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: PRODUCTION AND PURIFICATION OF MANNAN OLIGOSACCHARIDE WITH EPITHELIAL TIGHT JUNCTION ENHANCING ACTIVITY
Review round: 1
Reviewer: 2

Output:
Basic reporting: In this study, Nopvichai and colleagues produced and tested the MOS. They found MOS could enhance the tight junction integration T84 cells. In general, the authors need to do a better job in organizing and writing the text. To be specific, I have some concerns about this work that should be addressed before further consideration.

1. Previous study (Silvia Torrecillas et al., Fish & Shellfish Immunology, 2013) have shown that MOS could enhance intestinal epithelial barrier health status, including enhancing the integration of tight junction. The authors should discuss and compare previous study with the current one to show the improvement or importance of their work.

2. The authors should be more careful with the references. There are a lot of text missing references. Meanwhile, it is inappropriate to put all the references at the end of one paragraph. The authors should specify the references to each point.

3. Figure 5: The authors should provide following information of this figure. a) The concentration of MOS used in this experiment. b) N (biological repeats) for each group. c) The authors only mentioned the software for statistic analysis. They should provide the statistic method used in the analysis.

4. Since MOS had impact on tight junction, the authors should test the expression level of certain tight junction genes via RT-qPCR and westernblot.
Experimental design: no comments
Validity of the findings: no comments
Additional comments: no comments


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

Input:
Title: CONFLICT OF INTEREST EXPLAINS THE SIZE OF STUDENT EVALUATION OF TEACHING AND LEARNING CORRELATIONS IN MULTISECTION STUDIES: A META-ANALYSIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The coverage is good, but the paper omits a pivotal small multi-section study published in 2014 by MacNell et al. and a large multi-section study published in 2016 by Boring et al. (also reported by Boring in 2017: https://www.sciencedirect.com/science/article/abs/pii/S0047272716301591). Those studies are generally consistent with the findings of the current ms., though: negative association, and the authors were from Anthropology, Sociology, Economics, and Statistics. However, the methodology in the current paper would have counted the Boring et al. (2016) study as 2 COI and the Boring (2017) study as one COI (Boring and Stark held administrative positions as well as academic positions; Boring was responsible for SET at her institution). I think there were no COIs for the MacNell et al. study.
Experimental design: Non-experimental. Research questions were well defined. It was not clear whether the ordering of the severity of COI was developed a priori (before computing the correlations) or was simply reporting the order of the strengths of association. In particular, it was surprising to me that SET instrument authorship was less strongly associated with the correlation than was role as an administrator, if the COI hypothesis is correct (since it seems like a stronger COI).

If the ranking is intended to be a conclusion, a permutation test of the association between "strength of COI" and "strength of correlation" could be performed. Within a parametric framework, one could assess the confidence that the empirical ranking is "correct."

I have not used the R packages the paper mentions, so I do not happen to know whether there are parameter settings one needs to select, nor whether the authors used default settings. The paper should give the versions of all software packages and libraries used (R, meta, metafor, and metasens).

Other than that, the analysis seemed replicable from the tables.

Supporting data to show, paper by paper, the reasons the authors arrived at the particular COIs (columns 4--10 of Table 1) would be valuable, as would making Table 1 available in machine-readable electronic form (e.g., .csv).
Validity of the findings: The papers within the meta-analysis seem to be an appropriate collection to study, if not exhaustive.

The reliance on meta-analysis (with or without random effects) as a statistical technique does not make the paper more persuasive, because the assumptions of meta-analysis are baroque and disconnected from reality.

I think the correlations and figures tell the story on their own. If the authors want to assess some measure of statistical significance of the associations, a suitable permutation test would be more appropriate and convincing than meta-analysis with random effects. In particular, it could test hypotheses such as "year of publication is irrelevant to the correlation the study finds" or "number of COIs is irrelevant to the correlation the study finds," etc., without requiring the assumption that different studies represent independent samples from normal distributions of normalized effect sizes, with means that (may) depend on other covariates.

The tables need more informative captions, including explanations of the variables.
Additional comments: Boring et al. (2016) has two online reviews, one positive and one neutral. The neutral review is from an employee of IDEA, Jason Barr.  IDEA also blogged about the Boring et al. paper, also misattributing quotations from the literature to the authors: https://www.ideaedu.org/Portals/0/Uploads/Documents/Response_to_Bias_Against_Female_Instructors.pdf

The IDEA blog author evidently misread Boring et al., since most of the criticisms in the blog post are contradicted by the paper itself.

Hilariously, the IDEA blog criticized the fact that the Boring et al. paper was post-reviewed, despite (1) the fact that their own employee was one of the post reviewers, and (2) many of IDEA's papers used as evidence that there is a positive association between SET and learning are self-published tech reports that were not peer reviewed at all.


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: CONFLICT OF INTEREST EXPLAINS THE SIZE OF STUDENT EVALUATION OF TEACHING AND LEARNING CORRELATIONS IN MULTISECTION STUDIES: A META-ANALYSIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: .
Experimental design: .
Validity of the findings: .
Additional comments: Attached my review of the article by Uttl et al.


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: CONFLICT OF INTEREST EXPLAINS THE SIZE OF STUDENT EVALUATION OF TEACHING AND LEARNING CORRELATIONS IN MULTISECTION STUDIES: A META-ANALYSIS
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment.
Experimental design: # I still cannot endorse the statistical analyses:

1. The confidence intervals for correlation coefficients are based on the assumption that the data are samples from bivariate normal distributions, which is false. The confidence intervals are meaningless. Permutation tests of the hypothesis that the correlation is zero would be more appropriate for this part of the analysis.

2. The permutation tests for correlation between COI and strength of association are not valid because the COI scale was established post hoc, after examining the data. This could be accounted for by maximizing the correlation coefficient over partial re-orderings of the COI scale, in each iteration of the simulation. That will require some coding, but not a great deal.

# There is also a real question about data quality. Author affiliations generally do not include enough information to determine whether some of the COIs considered in the manuscript exist. For instance, in Boring et al. (2015), Boring had a research position but also had institutional responsibility for SET at SciencesPo, and Stark was department chair, responsible for evaluating faculty for employment decisions. Neither institutional role would be evident from looking at the paper. Boring's role would not be ascertainable from a web search, and a current web search would not uncover Stark's prior role. I have no reason to believe that there is no similar issue with the publications analyzed in the present manuscript. I think a cruder taxonomy would be easier to defend (works for a company that sells SET, designed an SET instrument, both, or neither). Within universities, faculty administrative roles are ephemeral and hard to ascertain.
Validity of the findings: I think the overall qualitative conclusions are true, but the quantitative conclusions are not valid: they are based on misapplications of statistical methods. They ignore distributional assumptions and do not take into account the post facto tuning of the COI scale.
Additional comments: I really like the hypothesis and the general approach--and I believe the conclusions--but I can't overlook the misuses of statistical methods. I'm truly sorry.


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: CONFLICT OF INTEREST EXPLAINS THE SIZE OF STUDENT EVALUATION OF TEACHING AND LEARNING CORRELATIONS IN MULTISECTION STUDIES: A META-ANALYSIS
Review round: 2
Reviewer: 2

Output:
Basic reporting: The manuscript is very readable and the authors responded well to criticism of their earlier version. I have only two comments. I think that the two money quotes at the beginning of the paper are inappropriate. First they would only apply to publications by SET publishers and not the other COIs, second they imply that COI resulted in deliberate biasing of findings. The ample literature about the influence of corporate funding on scientific results has never shown (nor assumed) that the bias was due to deliberate decisions by researchers. And the authors are careful in not suggesting this either. Therefore, I suggest that they skip the two quotations at the start of the paper.

I also wondered whether the authors did not engage in a bit of overkill in presenting their results in figures and tables. For example, in Figure 2, panels C and D appeared to me a bit redundant. I think that the bottom right panel could be eliminated without great loss of information. Similarly, the random effect meta-analyses are sufficiently discussed in the text as to make Tables 3 and 4 redundant.
Experimental design: not relevant
Validity of the findings: The findings are extremely relevant
Additional comments: The Uttl et al. (2017) meta analysis was an extremely important publication. Although there was overwhelming evidence that SETs were biased, the fact that they seemed to correlate with grades in between class comparisons suggested that there was some degree of validity, which probably could be uncovered, if one eliminated the biasing factors statistically (i.e., Greenwald & Gillmore, 1997). After their meta-analysis, this procedure seems unlikely to be successful. The present paper is not only and important addition to the literature on the (lack of) validity of the SET, it also adds to the growing literature on the influence of COI on research findings. Thus, I hope that it will be published soon so that I can cite it.


