being, however, it would be interesting to mention that other studies have already identified negative NDEs, and sometimes in relatively large proportions. See, for example, the following articles:
Cassol, H., Martial, C., Annen, J., Martens, G., Charland-Verville, V., Majerus, S., & Laureys, S. (2019). A systematic analysis of distressing near-death experience accounts. Memory, 1–8.
Knoblauch, H., Schmied, I., & Schnettler, B. (2001). Different kinds of near-death experience: A report on a survey of near-death experiences in Germany. Journal of Near-Death Studies, 20(1), 15–29.
Lindley, J. H., Bryan, S., & Conley, B. (1981). Near-death experiences in a pacific northwest american population: The evergreen study. Journal of Near-Death Studies, 1, 104–124.

Besides, authors cite Charland-Verville et al. 2015 who report ‘less blissful’ NDEs. Charland-Verville et al. 2014 might be more appropriate to cite in this case.

-Line 245, still regarding the valence of NDEs, authors indicate that “experiences with the cut-off score of ≥7 GNDES points were reported significantly more often as pleasant (49%) than experiences with a lower score (13%)”, written this way this seems a bit misleading. Indeed, one could interpret that more intense NDEs (i.e. higher GNDES scores) are also more positive. But actually, this result might be biased by the focus of the GNDES on positive emotions.

-Lines 313-314: this entire sentence is misleading, “The central point in NDE research is that NDE do not only occur in healthy individuals but also during resuscitation.”. What do authors mean by healthy individuals? Do they refer to NDE-like (similar experience happening in absence of life threat)?


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: MIGRAINE AURA, A PREDICTOR OF NEAR-DEATH EXPERIENCES IN A CROWDSOURCED STUDY
Review round: 2
Reviewer: 1

Output:
Basic reporting: The writing of this revision is clear and unambiguous.  Literature background is very thorough.  Article structure is professional.  Results are relevant to the hypothesis.
Experimental design: This original research has a well-defined research question that explores a new potential predictor of near-death experiences, which may have implications for understanding their underlying physiological mechanisms.  The investigation was performed ethically and the methods described in sufficient detail to permit replication.
Validity of the findings: As the authors suggested, their findings from this crowdsourced internet study should be replicated conceptually using other methods that do not rely on internet respondents.  Underlying data are adequately provided and appear robust and statistically sound.  Conclusions are well stated and limited to supporting findings.  Speculation is now identified as such,
Additional comments: This revision is a significant improvement over the original submission, in that several points are now clarified that were ambiguous or insufficiently supported in the original.


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: MIGRAINE AURA, A PREDICTOR OF NEAR-DEATH EXPERIENCES IN A CROWDSOURCED STUDY
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: Thanks to the modifications, the experimental design is now clearer.
Validity of the findings: no comment
Additional comments: The authors addressed the issues raised by the reviewers. The manuscript has been improved.


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: MIGRAINE AURA, A PREDICTOR OF NEAR-DEATH EXPERIENCES IN A CROWDSOURCED STUDY
Review round: 2
Reviewer: 3

Output:
Basic reporting: all comments have been addressed
Experimental design: no comment
Validity of the findings: no comment
Additional comments: all comments have been 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: PHOSPHOLIPASE A2 FROM KRAIT BUNGARUS FASCIATUS VENOM INDUCES HUMAN CANCER CELL DEATH IN VITRO
Review round: 1
Reviewer: 1

Output:
Basic reporting: In this paper, the authors evaluated the cytotoxic effects of a new Phospholipase A2 from krait B.  fasciatus venom on two cancer cell lines. The work was well done and the paper is well written. Literature references, as soon as, introduction and results demontrate the relevance of the work.
Experimental design: The authors must inform the Ethical statement (scientific committee of the university)
Validity of the findings: Because the authors did not tried to go further in analyzing some possible mechanisms for Phospholipase A2 effects, the results are still preliminary. In addition, there are some concerns that must be addressed before considering publication. My main concern is if the cytotoxicity may be similar for non-tumor cells and it may be a limitation of the potential applications, as no control with no-tumor cells are showed. A justification and at least a comment about it should be included in the discussion, and expressed as a potential limitation in the final conclusions, and in the abstract.
Additional comments: Minor comments:
Results
1- The authors revealed a protein with molecular mass of about 13 kDa possessing cytotoxicity. From my point of view, the authors should demonstrate the protein purity by using electrophoresis or Mass Spectrometry and not the way they did considering they isolated a new PLA2.

2-Figure 1. Please, full information should be provided in the figure legend regarding the purification of PLA2. For example: Gel filtration of Bungarus fasciatus crude venom on Superdex HR75 column. Crude krait B. fasciatus venom (30mg) separated by gel-filtration on the Superdex HR75 column 86 (1x50 cm) equilibrated with the 0.1 M ammonium acetate buffer (pH 6.2). Flow rate 0.5 ml/min and fractions (x mL) was coleted. The eluate was monitored by spectrophotometry (OD=280nm).
All figures for legends need to be standardized with complete information. So, we invite the authors to add the precise information.
3-Figure 2. B. fasciatus venom. The correct is B. fasciatus venom in italic.
4-Figure 4. Legend: Cytotoxicity of fractions obtained after reversed phase chromatography against human MCF7 and A549 cell lines: “The statistical results are reported in Tables 2 and 3”. This information can be removed and replaced by: All results were analyzed by t-Test and are presented as the mean ± SEM (standard error of the mean).
5-Figure 5
Legend: Authors say informed morphological changes of the A549 (A-B) and MCF7 (D-F) cells after treatment with “various fractions” of crude venom but they compared only two fractions (BF3 and BF3.3). Please, correct the information.