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

Input:
Title: THE ROLE OF DISPERSAL FOR SHAPING PHYLOGEOGRAPHICAL STRUCTURE OF FLIGHTLESS BEETLES FROM THE ANDES
Review round: 1
Reviewer: 1

Output:
Basic reporting: Comments on Munoz-Tobar & Caterino
These comments are numbered as per the numbered comments on the ms.
1.	The abstract mentions 4 species of Dercylus, yet the Introduction (line 91) lists 3. The numbers are complicated by the authors’ recognition of several undescribed species that were elucidated by the genetic analysis. You should go back through the manuscript and determine how best to list these species (e.g. “three described and two previously unknown, undescribed species”, or the like). Also, I have flagged various situations where there is conflict between the plurality of the subject and verb tense used. Also, lines 75-76 do not include a sentence. The final manuscript should be gone over for correct English usage.
2.	The authors focus on genetic heterogeneity, however the geographic aspect of genetic variation is more important. Many species across a higher taxon have similar levels of heterogeneity. It’s how that heterogeneity is partitioned geographically that is information about popluational differentiation.
3.	You should cite the original authors’ paper within which the Dercylus are described, i.e. include it in the refernces. It is only when taxonomists are given credit for their work that systematics will survive.
4.	The fossils used to calibrate the Dyscolus phylogenetic analysis are not adequate. Instead of Larsson (1978) you should cite Schmidt (2015, Zootaxa 3974: 573-581) who actually described a fossil Limodromus (= Platynus) species dated as Eocene. Also, Scudder (1890, NOT 1980) was a horrible taxonomist, and often incorrectly assigned his fossil taxa to genera. Among the two “Agonum” from the Green River Formation, Platynus caesus looks to me to actually be a Clivina! His Platynus senex (p. 519, Plate 7, Fig. 38) could be an Agonum. I would have no problem with that assignment, especially given Liebherr and Schmidt (2004, Deutsche Entomologische Zeitschrift 51: 151-206) who assigned the origin of Agonum as Eocene based on cladistic biogeographic information (see Figs. 97, 105). Examination of the MCZ photo of P. senex--https://mczbase.mcz.harvard.edu/guid/MCZ:Ent:PALE-1844--suggests that the beetle could be a Europhilus like species. It is not like P. variolatus as Scudder suggests as the humeri are broadly rounded, but it can credibly serve as an Eocene placemarker in the plylogenetic analysis. Finally, the citation of Scudder is incomplete as well as incorrect as to year. I suspect this is an artifact of over-reliance on Endnote, which would have trouble with such a long title, volume number, special periodical, etc. As this reference is available via BHL, there is no excuse regarding how to properly cite it.
5.	The neutrality tests for both genes have internally conflicting data. You state the populations are going through expansion, but a high proportion of rare alleles is not present. You cannot present both aspects. Better to delete these details. That is, skip mentioning whether the populations are expanding or not.
6.	You mention individual SIMT208H6 which appears in the COI tree. But it is not in the CAD tree, where SIMT208H5 assumes the outgroup position. Also in 5A, SIMT208H5 is labeled SIMT028H5. The specimen numbers need to be gone over carefully to get rid of this sloppiness. And why exactly should SIMT208H6 not be assigned to Dyscolus alpinus in 5A, especially given the vagaries of mtDNA in accurately circumscribing species limits? AND FINALLY, why is Dyscolus spp. SIMT208H5 not considered a D. alpinus in 5B whereas it is considered so in 5A. This entire figure creates more problems than it solves, either through sloppiness or incorrect interpretation of the interaction of genetic data and taxonomy. You cannot just remove individuals from species willy nilly, and then put them back again when it suits.
7.	There is much space utilized to present the individual COI and CAD trees for Dercylus, even their results are nonsensical. This is not surprising given shared ancestral polymorphisms in mtDNA. That the combined analysis results are credible means that only those should be presented. Delete Fig. 7 and its discussion.
8.	8. If you dump the COI tree for Dercylus this point of discussion disappears. Arguing that species are monophyletic or not based on mt DNA is indefensible.
9.	Only in the discussion are we presented with information (unpublished) concerning Pelmatellus beetles. All mention of Pelmatellus results should be struck.
10.	Fig. 4—if you are dealing with only 1 species, the abbreviation is “sp.” Also, Dercylus should be italicized in the CAD network.

I have looked at some of the documentation regarding permits, etc. and it seems genuine. However I view that as a role for the editor, not for a reviewer.
Experimental design: See above
Validity of the findings: See above.
Additional comments: This manuscript needs to be seriously streamlined, with only tree presentation of combined COI plus CAD data. Species limits must be carefully considered, rather than putting individuals into and out of species without taxonomic consequences. The entire manuscript should be carefully read for English usage after revision.


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

Input:
Title: THE ROLE OF DISPERSAL FOR SHAPING PHYLOGEOGRAPHICAL STRUCTURE OF FLIGHTLESS BEETLES FROM THE ANDES
Review round: 1
Reviewer: 2

Output:
Basic reporting: I find this study well executed, of interest and well-written. I have only one major issue with the methods and otherwise a few comments:

I find the paper fairly long considering the amount of data that is generated, with a particularly lengthy discussion. Perhaps an additional effort to summarize the main ideas could be made to save some space.
Experimental design: Each genus should be presented in the introduction to give a sense of their respective species richness, knowledge of their phylogenetic placement and potential proximity, and monophyly of the species groups the authors are focusing on.

The choice of outgroups should also be better justified.

The dating exercise seems relatively simple but in fact is probably severely biased by the choice of the taxon sampling. The authors rely on fossils placed in the outgroups to calibrate their clocks. This would be acceptable if the taxon sampling was denser. As it stands, the taxon sampling does not allow a correct placement of the fossils and the ages are very likely to be incorrectly estimated. I would recommend that the authors repeat their analyses with a denser taxon sampling to allow a more accurate placement of the fossils in this dating scheme. I would also recommend that the authors compare their results with a clock calibration based on published rates of molecular evolution for the CO1 of Carabidae which are available from Andújar et al. (2012, BMC Evol. Biol.). Even if the rates can be different between their Andean beetles and the genus Carabus, this would give some sense of how reliable the divergence time estimates are. Also this can be accommodated to some extent using relaxed clocks as the authors used in this submission.
Validity of the findings: no comment
Additional comments: No additional comments


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

Input:
Title: THE ROLE OF DISPERSAL FOR SHAPING PHYLOGEOGRAPHICAL STRUCTURE OF FLIGHTLESS BEETLES FROM THE ANDES
Review round: 1
Reviewer: 3

Output:
Basic reporting: Line 75: Remove the word Where

Line 130: change to …one taxon was collected…

Line 347: From figure 6 it appears the D. alpinus clade originated 6.32 Mya with a confidence interval of 1 – 14 MYA  not 5.1-8.0

Line 355: change to …each species represents a distinct clade…

Lines 372-376:  include the 95% confidence intervals for the dates  of Dercylus species

Figure 5.
-	Make the font size of bootstrap and Bayes PP a little larger. (Also in Figure 8)
-	Explain in the text or figure legend that only one sample of each haplotype was included in the phylogenetic analysis
-	Explain in the figure legend what is meant by North, North West, Mixed, etc. or maybe use the site names instead.  Make this more explicit in the text line 344-345.  Put the focus on the site and the direction in parentheses.

Figure 9.
-	I like the time labels for each species of Dercylus. Please also include a similar label for the genus in the Andes  30.13 (~12-47mya)
Experimental design: Line 133: IT would be good to include additional outgroups to the Dercylus phylogenetic and BEAST analysis as the deepest branches have low support.  I did not see any additional Oodine  or Licinine CAD sequences in GenBank, but there are a few CAD sequences in the Harpalinae subfamily that might be useful and there may be many COI sequences that would be useful in GenBank.

Line 159-194: Please include (probably as supplemental information) the details of the settings and parameters for each of the analyses for MrBayes, RAxML, DNAsp, TCS, BEAST, Arlequin, and Structure so one can replicate your results.  If you used only defaults, please say so.
Validity of the findings: Line 96 and 404: Aim of the study: I felt uncomfortable with the comparison of these to taxa with regard to how flightlessness has affected their phylogenetic structure and population connectivity.  I would not expect – a priori- so see similar patterns of genetic diversity and gene flow when comparing populations of a single species (D. alpinus) to species of a genus (Dercylus), especially if they are different ages.  There is more going on here than just flightlessness.  The history of these taxa is very different.  Make it clear that you are comparing apples and oranges.  It is not surprising that there is very little gene flow between different species that have been separated for a long time and that there is more gene flow or evidence of recent gene flow among populations of the same species that have been separated relatively recently.  I think you should put more emphasis on how these are two different scenarios and not try to connect them so much with flightlessness.
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: MITOCHONDRIAL GENOMICS OF HUMAN PATHOGENIC PARASITE LEISHMANIA (VIANNIA) PANAMENSIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: In this manuscript, the authors describe the assembly of the maxicircle coding region for Leishmania panamensis using NGS sequence reads. Additionally, the sequence of several minicircles was determined. Finally, comparisons with other previously reported sequences of maxicircles and minicircles are presented. The manuscript provides valuable information, but flaws in interpretation may exist. Moreover, the writing of the manuscript, mainly in the Results and Discussion sections, results unpleasant. The meaning of several sentences is really confusing. These deficiencies prevent to appreciate the consistency and relevance of this work. Therefore, the manuscript would benefit by an extensive editing, and this is a compulsory step before its evaluation for publication.

Major revisions.