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

Input:
Title: PHOSPHOLIPASE A2 FROM KRAIT BUNGARUS FASCIATUS VENOM INDUCES HUMAN CANCER CELL DEATH IN VITRO
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript entitled-“Phospholipase A2 from krait Bungarus fasciatus venom induces human cancer cell death” by Tran et al. describes the characterization of anticancer activity of a RP-HPLC fraction from Bungarus fasciatus venom against MCF-7 and A549 cells. The anticancer activity was assayed by MTT-based cytotoxicity assay. Although it is an interesting study; however, the manuscript in the present format shows some preliminary and incomplete data. To my opinion few new experiments have to be performed for a more meaningful and conclusive outcome of this study. Several potent anticancer drugs are available in the market; therefore, the opportunity of discovering a new anticancer agent from snake venom should be highlighted in the Introduction section of the manuscript.
Experimental design: 1.  The purity and mass of the most active RP-HPLC fraction 3.3 were not determined and therefore, purity of preparation is under a question.  Purity and molecular mass of the PLA2 enzyme must be determined by SDS-PAGE as well as MALDI-ToF-MS analyses.  Further, line 217-218. The LC-MS/MS data suggests that the sample is a heterogeneous mixture of several isoforms of basic PLA2s which further raised question on the purity of preparation.  This is a single PLA2 enzyme or mixture of several isoforms? Studies have shown that a minor contamination of some other toxins in PLA2 preparation can make the latter more cytotoxic and hemolytic. Therefore, purity of preparation must be established.
2. Multiple sequence alignment of tryptic fragment of B. fasciatus PLA2 enzyme with homologous proteins deposited in the NCBI database should be shown, may be as a supplementary Table.
3. It has been claimed that RP-HPLC fraction is a PLA2 enzyme. I suggest that the enzyme activity must be determined by biochemical assay. Further, purity of preparation in each step of fractionation, protein yield, and specific activity should be shown in a tabular form. The SDS-PAGE image of protein(s) from each fraction, starting from crude venom, gel filtration to RP-HPLC fractionations, should be shown.
4. Gel filtration chromatographic profile (Fig. 1). The separation of venom proteins was very poor and therefore, most of the proteins were eluted in a single sharp peak at around 60 min suggesting elution of 3FTxs of venom in this peak. Theoretically, PLA2 should elute before 3FTxs. However, elution of PLA2 with 3FTxs suggests complex formation of venom proteins. I suggest re-fractionation of venom to get a better separation and do the PLA2 assay of each fraction. Further, the gel-filtration column may be calibrated with protein molecular markers.
5. No positive control (anticancer agent) was used in this study. Literature survey shows that anticancer activity of chemical drugs are much higher than the PLA2 under study. Further, anticancer activity of this PLA2 is found to be much lower as compared to many snake venom proteins/peptides showing cytotoxicity against breast cancer cells. Therefore, it is difficult to envisage the anticancer potency of this PLA2 enzyme and its application as anticancer agent in future. Authors need to take a note of this point and discuss in their manuscript.
6. What is the cytotoxicity of this PLA2 enzyme against normal cells? This should be assessed to show that it does not have any harmful effect on normal cells. However, if this PLA2 also shows cytotoxicity against normal (non-cancerous) cells than its application as anticancer agent is doubtful.
7. Snake venom phospholipase A2 enzymes are known to exert toxicity.  I would suggest to determine the in vivo acute toxicity of this PLA2 in a rodent model; otherwise, the therapeutic use of this protein as anticancer agent, as proposed by authors, will raise questions and doubts in the mind of readers.
Validity of the findings: 1. The venom was obtained from a single snake or represents a pooled venom from several snakes? If it was a pooled venom please indicate the number of snakes. Who has taxonomically identified the snakes?
2. The concentrations of PLA2 should be shown in molar mass rather than in µg/ml.
3. Line 116. The results are mean of how many experiments (n)? Please indicate.
4. Tables 2 and 3 should be shown as supplementary Tables.
5. Figs. 1 and 3. The X-and Y-axes labeling are missing.
6. Figs. 2 and 6 should be shown as supplementary figures.
Additional comments: The manuscript in the present format is not suitable for publication. Author may consider my suggestions for further improvement of this work.


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

Input:
Title: WARM TEMPERATURES, COOL SPONGES: THE EFFECT OF INCREASED TEMPERATURES ON THE ANTARCTIC SPONGE ISODICTYA SP.
Review round: 1
Reviewer: 1

Output:
Basic reporting: The language is clear and unambiguous.

Introduction should include more detail about the choice of study organism and the temperature conditions experienced at the collection site.

Figures are relevant and are discussed well in the text.
Experimental design: Methods are detailed and well described.
Validity of the findings: Data are provided as supplemental information. A link to a table showing all available annotations and expression values for contigs in the reference transcriptome would be useful for corroborating results described in the manuscript. The transcriptome described here is a valuable addition to available sponge sequence resources.