1. In the abstract, at many places along the manuscript, and in figure 1, the authors claim that a complete maxicircle sequence is provided. However, the read coverage observed in the maxicircle divergent region (DR) is indicating that a collapse occurred during the assembly. This region is composed by low-complexity repeated sequences and complete sequencing of the maxicircle has not been attained yet for any Leishmania species (Flegontov et al. 2006. The Leishmania major maxicircle divergent region is variable in different isolates and cell types. Mol Biochem Parasitol 146: 173-179).
	The authors should be cautious, avoiding to conclude that a complete maxicircle sequence has been assembled.

2. Lines 41-42. It is read: ‘Maxicircles, with 20-40 copies per cell, around 20-25 kb in size…”. However, this reviewer failed to find these data about the maxicircle size in the quoted references (Liu et al, 2005; Simpson et al., 1987). Looking for articles dealing with the direct determination of the maxicircle size, I found that sizes close to 40-kb have been estimated for this molecule in several trypanosomatids (Maslov DA et al. 1984. Mol Biochem Parasitol 12: 351-364).

3. Line 143. Could the authors explain the meaning “encryption variations”?

4. Lines 163-165. This sentence is confusing. The authors should use direct language. It seems that the authors want to indicate that combining NGS reads they assembled a contig of 19,957 nucleotides. If this is the message, please, rewrite the sentence.

5. Lines 167-168. Please, specify how the PCRs confirmed the “circularity of the molecule”. When responding to this question, keep in mind the points 1 (above) and 6 (below).

6. Lines 173-175. According to this sentence, the authors seem to be suggesting that the assembled molecule might be incomplete. This point must be clearly stated in the text, and figure 1 has to be modified accordingly to avoid confusion regarding whether or not a full assemble was attained.

7. Lines 189-190. Figure 2B does not seem to correspond to a “synteny analysis”, but a phylogenetic analysis based on sequence alignments.

8. Lines 194-195. The authors indicate that the L. panamensis maxicircle share high sequence identity with L. braziliensis maxicircle, and the percentages of identity between the different pairs of genes are shown in table 1. However, the authors are not citing the work in which the L. braziliensis maxicircle was reported (Nocua P et al. 2011. Universitas Scientiarum 16: 29-50).

9. Lines 206-207. The sentence is confusing: Which are the six L. panamensis strains that share the two SNVs located in the ATPase6 gene? My suggestion would be rewritten the entire paragraph.

10. Line 213. The authors wrote: “the 21 minicircles assembled here”. This information is relevant, but it is not shown. The authors should provide the sequence of the 21 minicircles as a supplementary file (the sequences in figure 3A are not readable). Moreover, it should be indicated whether they are or not complete molecules.

11. Line 221. What is the meaning of “nodes without polyphyly”? In figure 3B, please, include the Leishmania species for the minicircles with the AF identifiers.

12. Lines 221-230. This part of the manuscript results hardly understandable. The authors should clearly indicate the value of this analysis and the achieved conclusions.

13. Lines 290-291. What is the meaning of “the two strains analyzed here have a lag of 16 years in terms of sequencing interval”.

14. The discussion is too long and disorganized. My suggestion is to reduce it, putting in value the information reported in this work.

15. No conclusions are in the conclusion section. A conclusion is a concrete finding derived from the experimental data.


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

Input:
Title: MITOCHONDRIAL GENOMICS OF HUMAN PATHOGENIC PARASITE LEISHMANIA (VIANNIA) PANAMENSIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The paper describes the complete maxicircle kDNA annotated genome of L (V) panamesis. The paper is very well written, with the appropiated refrences, the approach is sound and the conclusions clear.


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: MITOCHONDRIAL GENOMICS OF HUMAN PATHOGENIC PARASITE LEISHMANIA (VIANNIA) PANAMENSIS
Review round: 1
Reviewer: 3

Output:
Basic reporting: In this work, the authors report the assembly and annotation of the complete maxicircle sequence of Leishmania V. panamensis, and compare it with published genomes of other related parasites.

Overall clear and unambiguous writing, background sufficiently covered except where noted in general comments for the author.
Experimental design: The aim of the study is well-defined and the methods used are appropriate.
Validity of the findings: Conclusions are well stated and supported by the results shown.
Additional comments: 1)	From the introduction it is not clear what motivated authors to study L.V. panamensis assembly in particular. Introduction should be revised to include this information.
2)	On line 84, “..Here we report the sequencing, assembly, annotation, and ….”. This statement seems misleading and should be changed accordingly as authors are not reporting sequencing as part of this work rather working with sequences available through public repository as described in Methods.
3)	On lines 118-119, “…The mitochondrial genes were annotated through alignment between proteins and genes reported in databases”. It is not clear which database authors are talking about here.
4)	Authors should provide the version number of different programs described in Methods. For example, on line 159 “..Hotelling’s T-square in R environment” which R version was used?
5)	Some of the abbreviations, such as PE on line 100, are used without first defining it.


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

Input:
Title: MITOCHONDRIAL GENOMICS OF HUMAN PATHOGENIC PARASITE LEISHMANIA (VIANNIA) PANAMENSIS
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: After reading the revised manuscript and the authors’ responses, this reviewer considers that the authors have amended sufficiently the manuscript, addressing adequately previous concerns.


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

Input:
Title: RADIATION DOSE-RESPONSE (A BAYESIAN MODEL) IN THE RADIOTHERAPY OF THE LOCALIZED PROSTATIC ADENOCARCINOMA: THE RELIABILITY OF PSA SLOPE CHANGES AS A RESPONSE SURROGATE ENDPOINT
Review round: 1
Reviewer: 1

Output:
Basic reporting: The authors seek to correlate radiotherapy dose levels with PSA kinetics after therapy through a Bayesian approach. The authors call such PSA kinetics a "surrogate endpoint" but it is unclear for what endpoint they are seeking a surrogate. They rightly point out in the discussion that use of PSA velocity as a surrogate is controversial: without measurements of some endpoint in this study, the authors are unable to apply Prentice criteria and claim PSA slope as a surrogate in this treatment setting. If any follow-up data is available for these patients, analytic inclusion would greatly improve the paper.

The study would benefit from explaining how the current study complements the following studies, which address highly similar hypotheses (PSA kinetics after radiation therapy):
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2757130/
https://www.ncbi.nlm.nih.gov/pubmed/19825885

The raw data is in Excel format, and the columns appear in strange locations of the .xlsx file. It might be easier to instead share a .csv/.txt/similar.
Experimental design: The methods for calculating PSA slope and how the authors' calculation differs from existing notions of "PSA slope" and "PSA velocity" is unclear. To quote the paper:

"Another of these changes over time is the PSA slope. It differs from the above concepts and is another concept. In fact, it is the rate of PSA marker diminishing over time after radiotherapy."

The authors helpfully cite Suzman et al. 2015, where we get the following definitions:
* PSA velocity is the slope of the linear regression of PSA vs time
* PSA slope is the the slope of the linear regression of log(PSA) vs time
You can (and several authors have) compute either of these quantities before or after treatment, so claiming that a new concept is created here is inaccurate (if only timing with respect to treatment delivery defines the novelty). Related to the above definitions, it's not clear how the slopes were calculated in this study. Was PSA log-transformed? If not, why not?

From the paper:
"To avoid complexity in dose response models, the Bayesian model provides a flexible approach for accurate detection of the dose response curve."
- Can the authors describe how using a Bayesian model avoids complexity versus, for example, a simple linear regression as other authors have used?

Broadly, the details of the Bayesian dose-response model are unclear. Why is one needed? I think I see a prior distribution specified, but it's not labeled as such.

How were the cutpoints to create the four radiation dose categories chosen?

A helpful guidepost for the authors to consider when revising: could someone take the raw data and exactly reproduce the numbers (p-values etc) shown in the paper by following steps written in the methods section? As written now, I don't believe this is feasible.
Validity of the findings: The sample size is rather small, particularly within dose groups, so concluding that "the PSA slope changes association with dose increase was not clear and therefore it cannot be used as a reliable response surrogate endpoint" is not justified: lacking statistical significance in this study may simply be the result of low power. Changes in PSA kinetics as a result of varying radiotherapy doses have been robustly shown in larger cohorts (for example those I cited earlier in the review). The authors could calculate how much of a change in PSA slope their study was powered to detect.

There is very sparse data for the low-risk patient group within dose levels: showing boxplots in Figure 2 for the distribution of 3 points is inappropriate (this happens for the two dosage groups). Simply plotting the points might help resolve.
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: RADIATION DOSE-RESPONSE (A BAYESIAN MODEL) IN THE RADIOTHERAPY OF THE LOCALIZED PROSTATIC ADENOCARCINOMA: THE RELIABILITY OF PSA SLOPE CHANGES AS A RESPONSE SURROGATE ENDPOINT
Review round: 1
Reviewer: 2

Output:
Basic reporting: The writing is mostly clear and unambiguous. However, there are certainly sentences that I suggest the authors reword or clarify (see Grammatical Comments). I have also listed some minor grammatical errors. The introduction contains relevant background information and references. The structure of the paper seems appropriate and figures are relevant to the content of the article. The raw data can be opened. However, I would suggest including a key to explain this data (see comment #7 below). This study seems self-contained.
Experimental design: The work seems original. However, as mentioned below, I suggest the authors explain how this work differs to references given in the paper (see comment #5). The research question is clearly defined (What is the relationship between PSA slope and increasing radiotherapy doses?) and meaningful. Various changes in PSA characteristics are important in assessing response to radiotherapy. However, according to this paper,  the impact of radiation dose escalation on PSA slope is not clear and thus is of interest to study. The methods of the investigation seem appropriate and ethical. However, I would suggest the authors add more detail in order to be reproducible by another investigator.
Validity of the findings: The data in this study were from a previous study. The statistical analysis of the data seems appropriate (see comment #2), although I recommend that more details be provided.  The conclusion is stated clearly and is connected to the original question.
Additional comments: Below I list some specific comments.

Comments about content:
	1. Line 118. The authors divide the patients into four groups in terms of the total dose received. What is the standard dosage for radiotherapy? How were these categories chosen? Why did the authors choose a category exactly for 70 Gy? Why is 71 Gy not in a category? Do no patients have a dosage level of 71 Gy?
	2. How exactly do you calculate the slope? It seems that, for example, two patients might have the same PSA values (initial PSA and PSA after RT) but one patient was able to get their PSA measurement after RT earlier than the other. That would indicate that the first patient has a more negative (steeper) slope, when in fact, they may have responded the same. Does this cause a problem in the study?
	3. Figure 1.
	- Why aren’t there 195 circles? Doesn’t each circle represent a patient? If not, the authors should clarify what this figure represents.
	- Line 145 states that this figure is the mean of the PSA slope between the baseline pretreatment PSA and the first PSA measurement after RT. What are the authors averaging over?
	- If I am understanding this figure correctly, the authors could add more information into Figure 1 by having different shapes or colors to represent the different groups (risk, radiation dosage, etc.).
	4. Lines 146-147. The authors state that this indicated that the long interval to the first treatment after radiotherapy was accompanied by a slower slope. Isn’t this what you might expect? (See comment #2).
	5. Lines 165-168. How exactly does this study differ to the ones mentioned?
	6. Lines 178-181. Can the authors cite these studies?
	7. I suggest the authors create a key for the raw data. For example, what is “HT”? Does “year” mean age of the patient?, Does “No-mon of fallow” mean number of months of follow-up? Why are there missing values in this column? etc.

Grammatical Comments
	8. Line 49. The authors write RT before defining it. (It is defined on line 84).
	9. Line 89. I think the period between “radiotherapy. (Vollmer…” should be removed.
	10. Line 116, I think there should be a space between “orT.stage”.
	11. Line 147. I don’t think it is mathematically correct to say a slope is slower. Perhaps “the PSA is decreasing at a slower rate”, or “a smaller slope in magnitude”. Similarly with line 203 (“slope is faster…slower”).
	12. Lines 147-149. This sentence is a little confusing. Perhaps the authors can reword it.
	13. Lines 173-177. These lines are not exactly clear. What exactly do you mean by “The PSA slope dropped with a slow rate”? How does this sentence mean that although there is a better therapeutic response with higher doses of radiotherapy, the PSA slope changes are not significant? And why would the fact that PSA slope changes are not significant mean more benefit to the patient?
	14. Lines 186-187. I suggest the authors define the abbreviations.
	15. Line 188. The “an” should be removed in “In an another randomized…”
	16. Line 210-213. Do the authors mean “…our findings were restricted to patients without recurrence and with a short follow-up time”, or “…our findings were restricted to patients without recurrence and without a short follow-up time”? If it is the former: In lines 203-206, the authors state that outcome should not be relied on the values of PSA in the short term. Doesn’t this mean that your study isn’t reliable? If it is the latter, then why is a long term follow up needed (line 205)?
	17. There are different fonts throughout the paper.
	18. Table 2. In the second row, the dose level reads ≥50 Gy <. I think there should be a 70 after the <.
	19. Figure 2. Some words and symbols are in bold and others are not. I would suggest being consistent.


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: RADIATION DOSE-RESPONSE (A BAYESIAN MODEL) IN THE RADIOTHERAPY OF THE LOCALIZED PROSTATIC ADENOCARCINOMA: THE RELIABILITY OF PSA SLOPE CHANGES AS A RESPONSE SURROGATE ENDPOINT
Review round: 1
Reviewer: 3

Output:
Basic reporting: ABSTRACT
1.	The sentence beginning with “It is different from the PSA amount…” should be rewritten for clarity.”
2.	It is stated that the PSA changes were negative for 176, but the total number of patients is not previously mentioned.

INTRODUCTION
1.	It would be beneficial to add more details to the Introduction. The authors touch on one specific model that studied the relationship between PSA slope and time to recurrence, however the examples presented in the Discussion (lines 184-202) might help to motivate the study better in the Introduction.
2.	Additionally, the authors mention that “different statistical methods are used to investigate the relationship between dose and response in medicine,” however they do not state specific examples besides the Bayesian model. What are the other methods that could have been used to study this concept and why was this model used over other methods? Referencing additional methods or background on the Bayesian method would be useful due to the few details included in the statistical method subsection.

RESULTS
1.	Was there a correlation between positive PSA slope and dosage or risk-stratification?
2.	The second sentence of the second paragraph of the results section (lines 145-149) should be rewritten for clarity. The data in Figure 1 shows an inverse relationship between PSA reduction and follow-up time, but this is not clear from the text.
3.	The dose-response curve is mentioned (line 158), but is not shown in the manuscript.
4.	For Figure 1, it may be useful to differentiate doses to show relationship or lack or relationship between dose and PSA slope. It may also help explain why those 19 patients saw an increase in their PSA, rather than a decrease.
5.	For Figure 2, results should be shown on same scale for comparison purposes.
6.	What do the red numbers, as well as the horizontal line, signify in Figure 2?
7.	It is mentioned throughout the results that the differences were not significant (lines 158, 175) however it would be beneficial to include the p-values that lead to these conclusions both in Figure 2 and in the text.

DISCUSSION
1.	Although the paper referenced on line 166 fit a model of tumor kinetics, no relationships between PSA slope and clinical outcomes were described.
2.	It is mentioned on lines 178 through 180 that previous studies suggested that response would improve with increasing doses but references are not provided until three lines later.
3.	The results of Pollack et al. are said to be consistent with the authors’ study, but results presented earlier in the text do not support this statement. Though increasing the dose does show an increase in the PSA reduction, it has been said not to be statistically significant (lines 158 and 175). If the p-values do indeed show a significant difference, these values should also be reported on line 202.
4.	As previously mentioned, it may be beneficial to move the discussion of prior studies (lines 184-202) into the Introduction.

Minor remarks:
1.	Lines 87-89 should be combined into one sentence.
2.	On line 95, it states “The present study shows the impact of radiation dose…” but a clear relationship is not shown throughout the manuscript. Consider changing “shows” to “investigates.”
3.	The stratification of the patients based on the D’Amico system is not clear and consistent. Line 113 should state “iPSA <= 10 OR GS <= 6 OR T.stage = T1-T2a,… 10 < iPSA <= 20 OR GS = 7 OR T.stage = T2b,… PSA > 20 OR GS = 8 OR 10 OR T.stage = T2c-T3" for consistency.
4.	In Table 2, the “dose levels” label should be moved to top row with the others and line 4, column 2 should read [50,69) (or some other variation to show that 69 is the upper limit).
5.	On Figure 2, “mu” should be changed to the Greek symbol for mu.
6.	On line 174, omit “This means that”.
7.	Double-check statistics shown on line 185 and include the p-value.
Experimental design: The authors aimed to study the relationship between PSA slope to study the dose-response relationship in a retrospective study. The methods were described sufficiently, such that results may be reproduced. However, the Introduction did not serve as a clear motivator for why this research was filling a necessary gap.
Validity of the findings: The findings of the study suggest that there is not a clear dose-response relationship when using PSA slope as a response criterion. However, conflicting results were presented throughout the text (line 192) and the results were not presented with appropriate statistical methods (p-value).
Additional comments: Overall, the findings of the study are interesting, but could benefit from further analysis to clearly prove or disprove that a relationship between dose and PSA slope exists. Additionally, the English language should be reviewed to ensure that the text is clear to all audiences.


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: RADIATION DOSE-RESPONSE (A BAYESIAN MODEL) IN THE RADIOTHERAPY OF THE LOCALIZED PROSTATIC ADENOCARCINOMA: THE RELIABILITY OF PSA SLOPE CHANGES AS A RESPONSE SURROGATE ENDPOINT
Review round: 2
Reviewer: 1

Output:
Basic reporting: There are still numerous sentences throughout the paper that are hard to read due to grammatical errors. I have listed some of these sentences below.