Statistical analyses and thresholds used are suitable for low/no replication. However, the lack of a control replicate seriously limits confidence in the validity of the identified differentially expressed genes, as well as the strength of conclusions that can be drawn from the data. Therefore, interpretation of results needs to be carefully considered and limitations must be clearly stated where applicable.

While the analysis provides some interesting insights on the temperature response of this species, improvement of the annotation pipeline may allow more sequences to be assigned and included in the interpretation of temperature-responsive functions.
Additional comments: Line 87: Additional information on Isodictya is needed. In particular, why was it selected to test the effects of warming in the WAP? Also, what is the typical temperature range experienced by the sponge in its natural habitat?

Line 174 and 268: Were transcripts clustered after assembly to minimize redundancies?

Line 291: Very few genes (~23%) were annotated by blastp alignment at an evalue cutoff of 1x10^-6. This has resulted in much of the data being disregarded from the analysis. Have the authors tested annotation at less stringent evalue cutoffs to find a suitable setting that would allow more sequences to be annotated without necessarily compromising specificity?

Line 293: Were the Isodictya sequences compared to other available sponge sequences, particularly other cold-adapted species? Any similarities might provide clues to the repertoire of genes that are critical for survival in this type of environment or genes that may respond to temperature perturbations.

Line 309: Was domain annotation performed for all predicted peptides? What percent of the predicted peptides have recognizable protein domains. Analysis of domains present in differentially expressed transcript sets would also help in inferring temperature-responsive cellular pathways even in the absence of gene ontology annotation. A summary figure or table for all annotation data would be useful to show how much of the assembled transcriptome was translated, how many have recognizable domains, how many have matches against various databases, and how many have annotation assignments.

Line 313: Annotated peptides make up only 23% of the reference transcriptome (or ~41% based on KEGG annotation), which means that majority of the sequence data (~77%) is disregarded from analysis. How does this compare to other de novo assembled transcriptomes?

Line 333-334: The statement that “replicate samples at 3C are almost identical but there was a slight fluctuation in the 5C samples” is vague and is not apparent from the figures. Please rephrase.

Line 335: Clarify if figure 2A show the correlation based on the full transcriptome profile of each sample or from expression of just the differentially expressed genes.

Line 340: The major difference apparent in figure 2B is that some genes are present in the control but less so in the heated samples. It’s also apparent that there is high intercolony variability in terms of transcript expression. Thus, if there were another control replicate, the set of differentially expressed genes would very likely change and this should be emphasized here. Also, please revise the statement that “the initial findings are already compelling.”

Line 363: Could the similarity between the 3C and 5C treatments be due to the short duration of the treatment (4hr) rather than due to the transcriptional machinery operating at max capacity?

Line 416: “their” to “the”

Line 424: What would it mean for downregulation of TNF receptor binding genes at elevated temperature?

Line 429: This section is quite interesting. Perhaps the section header should read ‘target genes’ instead of ‘target signaling cascades’ to more broadly cover the types of genes discussed.

Line 430: Rather than “un-annotatable genes” one might say “genes with no gene ontology assignment.” Note that the number of annotatable genes likely depends on the stringency thresholds applied. This is why it would be useful to look at top refseq blastp hits at varying evalue cutoff.

Line 496-500: Need more detail on how these genes relate to the temperature response and processes of molecular adaptation. Are these usually identified in heat stress studies?

Line 501-512: This paragraph could be moved elsewhere as it seems out of place between paragraphs discussing transcription factors and morphogens.

Line 588-591, 617-619, 627-619, 639-639: It’s an interesting idea that cold-adapted organisms have an “all or nothing” response to warming. However, it would be good to provide alternative explanations for why not much difference is seen between these temperature treatments. Are 3C and 5C within or outside the normal range experienced by the sponge in the WAP? If these are both extreme temperatures for the sponge, perhaps the molecular and cellular effects of 3C or 5C exposure would be similar. As noted previously, the similarity in the response of the sponge to 3C and 5C treatments could also be due to the short duration of the experiment, rather than a diminished ability to deal with high temperature exposure, and should be indicated.

Line 632: Remove “excellent”

Line 634: change “begun to identify” to “identified” and remove “will”

Figure 2: Please check legend. Based on the methods, computation of expression values was done by RSEM but differential expression analysis was conducted using edgeR.


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: WARM TEMPERATURES, COOL SPONGES: THE EFFECT OF INCREASED TEMPERATURES ON THE ANTARCTIC SPONGE ISODICTYA SP.
Review round: 1
Reviewer: 2

Output:
Basic reporting: DNA data checks: fulfilled
Field Study permits: fulfilled
Basic reporting:
This manuscript is well written, with a very good standard of English. In places the manuscript is over-written, in terms of excessive explanation or over-justification given in some places, but specific examples will be listed in general comments below. The referencing is generally OK, but I have a few suggestions as to more suitable references in certain places (again noted in general comments below). There are a lot of references (90), which is rather excessive for this type of manuscript and could be cut back a little to, I would suggest 60-70 at most (which is recommended by a lot of journals, such as Global Change Biology). I appreciate this is sometimes difficult, but it does help to critically evaluate whether all the references are needed and I would encourage the authors to cut back a few references. The structure of the manuscript conforms to PeerJ standards, but fails with respect to statistical rigour.
Experimental design: Experimental Design:
The authors, correctly state this this experiment is an acute heat shock experiment. However, they then go onto discuss this with relevance to IPCC predictions and climate change. These are simply not equivalent. With a 4 hour experiment, the animals could still be in a state of resistance and this does not allow extrapolation to long-term chronic responses to climate change. The authors are correct when they state that little is known about responses to change in Antarctic species, therefore there is some novelty in these data, particularly with regard to the presence of a heat-shock response. What they should have done, which is much more appropriate is to compare these data with other acute heat shock experiments in temperate and tropical corals: do you get the same responses? Is something different? However, this does require similar acute experiments to have been performed in other coral species.
On a more serious note, which the authors admit: there is insufficient replication in this experiment with only 1 control and 2 each of the temperature treatments. This somewhat invalidates the statistical analyses, especially as there looks to be quite a few differences between individuals at 3°C and 5°C and they are only comparing against ONE control sample. There is clearly variation between individuals and at least n = 3 should have been used for each treatment in this experiment. This is a serious experimental flaw and significantly reduces the quality of the output and validity of the findings.
Validity of the findings: I appreciate that a lot of work has gone into this article, but the limitations of the experimental design are not reflected in the results and discussion. The latter would work much better if focussed specifically on comparisons of similar experiments performed in temperate and tropical sponges, not climate change.
Additional comments: Specific comments:
L60. Ref 9 rather outdated and both are best replaced by Turner et al (2016) Nature535, 411-415
L61-64: description of IPCC 2001 results not necessary, as replaced by IPCC 2014.
L69: Suggest ref 14 replaced by Somero (2010) JEB 213, 912-920 which is more appropriate as he refers to importance of long-lived species, such as those in the Antarctic.
L82-85: duplication of line starting on line 64.
L86: refs 17,28,29 are not the best as these do not refer to processes over evolutionary time.
L92-97: not needed, as we are now all fully aware of the power of NGS.
L110-112: statement not justified as you cannot use results from a single 4 hour heat shock to predict chronic responses.
L139-141: again erroneous reference to comparability to climate change responses.
L236-242: No need to describe results or samples not included in the study.
L256: reference transcriptome: surely need to refer to Table 2 here?
L267-278: over wordy reads rather like a thesis including justifications. You can just refer to Table 2 which has all the relevant data. Reference to samples having little genetic variability not needed given sample sizes are severely limited.
L279: suggest you add BUSCO output to supplementary tables to avoid having to explain in detail (or add as a manuscript figure).
L285-287: no need to justify results in results section.
L292-299: not really needed. Lack of sequence similarity to other species almost certainly due to lack of closely related sequenced species.
L308-309: you can just refer to the table.
L311-315: again superfluous explanation.
L330-332: acknowledgement of lack of replication: a really critical issue.
L33: mention of “replicate” samples and correlation: there look to be quite big differences between samples in Fig 2B. Not sure that Fig 2A really shows anything, especially as 2B is far more detailed. Fig 2A could easily be deleted.
L337-339: disagree: there is variation between samples.
L340: disagree results are not compelling with lack of replication.
L356-362: No pint including Table 3 as most sequences have no annotation. This does not help with result interpretation.
L363-370: lack of difference between 3°C and 5°C is interesting, but given such little differential expression, little significance can be placed on the annotation of those genes.
L399: cytoskeletal proteins can also be involved in the stress response: see Tomanek et al. (2012) JEB 215, 3905-3916 and Serafini et al (2011) Comp Biochem Physiol D 6, 322-334 as examples.
L384-413: just a description of GO categories which are very vague and do not explain an awful lot and Fig 3 not very informative. Much better to concentrate on specific biological results coming out of data.
L430: disappointing to see emphasis on candidate gene approach as RNA-Seq is supposed to be discovery –led. Needs better justification.
L461: see Cascella et al (2015) PLoSONE 10, e0121642 for better description of inducible and constitutive forms of HSP and the issues of classification.
L496-500: does not really say anything concrete.
L501 onwards: need better comparison to temperate and tropical sponge data and include the relevant experimental regimes used for them to give context.
L532: reference to “slight error”: this can easily be checked by manual annotation and needs validation before you start to describe significance.
L561: careful on references to GSTs: big gene family and function depends on orthology assignments.
L572: Is this whole sequence, or just match to functional domains? Please check as this can make a big difference.
L611: more on GPCRs would have been useful
L618-619: “all or nothing” response: the animals could be in a resistance phase, as the acute shock was for such a short period.
L639: comment on “little wiggle room” difficult to justify given experimental design. These data document an acute response only.