The following phrases have grammatical errors or are difficult to read. I would recommend that the authors read through the paper again as I have not listed every grammatical error.
1.	Sentence on line 90-93, “Both the PSA amount … surrogate end point”. This sentence is very long and hard to understand. I would recommend splitting it into two sentences.
2.	Figure 1 title, “slpoe” should be “slope”
3.	Figure 3, part of the legend is “…”
4.	Figure 4, “gy” in the legend should be “Gy”
5.	Lines 201 – 204, “As table 2 indicates, … Kruskal-Wallis Test”. These sentences are hard to understand and are grammatically incorrect.
6.	Lines 214 – 216, “Although the changes in PSA slope…”. I am not sure what this sentence is saying.
7.	Line 227, “were significantly less likely to involve with PC recurrence…”. What does “involve with PC recurrence” mean?
8.	Line 252, it is still not clear to me what “PSA slope dropped with a slower rate in most patients” means. Doesn’t the slope become more negative with increasing dose level, in which case “PSA decreased at a faster rate”?
9.	Line 273-275, “…revealed that however doses equal”. I am not sure what this is trying to say.
10.	Line 277-278, the phrase “using a simplistic approach as linear regression to time indeed nonlinear functions” is confusing.
Experimental design: It is still not obvious to me that the results are reproducible by another investigator (see comments #1, 5 below).
Validity of the findings: See comment #5 below.
Additional comments: 1.	The improvements to the data headings make it easier to read. However, I would still recommend a key to explain the data. For example, there is a section called mean of psa. What is this averaged over? What is the difference between iPSA (baseline PSA measurement before treatment) and IniPSA? I assume this isn’t the raw data as there is a category for number of repeated PSA, but there isn’t the actual  repeated PSA measurements. In order to reproduce the results, would we not need these values to calculate the slope?
2.	Line 98, the authors state that BF has various definitions, but rising PSA was considered for all of them. Can the authors cite the “them”?
3.	Line 109, why do dose escalation advantages and dose effect need to be investigated?
4.	Line 123, I would recommend that the authors add something like “in this situation” unless the authors believe that it is always advantageous to use MCMC over MLE.
5.	It is still not clear to me exactly how you calculate PSA slope. On line 147, the authors state that PSA slope is the slope of the linear regression of PSA repeated measurements vs. time and was calculated by the slope function in Excel. Are these repeated measurements in the excel file (see Comment #1)?
6.	The authors should describe what j is on line 154.
7.	Line 193, should “PSA is decreasing at a slower rate” be “PSA is decreasing at a faster rate”?
8.	Line 235-256. In this paragraph, the authors state that there is controversy regarding the starting time and end of follow-up time for calculating the PSA slope. The authors then explain various studies. How do the author’s study fit into this?


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: RADIATION DOSE-RESPONSE (A BAYESIAN MODEL) IN THE RADIOTHERAPY OF THE LOCALIZED PROSTATIC ADENOCARCINOMA: THE RELIABILITY OF PSA SLOPE CHANGES AS A RESPONSE SURROGATE ENDPOINT
Review round: 2
Reviewer: 2

Output:
Basic reporting: The manuscript is mostly clear and unambiguous, however there are several areas that need to be rewritten for clarification (see general comments). The introduction and background show relevance, however they could benefit from some revision to improve the overall flow of the manuscript. On lines 92-93, the authors state that “some of these PSA metrics are surrogate…some of them are not surrogate end point.” Please provide specific examples of those that are. From line 115, what specific statistical methods are used to investigate the relationship between does and response?

For the raw data, it would be beneficial to include a legend for each column label.

The figures are relevant to the study. The authors state that “The purpose of this study was to evaluate the relationship between increasing RT doses and PSA slope as a potential surrogate for PC recurrence.” This point could be clearly driven by the data shown in Figure 1 if the authors differentiate between dose and PSA slope. In their response, they stated that they could not differentiate between doses. Why not?

The authors state in their response regarding Figure 2 that “This figure was corrected but we could not correct it.” Why not? This leads me to question the reproducibility of the study.

In Table 1, the dose group labels need to be revised for clarity. Perhaps using interval notation for the second group might be clearer (i.e [50,70)). The same should be done in Table 2. The table caption for Table 1 should also be revised.
Experimental design: The research question is well defined and relevant. The methods are described with sufficient detail and information to replicate.

The dose groups (lines 145-146) are not consistent with the abstract or Tables 1 or 2. The abstract has the doses ranging from 50 to 69 but the table seems to include any level below 70. Please revise. “Statistical method:” on line 151 should be bolded and lines 167-168 are not clear.
Validity of the findings: The authors have found that increasing dose results in a lower PSA slope, though the overall difference was not significant between dose groups. Lines 189-194 should be revised to clearly get this point across. Saying that PSA is decreasing at a “slower rate” contradicts their main findings. From line 179, “the overall trend changes were reduced” is not clear.

On lines 199-200, the authors state “the mean rank of dose is difference [different] in two groups with or without recurrence.” Should this be between the four groups? The final sentence of the results (lines 228-230) do not clearly flow from the previous results. How do the previous lines imply that the PSA slope cannot fully explain the effect of dose?

There is not a clear association of the first paragraph of the Discussion to the conclusions of the study. Did the authors find an issue in their calculation of PSA slope based on the interval used? The authors state that the follow-up time ranged from four to 67 months. Did this have an impact on the PSA slope? The second paragraph of the Discussion should be revised. In particular, lines 252-253. It is not clear to say that the PSA slope dropper with a slower rate. If the authors mean that the PSA slope is lower, then an increase in disease-free survival should be expected. The sentence following this, “This means that although…the PSA slope changes cannot fully explained [explain] it” does not clearly flow from the previous sentence and should be revised. On line 273, it’s stated “…doses equal to or greater than 70 Gy decreased PSA more than other dose levels but significantly increased free-disease survival time similar to above studies.” Using “but” would imply that this is a negative result. The “but” should be changed to “and”. Lines 277-281 are not clear.
Additional comments: Please see below for general grammatical comments:

Abstract
1.	Line 42 should be revised to say “It is the PSA rate of change over time…”
2.	Line 46 and line 134, change “during” to “from.”
3.	Lines 49-51 are not clear.
4.	The dose listed on line 54 is not consistant with the main body of the manuscript or Tables.

Introduction
1.	Lines 94-97 should be revised for clarity.
2.	Line 98 change “for all of them” to “in these studies.”
3.	Line 102, change “investigated” to “found.”

Results
1.	Please remove “First”, “Second”, “Third”, “Fourth” in the Results.
2.	Line 202, “because the all of” and line 203, “significant differences there are among” should be revised.
3.	Please use a consistent categorization of the slopes. For instance, on line 209, “positive and no positive” is used but then on line 223 “zero or less (decrease) vs. positive” is used.
4.	Line 219 is not clear.
5.	Line 220 should be revised to. Perhaps say “In a multivariate logistic model with PSA slope and dose level as inputs to the model…”

Discussion
1.	Lines 234-235 should be revised since PSA slope can be zero, positive, or negative.

Figures
1.	Typo in title of Figure 1 (slpoe).


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: RADIATION DOSE-RESPONSE (A BAYESIAN MODEL) IN THE RADIOTHERAPY OF THE LOCALIZED PROSTATIC ADENOCARCINOMA: THE RELIABILITY OF PSA SLOPE CHANGES AS A RESPONSE SURROGATE ENDPOINT
Review round: 3
Reviewer: 1

Output:
Basic reporting: Further requests that follow from Reviewer 2's comment numbering:

## Basic reporting
1. This sentence remains unclear to me due to grammatical errors: "Only some of PSA metrics such as: PSA velocity greater than 1.5 ng/ml/yr and PSA doubling time <6 months are surrogate for PC mortality or overall survival (OS) (D’amico 2005) but others as PSA decline >=30% and PSA doubling time >12 months are not surrogate end point. (D’amico 2012, Collette 2008, Halabi et al 2013 and Valicenti  et al 2006)."
5. Still not grammatically correct: "by using Bayesian model. The all of Bayesian credible intervals for all dose groups are overlapping."
6. This sentence remains redundant; additionally, a very brief description of what the 3rd (and other) Prentice criteria are could be helpful for this manuscript. The ordering of Prentice criteria are mostly consistent across the literature, but clarity would greatly assist the reader in this paper. "Because of this the third Prentice criterion was not met, consequently PSA slope as a continuous outcome did not meet the third criterion of Prentice."
7. Still unclear: does this sentence mean -- within the subset of patients with negative or 0 PSA slope, PC recurrence was significantly less frequent among patients with high doses compared to patients with low doses? "This result indicates that patients with negative or zero PSA slope had significantly PC recurrence less than others if they had been previously treated with higher doses rather than low doses."

A continuation of a request from Reviewer 3:
The following edited sentence in response to a lack of clarity in the discussion has grammatical errors that make it unclear what the authors are claiming. This is a critical point in the manuscript describing why the study is important, and the work would be improved if this section was easy to understand.
"Antonarakis et al (2012) has compared the pre- PSA slope and post- PSA slope. This issue may effects on the evaluation of PSA slope as a predictor or response variable in various studies. Incorporating pre-treatment PSA level in slope calculation indirectly joints a time-dependent covariate to post-treatment PSA behavior. Also it takes into account the PSA change at during treatment and reduce the time dependent interval censoring bias."
Experimental design: From Reviewer 2's comment to the author, item number 3: The response still didn't answer "why"
Validity of the findings: Broadly, some vagueness in the methods description remains. One solution would be to include code (the Statistical Method section implies that R was used) in the supplement alongside the data file. Ideally, this code would read and manipulate the .csv and/or .xlsx files therein and produce the reported numbers and figures directly. Such an effort would greatly improve the reproducibility and interpretability of this work.
Additional comments: The authors did a commendable job in responding to the previous round of reviews. The requested additions to the data file and figures are helpful. Some requests, primarily related to grammatical changes and clarity, were not fully 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: AN IMPROVED FRAMEWORK TO PREDICT RIVER FLOW TIME SERIES DATA
Review round: 1
Reviewer: 1

Output:
Basic reporting: There are several English and grammatical mistakes which should be addressed in the revised manuscript. Some of them are as follows.