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: WARM TEMPERATURES, COOL SPONGES: THE EFFECT OF INCREASED TEMPERATURES ON THE ANTARCTIC SPONGE ISODICTYA SP.
Review round: 1
Reviewer: 3

Output:
Basic reporting: The authors do a great job of providing extensive raw data files as well as tables and figures that describe particular aspects of the data set. Figure 6, however, needs to be enlarged with increased quality. In its current form, it is not possible to read the alignment, phylogeny, or amino acid identities. Also, for Table 4, it is unclear from the text how these genes were identified. Was a specific threshold change of expression used? It might help to include relative up and down regulation levels in the table.
Experimental design: The authors attempted to produce a transcriptomic data set with two technical replicates per treatment (control, 3oC, and 5oC). Their transcriptomes were of high quality and their assembly, testing for completeness, and annotation were well executed. It is unfortunate that one of their control replicates turned out to be a sample from a cryptic related species, but good that this was determined before moving forward with their transcriptomic analysis. This did mean, however, that for the control treatment, only one sample was sequenced. The authors are clear about this aspect of their experimental design and I don’t think that it invalidates their subsequent analyses, but I do think that more care should be taken in the interpretation of the data analyses (see below).

I am left wondering whether or not any other observations about the sponges in each treatment were recorded?  Did the authors note what happens when sponges are left at those temperatures for longer periods of time – how quickly do they die compared to the control? Lines 569-571 states that “It seems likely that these sponges are under severe oxidative stress, and the expression of these genes reflects this.” Are there other metrics from the experiment that were measured or observed?
Validity of the findings: RSEM was used to quantify gene abundances and this is a preferred method when a reference genome is not in hand. Thus, even with the one control sample, the software can quantify transcripts in reference to the de novo transcriptome assembly. However, the lack of replication in the control treatment precludes robust statistical analysis. For example, in lines 341-345 the authors discuss “significant” changes in gene expression. More occurrences of “significance” appear in lines 364, 379, 380, 385, 387, 398, 399, 405, 407, and elsewhere.  These sections should be clarified because it seems to me that statistical significance cannot be inferred when comparing the control (with a sample size of one replicate) to the 3oC or 5oC treatments. If the authors are not referring to statistical significance, then please define what is meant when the word “significant” is used.

The authors make good effort to connect their data to outcomes from other studies regarding thermal stress in sponges and other invertebrates. Further, there are interesting patterns of gene expression changes observed between the control sponge and the high temperature treatments. I do not agree, however, that the claim stated in the abstract and in the manuscript text, that the authors have demonstrated or studied how the sponges “adjust to environmental insult” is valid. Unless other metrics were measured that are not reported here, it seems to me that this study shows how gene expression patterns change when sponges are thermally stressed for four hours. Perhaps this could be referred to as a short-term thermal response of the sponges, but not an adjustment or “capacity to respond to thermal stress.” Further, it is interesting that the changes in gene expression between the 3oC and 5oC treatments were minimal (at least compared to the control). This is an important observation, but I’m not sure that it can be said that the data suggests “that moderate rises in temperature could cause stress at the limits of this organism’s capacity.” I don’t see how this was tested. I suggest revising this language to reduce the speculation (or state more clearly that this is speculation). Can you really say that changes in gene expression patterns (or lack thereof) are directly the result of cold-adapted sponges having limited range of tolerance? This may be a correlation, but causation has not been demonstrated.

Along these lines, the authors may want to modify their level of speculation regarding the role of particular gene products or pathways in the thermal response. Given that there are no functional studies related to the gene expression changes observed here, the most that can be said is that the data suggests particular genes may be involved in “thermal response.” For example, see lines 469-470, 479-482, 540-541, etc. I might suggest changing “likely” to “possible” and “confirms” to “suggests” to more accurately describe the data.

In reference to lines 632-635, I agree that the authors “generated an excellent reference transcriptome from a small number of replicate samples exposed to acute thermal stress,” and that they have “begun to identify the molecular responses” to short-term increased temperatures, but it is a stretch to say that these are the molecular responses “that these species will use to adjust to warmer ambient temperatures.” This part is quite speculative and not tested in this initial study. Please consider revising this sentence as well as 638-639. Again, without further testing, I’m not sure how this conclusion can be made.
Additional comments: This study is of clear importance and meaningful data sets were produced. The evaluation of gene expression changes was thorough. My main concern is regarding how the data are described in some places (e.g., whether or not “significance” should be used), and that some of the conclusions about the data are too speculative or have not been directly demonstrated by the study. If these issues can be revised, the manuscript will be improved.


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: WARM TEMPERATURES, COOL SPONGES: THE EFFECT OF INCREASED TEMPERATURES ON THE ANTARCTIC SPONGE ISODICTYA SP.
Review round: 2
Reviewer: 1

Output:
Basic reporting: The paper is well-written and pleasant to read. Sufficient background on the study has been provided. Figures and tables are clear and supplementary data is available.
Experimental design: Research question is well defined and methods are sufficiently detailed.
Validity of the findings: Although general concerns on replication remain, the authors have done a great job of incorporating the limitations of their study (e.g. short-term exposure and limited replicates) into the results and discussion sections. They have also sufficiently justified their choice of parameters for certain methods (e.g. use of refseq database and e-value thresholds for annotation). The conclusions of the paper are well-stated with limitations clearly indicated, as necessary.

The comparison of differentially expressed genes in Isodictya versus other sponge species is interesting as it confirms some common temperature-response pathways. This is a valuable addition to our current understanding on the temperature-induced responses of diverse sponge species.
Additional comments: The authors have sufficiently addressed reviewer comments and suggestions. They have managed to clarify the limitations/challenges of the study without minimizing the value of their findings. They also present recommendations for future studies that are certainly worth pursuing.