--Line 34-35: Authors are suggested to differentiate hydrological time series data and meteorological factors. For example, the authors mentioned rainfall as meteorological factors. Is rainfall not a hydrological data? What is meant by horological data? Authors need to provide references otherwise. What is underlying surface? It might be changes in underlying surface or underlying surface changes.

Line 37-38: Revised by making connections from literature. For example, authors are suggesting reformulation as “In past research, several hybrid models have been developed to address these issues. In addition, explain more about the hybrid model (mention their names and application).

--Line 37-38: revise as a hybrid model “uses” instead “used”. In addition, citations need to be refined and relevant to study/result. Authors are suggested to replace with:

Zhang, X., Peng, Y., Zhang, C., & Wang, B. (2015). Are hybrid models integrated with data preprocessing techniques suitable for monthly streamflow forecasting? Some experiment evidences. Journal of Hydrology, 530, 137-152.

-needs connection between lines 38-39 and 40-42.

--Line 40-48: need more compact and concise literature instead.

--Line 42: What is the difference between multi-scale component and time scale components? In addition, the authors did not mention where and whom derived the time scale components? Please reformulate sentence.

--Line 42-44. Too long sentence: need  reformulation.

--Line 46-49 # Citation is required.

--Line 49: Incorrect citation format: Campisi-Pinto et al. [8] forecasted

--Line 49-53 Revised the sentence. In addition reference style is wrong. Please check.

--Line 55-59:  The findings are already published. I suggest removing or make it concise.

--Line 49-60 Revised the sentence. In addition reference style is wrong. Please check.

--Line 49-53: Revise the statement. Reference style is wrong. Too poor English is observed. The whole paper requires proofreading from native English speakers before publication.

--Line 79-83. Too long sentence. Please reformulate it.

 –Line 108. Please reformulate it.

-- There are a lot of grammatical mistakes, for example, in line 167-169, what is key, I think some thing is missing.... like key importance??? What is smother? See also 172, 263-266, 306-310, 310-313, and 335-339 also.  Authors should need to proofread carefully.

--Line 212-2014:  Please check English and Grammer.

--Line 239-243: Similarity issue. It should be rephrased and cited accordingly.

--  Abbreviations need to be defined and used properly. In many places abbreviations are not defined, replicated and wrongly defined. For example, in line 2010, what is ANN?

Line 282. Figure 1. Wrong citation of figures. Need proper and accurate indexing and citation of figures/ tables  thorought the Paper.
Experimental design: --Line: 85-86: Need consistency of between the title of manuscript and the aim of research. Are you developing an estimator? Or suggesting a predictive framework? The,title should be revised accordingly.

Line 180-181: By inspecting the nature of IMFs, authors select Laplace distribution. Where are the results of the inspection? How author inspect the nature of IMF and determine the distribution before application and case study? It needs a strong argument to support the rationale for the proposed method.

--Line 192-194: Authors should need to mention these estimators in the earlier part of the manuscript.
Validity of the findings: -- In line line 176: The authors assumed that IMFs follows a normal distribution before proposing. In the case study, I did not see the statistical result proving normality behavior of IMFs.

-- There is much difference among SNR values. For example, Table 1, CEEMDAN-EBT (the proposed method) gives very high values of SNR. Parallel to this, there is too much difference in the existing method, for example, in Kabul Inflow, EEMD-HT gives -2.4276, while EEMD-ST gives -32.0408.

That is why my major concerns are related to the validity of the assumptions, the length and type of time series being used.  In addition,to assess the efficiency of the proposed methods, too much attentions are required on literature review and appropriate selection  about existing methods.
Additional comments: To resolve multi-scale and noisy complexity in time series data, the authors adopted an empirical Bayesian threshold estimator in CEEMDAN and compared it with existing methods. My first concern is that whether author giving a new framework or proposing a new method? It should be clear and consistent in title, objective, methodology and in other parts of the text.
Secondly, the authors stress on hydrological time series, but used only river data. What is multi-scale? Give a clear background of multi-scale and noisy complexity in hydrological time series particularly. How author assessed the multi-scale and noisy complexity in river inflow data which they are used? This really needs attention. In addition, instead of using only river data, the authors are suggested to include results from other hydrological related time series before generalizing their method/approach. Otherwise, reduce the scope of the method by substantial text changing and literature particularly for river deta.
Thirdly, the authors are suggested to include statistical result (P-value) proving non-stationary, non-linearity before working or proposing a method / framework for handling multi-scale and noisy complexity (see lines 276). In section Proposed method (line 180), how authors inspect IMF and proposed Laplace distribution before working with data?
Fourthly, I would recommend adopting a more focused title and to increase the length of time series data for the better evaluation.


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: AN IMPROVED FRAMEWORK TO PREDICT RIVER FLOW TIME SERIES DATA
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1. The manuscript presents a novel framework to improve prediction from inherently complex hydrological time series data, which is interesting. The subject addressed is within the scope of the journal.
Experimental design: It appears ok.
Validity of the findings: It appears ok.
Additional comments: 2.	However, the manuscript, in its present form, contains several weaknesses. Appropriate revisions to the following points should be undertaken in order to justify recommendation for publication.
3.	For readers to quickly catch your contribution, it would be better to highlight major difficulties and challenges, and your original achievements to overcome them, in a clearer way in abstract and introduction.
4.	It is mentioned in p.1 that a framework comprising complete ensemble empirical mode decomposition with adaptive noise-empirical Bayesian threshold is adopted to improve prediction from inherently complex hydrological time series data. What are other feasible alternatives? What are the advantages of adopting this particular framework over others in this case? How will this affect the results? The authors should provide more details on this.
5.	It is mentioned in p.3 that an empirical Bayesian threshold estimator is adopted to optimally reduce noises from time-scale components. What are other feasible alternatives? What are the advantages of adopting this particular estimator over others in this case? How will this affect the results? The authors should provide more details on this.
6.	It is mentioned in p.7 that Laplace distribution is adopted Intrinsic Mode Functions. What are other feasible alternatives? What are the advantages of adopting this particular distribution over others in this case? How will this affect the results? The authors should provide more details on this.
7.	It is mentioned in p.7 that marginal maximum likelihood approach is adopted to estimate weights. What are other feasible alternatives? What are the advantages of adopting this particular approach over others in this case? How will this affect the results? The authors should provide more details on this.
8.	It is mentioned in p.8 that soft threshold, hard threshold and improved threshold function are adopted as benchmark for comparison. What are the other feasible alternatives? What are the advantages of adopting these particular thresholds over others in this case? How will this affect the results? More details should be furnished.
9.	It is mentioned in p.9 that “…To train model, 80% data is used, remaining 20% data is used to test the accuracy of models.…” That means no cross-validation is made. How can the problem of overfitting be resolved then? More justification should be furnished on this.
10.	It is mentioned in p.9 that multi-layer perceptron neural network is adopted for the prediction of denoised IMFs. What are other feasible alternatives? What are the advantages of adopting this particular soft computing technique over others in this case? How will this affect the results? The authors should provide more details on this.
11.	It is mentioned in p.9 that logistic function is adopted as activation function. What are other feasible alternatives? What are the advantages of adopting this particular activation function over others in this case? How will this affect the results? The authors should provide more details on this.
12.	It is mentioned in p.9 that back-propagation algorithm which has the drawbacks of local convergence and slowness is adopted. Some justifications should be furnished on why other algorithms are not selected.
13.	It is mentioned in p.10 that an autoregressive moving average model is adopted to predict the noise free IMF’s and residual. What are other feasible alternatives? What are the advantages of adopting this particular model over others in this case? How will this affect the results? The authors should provide more details on this.
14.	It is mentioned in p.10 that four daily rivers inflow time series data of the Indus Basin System are adopted as case studies. What are other feasible alternatives? What are the advantages of adopting these particular case studies over others in this case? How will this affect the results? The authors should provide more details on this.
15.	It is mentioned in p.11 that four statistical criteria are adopted to gauge the performance of the proposed model. What are the other feasible alternatives? What are the advantages of adopting these particular evaluation metrics over others in this case? How will this affect the results? More details should be furnished.
16.	Some key parameters are not mentioned. The rationale on the choice of the particular set of parameters should be explained with more details. Have the authors experimented with other sets of values? What are the sensitivities of these parameters on the results?
17.	Some assumptions are stated in various sections. Justifications should be provided on these assumptions. Evaluation on how they will affect the results should be made.
18.	The discussion section in the present form is relatively weak and should be strengthened with more details and justifications.
19.	There are some occasional grammatical problems within the text. It may need the attention of someone fluent in English language to enhance the readability.
20.	Moreover, the manuscript could be substantially improved by relying and citing more on recent literatures about contemporary real-life case studies of soft computing techniques in hydrologic engineering such as the followings:
	Yaseen, Z.M., et al., “An enhanced extreme learning machine model for river flow forecasting: state-of-the-art, practical applications in water resource engineering area and future research direction,” Journal of Hydrology 569: 387-408 2019.
	Taormina, R., et al., “Neural network river forecasting through baseflow separation and binary-coded swarm optimization”, Journal of Hydrology 529 (3): 1788-1797 2015.
	Moazenzadeh, R., et al., “Coupling a firefly algorithm with support vector regression to predict evaporation in northern Iran,” Engineering Applications of Computational Fluid Mechanics 12 (1): 584-597 2018.
	Wu, C.L., et al., “Rainfall-Runoff Modeling Using Artificial Neural Network Coupled with Singular Spectrum Analysis”, Journal of Hydrology 399 (3-4): 394-409 2011.
	Ghorbani, M.A., et al., “Forecasting pan evaporation with an integrated Artificial Neural Network Quantum-behaved Particle Swarm Optimization model: a case study in Talesh, Northern Iran,” Engineering Applications of Computational Fluid Mechanics 12 (1): 724-737 2018.
	Chau, K.W., et al., “Use of Meta-Heuristic Techniques in Rainfall-Runoff Modelling” Water 9(3): article no. 186, 6p 2017.
21.	Some inconsistencies and minor errors that needed attention are:
	Replace “…proposed a novel framework comprised on Complete Ensemble Empirical Mode Decomposition…” with “…proposed a novel framework comprising Complete Ensemble Empirical Mode Decomposition …” in lines 11-12 of p.1
	Replace “…to cope up the simple…” with “…to cope with the simple…” in line 76 of p.3
	Replace “…method is comprised on four stages…” with “…method is comprised of four stages…” in line 93 of p.4
	Replace “…method is proposed by [12] is used…” with “…method proposed by [12] is used…” in line 108 of p.4
	Replace “…it must belongs to…” with “…it must belong to…” in line 179 of p.7
	and many more…
22.	In the conclusion section, the limitations of this study, suggested improvements of this work and future directions should be highlighted.


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: AN IMPROVED FRAMEWORK TO PREDICT RIVER FLOW TIME SERIES DATA
Review round: 2
Reviewer: 1

Output:
Basic reporting: Authors have addressed all of my comments and resolve issues in the manuscript structure such as appropriateness of title with respect to data used in this paper, grammar mistakes, reference style, citation, text flow, etc. I am very happy to see the improved version of this paper. I am thankful to the authors to consider my comments relevant to the data and their types. From my side, the paper is fully ready to publish under the PeerJ forum. However, a little improvement if possible can be made to enhance the quality of labels in the graphs that are presented in figure 5, 6, 7 and 8. It will surely strengthen the reading quality of the paper.
Experimental design: I agree with the responses of authors which are given against my previous review.
Validity of the findings: The authors have now provided sufficient answer to all those questions which I raised in the first review of this paper.
Additional comments: Congratulations! The manuscript has been improved considerably. I recommend this paper for its publication in PeerJ forum.  However, I suggested improving the label of figure 5, 6, 7, and 8.


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: AN IMPROVED FRAMEWORK TO PREDICT RIVER FLOW TIME SERIES DATA
Review round: 2
Reviewer: 2

Output:
Basic reporting: ok
Experimental design: ok
Validity of the findings: ok
Additional comments: The revised paper has addressed all my previous comments, and I suggest to ACCEPT the paper as it is now.


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

Input:
Title: THE EFFECTS OF RESVERATROL FEEDING AND EXERCISE TRAINING ON THE SKELETAL MUSCLE FUNCTION AND TRANSCRIPTOME OF AGED RATS
Review round: 1
Reviewer: 1

Output:
Basic reporting: See general comments
Experimental design: See general comments
Validity of the findings: See general comments
Additional comments: The manuscript entitled ‘The impacts of resveratrol feeding and exercise training on skeletal muscle transcriptome and function of old rats’ by Zhou et al., has investigated the role of resveratrol as a potential treatment for sarcopenia.

The introduction is concise and clearly outlines the relevance of this study.

The methods are well thought out and extensively described in the materials and methods. The authors carefully describe the qRT-PCR experiments carried out (Line 148 on), however the authors do not include the primers used for these experiments and these should be included.

In the first paragraph of the results the authors state that there is a change in body mass of each experimental group, however no data is actually presented. It is suggested that the data be elaborated upon in the main text (i.e. such as percentage changes etc). Additionally, Table S1 is important data and should be included as a main table rather than supplement.

Moreover, more details of the actual data for the grip strength tests should be included in the results section to demonstrate there is or is not a difference. While this is presented in the figure, this should also be addressed in the main body of text.

In the discussion the authors introduce the term AMPK, however this should be defined and why it is important.

There has been a recent study (Bosutti and Degans, Sci. Reps. 2015, 5) which has shown that there is a dose-dependent effect of resveratrol on muscle cell plasticity. This is an important study and the dose of resveratrol given may alter the overall outcome of this study. While it is not expected that the work be repeated to take this into account, it is important to mention this in the discussion.

The authors seem to have missed an important point in the data presented. While the treatment of resveratrol did not change the overall muscle function, it did show that there was an overall decrease in body mass index compared to untreated sedentary control animals. These data suggest that while the dose given may not be useful for muscle function, it may be useful as a treatment for maintaining weight or for weight loss. This is consistent with previous studies where there is potential protection from high fat diets (Momken, et al., FASEB J, 2011, 25, 10) but does not improve metabolic function in non-obese individuals (Yoshino et al., Cell Metabolism, 2012, 16, 658). Interestingly, a large number of the genes found to be altered are normally thought of as neuron specific (with some lower expression in other tissues in the EST database), but these are linked to diabetes and obesity. For example: Slc17a7 (Ly et al., Diabetologica, 2014, 57, 192), TRIM67 (Comuzzie et al., PLos One, 2012, 7, e51954).

It is suggested the authors include a section in the discussion to address this and the potential for further study.

Specific comments:

Overall the language is fine throughout the manuscript but there are places where it is difficult to interpret the precise meaning (highlighted in annotated manuscript).

Line 105: The term executed should be replaced with euthanized.

Line 221-223 – there is a change in font compared the rest of the manuscript.


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

Input:
Title: THE EFFECTS OF RESVERATROL FEEDING AND EXERCISE TRAINING ON THE SKELETAL MUSCLE FUNCTION AND TRANSCRIPTOME OF AGED RATS
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Zhou et al. sought to characterise the functional and transcriptomic response to resveratrol and exercise on old rats. Identifying effective treatments for the aging process is an important area of research, with sarcopenia contributing to aging-related mortality. Old rat studies are logistically difficult, and the authors should be commended for their efforts in this study. There are some limitations that should be addressed before the manuscript is published.
Major
It is hard to conclude from the transcriptomic data whether the exercise or resveratrol interventions were effective. It is not unsurprising that the authors did not observe differences in the classical markers of exercise via transcriptomics as the majority of the transcriptomic response to exercise is acute. The authors should assess the protein expression of some of the classical exercise markers. Since several of the pathways are somewhat similar in response to resveratrol, the authors should also assess these markers in the resveratrol treated group.
Please specify how long after the last exercise bout/dose of resveratrol the muscles of the rats were harvested.
Several of the enriched GO and KEGG pathways/terms appear to have very few altered genes. A cut-off should be used to exclude pathways with only 1 differential gene.
The most striking effect of the interventions appears to be weight-loss. It would benefit the manuscript if the authors were able to provide further characterisation of this weight-loss. E.g. lean-mass or fat-mass changes. Also beneficial would be plasma concentration of hormones associated with adipose e.g. adiponectin, leptin, insulin; or appetitive eg. GLP-1.
Minor
Conclusions in the discussion regarding the effects of exercise in older rats are overstating the data given the limitations of the study. Please temper these assertions.
The authors appear to treat ‘exercise’ as a homogenous intervention. However, it is extremely varied with many modalities and intensities, please take this into account when discussing the predominantly negative data in the current study. I.e. it would be pertinent to discuss more intense or longer duration forms of exercise interventions.
It is not necessary to abbreviate the names of the intervention groups and does not aid the reader. A suggestion of the names would be ‘Old’, ‘Trained’, and ‘Resveratrol’.
Please proof-read the manuscript carefully for grammatical and spelling errors. Some examples of grammatical mistakes below:
•	Line 57: This sentence is not grammatically correct.
•	Line 60: ‘Diseases’ should not be pluralised
•	Line 65: this sentence is not grammatically correct.
•	Line 233: ‘literatures’ should not be pluralised.


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

Input:
Title: THE EFFECTS OF RESVERATROL FEEDING AND EXERCISE TRAINING ON THE SKELETAL MUSCLE FUNCTION AND TRANSCRIPTOME OF AGED RATS
Review round: 2
Reviewer: 1

Output:
Basic reporting: See below
Experimental design: See below
Validity of the findings: See below
Additional comments: The manuscript language has greatly improved over the last submission. It is now in standard English and easy to follow.

The authors have addressed all the comments from the review and incorporated the changes into their manuscript.