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: EFFECTIVENESS OF WATER-SAVING TECHNOLOGIES DURING EARLY STAGES OF RESTORATION OF ENDEMIC OPUNTIA CACTI IN THE GALÁPAGOS ISLANDS, ECUADOR
Review round: 1
Reviewer: 1

Output:
Basic reporting: This article deals with an approach to try to increase the probability of survival of different Opuntia species by using two different water-saving systems (Groasis and Cocoon)and in four Galapago islands. The manuscript is well written and clearly organized. Literature references are sufficient and cover quite well the state of the art and also facilitate the discussion of results. In general, the manuscript is well structured and figures and tables are clear and well prepared. This article is technical and refers to the application of previous tested water-saving systems, but applied to the establishment of different Galapagos-threatened Opuntia species, whose populations have been diminished by different factors and need to be re-established in their natural ecological sites; this is the main importance and contribution of this article.
Experimental design: The experimental design of this work is right, and was focused to test two water-saving as an approach to try to increase the survival of different threatened Opuntia species in their ecological environments. The application of these systems is perhaps no quite original since they have been previously tested in some other plant species; however, their application as an alternative strategy to try to increase the survival of endangered or threatened Opuntia species is the important issue. This research was carried out with rigorous technical and scientific standards and the methods were clearly and sufficiently described to be replicated, if necessary.
Validity of the findings: The findings of this work were in some way contradictory when compared with previous reports cited by the authors since, in general, only partial success was attained by using the tested water-saving systems, and in some cases detrimental effects on the survival of Opuntia species were observed; however, the partial success can be applied to improve the rescue of Opuntia species in specific sites. In general, the results are robust, statistically sound and controlled. However, perhaps they should be used to get more information; for example, the authors mentioned in Table 2 that 349 individuals of Opuntia echios var. echios were planted in Baltra and 601 in Plaza Sur, and no any observation or comparation between the behavior or the survival of the same species in these two sites was mentioned. This should enrich the findings. Conclusions are well stated and limited to support the results.
Additional comments: The authors mentioned in Table 2 that 349 individuals of Opuntia echios var. echios were planted in Baltra and 601 in Plaza Sur, and no any observation or comparation between the behavior or the survival of the same species in these two sites was mentioned. Some minors corrections and observations are indicated in the text of the attached file.


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: EFFECTIVENESS OF WATER-SAVING TECHNOLOGIES DURING EARLY STAGES OF RESTORATION OF ENDEMIC OPUNTIA CACTI IN THE GALÁPAGOS ISLANDS, ECUADOR
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript by Tapia et al. is well written and should be published after minor editing. The introduction does a good job at providing background into why it is important to develop technologies to restore the endemic Opuntia cacti in the Galápagos archipelago, Ecuador. The authors also provide justification that the type of technologies should focus on improving water availability to the cacti. The Groasis Waterboxx and “Cocoon” are the technologies evaluated in the study to help improve water availability. Despite the importance of these technologies to the study, they are not mentioned in the introduction. I feel the paper would be improved by providing specific justification for testing these two technologies. Either in the introduction or the methods it would also be of value to explain in greater detail than what is in the paper how the two types of technologies work to improve water availability.
Experimental design: This research is original based off of the location it is being applied and the species of focus. The methods employed is also highly rigorous.

The study design is somewhat challenging to understand. The author’s state: “The number of controls was maintained at one control for every five Groasis or Cocoon technology treatment replicates.” Why was there not an even number of controls as the other treatments in the study? This should be clearly explained in the manuscript. Consider incorporating this information into the text or Table 1 and let the reader know how many replications of each treatment there is. I also do not understand how you randomized where you planted the different treatments. I assume the study utilized a completely random design? How the different treatments (control, Groasis, or Cocoon) were applied at different islands is also not clear in the methods section
Validity of the findings: The authors do a good job at explaining the results of their research and in describing the general impact this research has on the study of restoration ecology.

I feel the authors have correctly interpreted the results of their data and their conclusions are well stated.
Additional comments: Lines 138 -139 Please define what these terms mean “good,” “regular,” “poor,” and dead. Also, note you did not put quotation marks around “dead” like the other descriptive units.

Figure 4 caption does not provide an adequate description data being displayed. Please explain define what each of the categories mean


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: EFFECTIVENESS OF WATER-SAVING TECHNOLOGIES DURING EARLY STAGES OF RESTORATION OF ENDEMIC OPUNTIA CACTI IN THE GALÁPAGOS ISLANDS, ECUADOR
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript is generally well written but contains a few minor English mistakes and typos to be corrected (E.g. ‘parenthases’; ‘Opunitas’). The title of the manuscript may somewhat be currently overselling the findings in the sense that it seems “restoration” should really be replaced by something more like “early stages of establishment” (unless of course authors can clarify this point to satisfaction). On this topic, it seems also important for the authors to give the reader some context about the life history of the cacti species in nature, like for example, how long would it take for the life cycle to be completed? (from germination to seeding). This would give a better appreciation of the significance of the results in the broader scheme of things (how much more time would it take for the planted individuals to recruit into reproductive individuals, and therefore how close really are we to really achieving restoration of a reproductive population from the planted seedlings?).
The manuscript generally makes use of many references although certain specific statements lack reference support. E.g. line 73 should be backed by a reference as it is not automatically understood that active planting should necessarily be critical in the current case. There are however about 20 references that are listed in the reference section but which not find in the text. I suggest that authors consider using a bibliographic software, e.g. the freeware Zotero when preparing their reference section.
Abstract: About “restoring Opuntia population”: Is it really what is being achieved or is it rather “early stages of establishment of Opuntia” that should be written. How long does it take for a plant to mature and what is the longevity of these species?
Ok, I don't necessarily feel too strongly about it, but I feel that the number of display items used (five figures and two tables) is slightly on the high side and that the authors could consider moving one or two, of the least important ones, to supplementary materials.
Experimental design: No comment
Validity of the findings: No comment
Additional comments: How did human activities substantially reduce Opuntia population? Is it by direct destruction or changing the environment away from what is optimal for the Opuntia? For example, has human activities changed the soil parameters away from what is required by Opuntia? Why for example are the Opuntia not simply able to regenerate by themselves?

The authors mention that extreme aridity poses a major obstacle to Opuntia restoration, but are not these Opuntia precisely used to extreme aridity?  Or do the authors mean that aridity has increased due to human activities, and that consequently it is now harder for Opuntia to regenerate in the wild? (Its mentioned that increased El Nino events pose a threat, but are these not making for wetter episodes rather than more arid ones?). I think it would be helpful to detail a bit why ‘extreme aridity’ poses such an obstacle to restoration, because if Opuntia are adapted to this, then it should be the wetter conditions posing a threat rather.

Minor comments:
L 34: Please specify the period over which this increased survival has been achieved, in the abstract.
L 70-71: The pronounced declines: Please can you specify at least roughly by how much and over which period, as these can be variously interpreted.
L 74: Please specify if such ‘severe aridity’ is something new (is it getting drier, and if so at what rate?) or whether it is just what the cacti are normally subjected to.
L 76: About ‘historically abundant’: It is a bit hard to figure out as it is a relative term. Can the author quantify at least roughly how much more abundant it  was?
L 124: Can the authors briefly give the reader at least a rough appreciation of such declines? (e.g. halved in X years?)
L 134: Please specify whether this was done because only on those two islands, (among all those where the planting were done), had iguanas (or whether iguanas were also present on the other islands where the study was done).
L 125: ‘seedlings’ were planted rather than sown.
L 142: Please give the reader an idea of the extent of mortality in that age class, relative to later classes.
L 172: Write ‘seedling’ instead of ‘seedlings’
L 265-266: About ‘more than doubling the population’: Do you mean that the plants reached maturity? Or was it more just an increase in number of juveniles?
L 266: Any idea that could be given to the reader about what would have been the original natural densities? And what are the likelihood that these plants would eventually reach maturity (or are they already mature?)
L 295: What is the tune of these costs?

About the Cocoon and Groasis technologies: It is hard to visualize what these look like. Could the authors please provide some description, for example in supplementary material? This may be particularly relevant as size of cocoon is mentioned in discussion to explain the performance of plants treated with this technology.

I would suggest that ‘altitude’ be changed to ‘elevation’.

Referencing (in the body of the manuscript)
A) et al., should be italicised (= et al.,)
B) Line 80: Jaramillo, Cueva, Jiménez, & Ortiz, 2014 should be Jaramillo et al., 2014 [as cited later in the ms]
C) Line 133: Jaramillo et al., 2017 does not appear in reference section
D) Line 329: Jaramillo pers. obs. Should be P Jaramillo, date?, pers. obs.)

Reference section:
Please review, using the journal's style with a referencing software such as Zotero and Endnote would solve most problems found.
A) Spacing is not standardised
B) Add DOI (when available)
C) Section do not follow journal format of referencing: 1) Author initials should not have dots, 2) for 2 more authors, do not add & for the last one; 3) year should not be within parenthesis. For example Aldridge, C.L., Boyce, M.S. & Baydack, R.K. (2004). should be Aldridge, CL, Boyce, MS, Baydack, RK. 2004.
D) Journal name is not always italicised (line 389)
E) Article title should not have all words starting with capital letters like reference starting on line 404
F) Line 413: change var. Orientalis to var. orientalis
G) Line 471: change ‘(ed.).’ to ‘ed,’
H) Line 472: pp. 50-5. There is a missing number (50-55?)
I) Line 481 and 521: Remove italics from the publisher
E) Line 506: Second author is Li Z and not Zhiyang L
J) Line 506-507: Please italicise binomial.
K) Line 526: Remove volume and page as it is a software
L) Lines 406, 533 and 537: Add italics to journal

Many listed references are not cited in the text:
1) Aldridge, Boyce & Baydack (line 378)
2) Bensted-Smith (line 382)
3) Bisconti (line 384)
4) Campbell et al (line 396)
5) Cardinale et al (line 400)
6) Chapin et al (line 410)
7) Cruz et al 2005 (line 417)
8) Cruz et al 2009 (line 420)
9) Defaa et al (line 423)
10) Eckhard (line 432)
11) Ferwerda (line 437)
12) Ferwerda & Moolenaar (line 439)
13) González-Pérez, & Cubrero-Pardo (line 448)
14) Hamman (line 455)
15) Hanson & Campbell (line 457)
16) Helsen et al (line 459)
17) Lavoie et al (line 501)
18) Larson et al (line 503)
19) McDonald-Madden, Baxter & Possingham (line 511)
20) Poulakakis et al (line 525)
21) Snell, Stone & Snell (line 542)