The authors have expanded their discussion to include more references that are relevant to the study. They have also re-written the discussion to be broader and more relevant to their work. This study does contain some interesting results that warrant further study in the future (i.e. the role of neuronal markers in muscle function). The authors have also stated other important aspects that need to be taken into account in addition to genomic approaches - i.e. gene expression, hormone response etc.

Overall, this version is a significant improvement and provides interesting data that will contribute to the field.


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

Input:
Title: THE EFFECTS OF RESVERATROL FEEDING AND EXERCISE TRAINING ON THE SKELETAL MUSCLE FUNCTION AND TRANSCRIPTOME OF AGED RATS
Review round: 2
Reviewer: 2

Output:
Basic reporting: n/a
Experimental design: n/a
Validity of the findings: n/a
Additional comments: I am satisfied with the amendments and recommend this manuscript be published.


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

Input:
Title: GENOME SEQUENCING AND ANALYSIS OF SALMONELLA ENTERICA SUBSP. ENTERICA SEROVAR STANLEY UPM 517: INSIGHTS ON ITS VIRULENCE-ASSOCIATED ELEMENTS AND THEIR POTENTIALS AS VACCINE CANDIDATES
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1.	The language needs some polishing e.g.
a.	‘Two fimbrial virulence clusters, tcf and ste, which were absent in S. Typhimurium LT2, were uniquely distributed in S. Stanley UPM 517 genome.’ – what does uniquely distributed mean? Uniquely found?
b.	‘has shown resistant towards many antibiotics’ – you mean ‘shown resistance’
c.	‘There is no death reported yet, however, this outbreak results in serious clinical signs and symptoms, such as diarrhea, abdominal cramps, vomiting, dehydration, nausea, muscle and joint pain and also asthenia (Pastore et al., 2008). Severe gastrointestinal colonization may occur, leading to severe systemic infection and can cause death (Guiney, 2005)’ – so, no known deaths but death could occur? You should remove the ‘Severe gastro…’ sentence.
d.	‘Our aim is to characterize the virulence associated-elements of S. Stanley UPM 517 draft genome to become a potential vaccine candidate’ – you mean ‘to identify a potential vaccine candidate’

2.	It is not clear from your language what work was done here and was done previously
a.	‘These clusters were associated with human and broilers intestinal colonization respectively.’ – where are these data presented? Or is this from previous work?

3.	The raw sequence data is not available. NQWD00000000.1 links to the assembled (unannotated) contigs. You should upload your raw sequence data (European Nucleotide Archive is easier than NCBI) and you definitely have to upload your annotated genome, as this is the whole subject of the paper.

4.	In the discussion, please give some examples of genomics being used for the rational design of vaccine candidates?

5.	‘highly homologous’ you cannot say highly homologous, something either is homologous or it is not. Like being unique. Replace with just homologous, or ‘highly similar’.

6.	In the discussion, it is sometimes difficult to determine what you have done and what is from the literature.
a.	‘These effector proteins were regulated by a major transcriptional regulator of SPI-1, hilA.’ You don’t show any data concerning this, so you should say ‘The effector proteins are regulated’ and then give the reference.
b.	Another example, you need to provide a reference or make it clear that this info is from the literature - ‘SopB/sigD effector protein of SPI-5 also promoted the Salmonella invasion and membrane ruffling of the host cell in phosphoinositide phosphatase manner.’
Experimental design: 1.	How was this isolate selected for sequencing? Random? Because of the large number of antibiotic resistance genes?
Validity of the findings: 1.	Would be good to have some phenotypic antimicrobial resistnace testing to help validate the in silico findings.
2.	Why do you have more coading sequences in UPM than in LT2? Despite the fact that your assembly is > 100 kbp shorter. Please address in the text.
3.	Genome alignment – do you think it is valid to compare your genome versus LT2 when you have re-arranged your draft genome vs LT2? If you want to talk about genome re-arrangements, you have to show me the contigs with the re-arrangement in. When you map reads back to that contig, do the reads support the re-arrangement, or is it possibly a mis-assembly?
4.	‘Of these resistance genes, 16 genes encode for multi-drug resistant genes’ what is a multi-drug resistant gene? A gene which gives resistance to multiple drugs?
5.	L203 ‘Two unique clusters of fimbrae’, unique compared with what?
6.	‘five functional prophage regions’ how do you know that they were functional? You haven’t provided your annotated genomes so I can’t judge for myself whether the pro-phage were intact or not.
7.	You need to provide more details of the potential plasmids you identified? How much of the reference plasmids were present in your Stanley assembly?
8.	‘These results indicated that S. Stanley UPM 517 was able to provide immunity against a number of foreign nucleic acids such as viruses and plasmids’ – this review says that there is no convincing evidence of recent spacer acquisition in Salmonella, did you find something different? https://www.ncbi.nlm.nih.gov/pubmed/25479838
Additional comments: Ashari et al have carried out a detailed examination of the genome of a Salmonella Stanley isolate from broilers. The work appears largely technically sound, and is very thorough. The authors do a good job of discussing the genes which could be knocked out in potential vaccine candidates. However, I have numerous issues with the manuscript, detailed in the provided sections. The most pressing issue is that the annotated genome has not been made publicly available.

Some of the language in the manuscript is potentially misleading and this needs a lot of attention. One example is the title:

'In silico identification of potential vaccine candidate of Salmonella... '

You have not identified a potential vaccine candidate, this appears to be a normal, virulent Salmonella Stanley. You have identified genes which should be considered for removal from a potential vaccine candidate. The title therefore requires modification.


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: GENOME SEQUENCING AND ANALYSIS OF SALMONELLA ENTERICA SUBSP. ENTERICA SEROVAR STANLEY UPM 517: INSIGHTS ON ITS VIRULENCE-ASSOCIATED ELEMENTS AND THEIR POTENTIALS AS VACCINE CANDIDATES
Review round: 1
Reviewer: 2

Output:
Basic reporting: See comments in "General comments for the author" section
Experimental design: See comments in "General comments for the author" section
Validity of the findings: See comments in "General comments for the author" section
Additional comments: In this study the uthors report the draft genome sequence of a broiler-isoleted strain of Salmonella Stanley. From this data, the authors propose some potential vaccine candidate to control the pathogen, which frequently causes outbreaks to humans.

First, the quality of writing is not suitable for publication unless extensively edited, making the paper very hard to read. I strongly suggest the authors to use a language editing service for their manuscript.

Several researchers have tried to develop vaccines through the use of attenuated strains. Given that the authors of this study ultimately discuss a similar approach, it is not clear why it was necessary to obtain a complete genomic sequence for this: it would have been sufficient to obtain mutants in the virulence factors. In other words, it is worth asking what is the novelty that can be deduced from the data about the biology of this serovar. The authors should discuss in more depth what is the real contribution that, eventually, makes this serovar different from the others.

Probably the most interesting finding of the study is the lack of the secretory apparatus encoded in the SPI 2. A question that arises from this is how the isolate could survive in the animal without the protection provided by the effectors secreted by this system of serción. A simple experiment to perform that could give information about it, would be to measure the intracellular survival capacity of the bacteria in macrophages.

I am sure the authors put serious efforts in this study. However, I feel that the manuscript deserves an equal attention.


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: GENOME SEQUENCING AND ANALYSIS OF SALMONELLA ENTERICA SUBSP. ENTERICA SEROVAR STANLEY UPM 517: INSIGHTS ON ITS VIRULENCE-ASSOCIATED ELEMENTS AND THEIR POTENTIALS AS VACCINE CANDIDATES
Review round: 1
Reviewer: 3

Output:
Basic reporting: In the manuscript "In silico identification of potential vaccine candidate of Salmonella enterica subsp. enterica serovar Stanley UPM 517 draft genome sequence and its virulence-associated elements" the authors have sequenced the genome of a strain isolated from a broiler chicken and have performed computational analysis in order to identify potential new virulence factors, genomic and pathogenicity islands, antimicrobial resistance genes and CRISPR/Cas systems in this bacteria.

The manuscript is well-written, scientifically sound and suitable for publication in this journal. It also adds interesting data to the Salmonella community. I have only minor comments before the manuscript is suitable for publication in PeerJ.
Experimental design: no comment
Validity of the findings: My only comments and suggestions are the following:

1)	The current title "In silico identification of potential vaccine candidate of Salmonella enterica subsp. enterica serovar Stanley UPM 517 draft genome sequence and its virulence-associated elements" overstates the main findings of the manuscript and my suggestion is to modify it to avoid confusion. The current work corresponds more to the genome sequencing and analysis study of this bacterial strain rather than a systematic study to identify vaccine candidates in silico. A more suitable title would be: ¨Genome sequencing and analysis of Salmonella Stanley UPM 517a: insights in its virulence-associated elements and potential vaccine candidates”.

2)	The same applies to the main conclusion (Line 45-446) “our study revealed that the virulence-associated elements identified in S. Stanley could potentially be used and modified as candidates for vaccination to control and reduce the prevalence of salmonellosis as well as an alternative to antibiotics”. I think the language can be softened highlighting the main aspects of the paper without overstating and overreaching its main findings.
Additional comments: No additional comments