Appendix (Opuntia_data.csv)
A) Add ‘n’ in the heading of column A.
B) Change ‘altitude’ to ‘elevation_m’ [column L]
C) Do you have such resolution to have elevation with two decimals?
D) Treatment cocoon from 2016-2018, treatment groasis and control 2013-2018


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: DIFFERENT DISTANT METASTASIS PATTERNS BASED ON TUMOR SIZE COULD BE FOUND IN EXTENSIVE-STAGE SMALL CELL LUNG CANCER PATIENTS: A LARGE, POPULATION-BASED SEER STUDY
Review round: 1
Reviewer: 1

Output:
Basic reporting: The quality of written English is poor and needs significant language editing to facilitate reading.
Abstract:
Abstract needs to be rewritten:
Clearly formulate the aim of the study.
Line 28: you should write “11058 ES-SCLC” instead of  “11058 SCLC”.
Line 31: “subtle differences” – please specify compared to what?
Lines 32, 33, 34: The results are scarce and need to be rewritten to better present your findings. Also make sure to specify the units of tumor size.
Line 35: The conclusion does not match your results and it is unclear how you can find difference “between tumor size and distant metastasis patterns”. Logistic regression shows associations, not differences.

Introduction:
Lines 44-45: “fewer study explored about the association between tumor size and the sites of distant metastasis”. Please add references there, and expand on the findings of those previous studies. This is essential to show the gaps in the literature and justify the need for your study.
Line 47-48: Sentence is not clear enough; please rephrase.
Line 50: “...evaluated the effects of several clinical features on the metastasis distribution in ES-SCLC. “ Specify what you meant by “several clinical features“.
The aim was not clearly formulated.
Provide units in all tables and figures.
Raw data is not provided.
Experimental design: In the section on patient selection, you should specify that this was a retrospective study.
Inclusion/exclusion criteria for patients are not well defined.
On which samples was the diagnosis made? (Surgical or biopsy/cytology?)
You need to specify at which time point the presence of metastases was noted (at the time of initial diagnosis or during the entire course of disease).
How many patients had one, two or more metastases? Please specify.
Which type of staging was used? Pathological or clinical TNM classification?
Provide information about the smoking status of patients.
No information about survival was provided. This would be really useful to add to the manuscript.
It is not clear why Ethics Committee of the Shandong Cancer Hospital gives the approval for using US data? Please explain. If there is a number of the ethics approval, specify it in the Methods section.
In the section “Statistical analysis” you wrote that univariate and multivariate logistic regressions were used, but there is no data for univariate analysis in the paper. Moreover, clearly state what was considered as dependent variable and what were potential predictor variables. Specify whether you used binomial or multinomial logistic regression.
In the table 2 (logistic regression) you could have used age as a continuous variable. What was the rationale for binarizing age to two groups (<65 and >65)?
What was the rationale for choosing three tumor size categories (<3, 3-7, >7 cm)?
Validity of the findings: Results are written with a lot of repetitions.
Specify the units of tumor size.
The text in the section “Metastasis pattern on different tumor size” completely repeats the data from Figure 1. This should be avoided.
You wrote that tumors smaller than 3 cm were “unlikely to metastasize”, but your data shows that they metastasized in more than 20%. Please check and correct.
Results of the statistical analysis (logistic regression primarily) are misleadingly presented.  For example, the sentence “The results showed that the incidence of patients with tumors 3-7 was significantly higher than patients with tumor 104 size ≤3(OR 1.165, 95% CI 1.055-1.287，P=0.003)” is misleading; namely, logistic regression does not compare the incidences of patients but shows how much higher or lower the risk (odds ratio) of certain event (in this case presence of a particular metastasis) is depending on the specific predictor variable.
There are no comments on the relationship between the metastasis pattern and age, gender, race and N-status.

Discussion:
Line 121: Add citation for “The incidence of distant metastasis at the time of the initial diagnosis of SCLC was more than 60%”. Also add more citations for “most common metastatic sites were the liver, bone, brain, lung and adrenal glands[3]”. I suggest the authors to consult the following recent articles:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5541967/
Lines 132-134: This should be moved to the Introduction section.
Line 143-145: Add reference. Moreover, this does not explain why specifically tumors in the size range between 3 and 7 cm spread to bone. Comment on the possible explanation for this observation.
Line 157: Please revise the sentence starting with “The predictive value...“.
Line 167-170: The text “except lung” contradicts the table 2 where it is clear that high N status (N2 or N3) reduce the risk of metastasis only for brain, while N3 increases the risk of lung metastasis (cf. your table 2).
Lines 173-174: Please check the sentence and rephrase for improved clarity.
Line 177-178: Please cite appropriate recent articles, such as:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3698258/
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5541967/

Conclusions:
Other clinical factors (except tumor size) were neglected.
Additional comments: No additional comments