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: HIF INHIBITOR TOPOTECAN HAS A NEUROPROTECTIVE EFFECT IN A MURINE RETINAL ISCHEMIA-REPERFUSION MODEL
Review round: 1
Reviewer: 2

Output:
Basic reporting: The article evaluated the neuroprotective role of topotecan, a HIF inhibitor. The language used is clear, not ambiguous. Introduction and background show context. All references used are relevant. The structure of the manuscript is in accordance with the PeerJ standard.
The article has an important contribution to ophthalmology. However, my recommendation is that the authors should clarify some points of the study before it is accepted for publication.
Experimental design: Why did the authors inject topotecan prior to the induction of retinal injury? As it is a possible therapeutic candidate, it would be interesting to evaluate the protective effect of topotecan after induction of the lesion.
Please define normal saline. Is it 10% NaCl?
Define IOP at first indication (intraocular pressure) - line 79
Which cocktail inhibitor protease was used in this study? Please enter the manufacturer's catalog number
Enter manufacturer's catalog number for all kits cited in the study
The methods were presented in the sequence in which they were performed? Rearrange the M&M section, if necessary.
Was the electrophysiological evaluation done according to the previous literature? Provide a reference, please.
Detail the preparation or acquisition of the primers.
Inform if the data were tested for statistical normality
Validity of the findings: The results were not discussed correctly. The authors did not relate their findings to previous literature. Please review this section carefully
The figures are adequate
Standardize the terminology. Please use RGC degeneration (not RGC neurodegeneration)
This reviewer understands that further studies are needed to elucidate the mechanisms of HIF as well as its inhibitory drugs. However, speculations would be welcome in the discussion.
Review the sentence - "This study suggested that pharmacological HIF inhibition may be candidates for RGC degeneration induced with renal ischemia or high intraocular pressure."
Additional comments: The article evaluated the neuroprotective role of topotecan, a HIF inhibitor. The language used is clear, not ambiguous. Introduction and background show context. All references used are relevant. The structure of the manuscript is in accordance with the PeerJ standard.
The article has an important contribution to ophthalmology. However, my recommendation is that the authors should clarify some points of the study before it is accepted for publication.
Why did the authors inject topotecan prior to the induction of retinal injury? As it is a possible therapeutic candidate, it would be interesting to evaluate the protective effect of topotecan after induction of the lesion.
Please define normal saline. Is it 10% NaCl?
Define IOP at first indication (intraocular pressure) - line 79
Which cocktail inhibitor protease was used in this study? Please enter the manufacturer's catalog number
Enter manufacturer's catalog number for all kits cited in the study
The methods were presented in the sequence in which they were performed? Rearrange the M&M section, if necessary.
Was the electrophysiological evaluation done according to the previous literature? Provide a reference, please.
Detail the preparation or acquisition of the primers.
Inform if the data were tested for statistical normality
The results were not discussed correctly. The authors did not relate their findings to previous literature. Please review this section carefully
The figures are adequate
Standardize the terminology. Please use RGC degeneration (not RGC neurodegeneration)
This reviewer understands that further studies are needed to elucidate the mechanisms of HIF as well as its inhibitory drugs. However, speculations would be welcome in the discussion.
Review the sentence - "This study suggested that pharmacological HIF inhibition may be candidates for RGC degeneration induced with renal ischemia or high intraocular pressure."


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: HIF INHIBITOR TOPOTECAN HAS A NEUROPROTECTIVE EFFECT IN A MURINE RETINAL ISCHEMIA-REPERFUSION MODEL
Review round: 2
Reviewer: 1

Output:
Basic reporting: The manuscript is well organized and is easy to read.  Literature references are valid, and figures are clean and well described.
Experimental design: Research fills a knowledge gap, and is translatable to human disease.  Methods are well described in this version.
Validity of the findings: Data are robust and conclusions are well stated.
Additional comments: The manuscript is much improved.


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

Input:
Title: HIF INHIBITOR TOPOTECAN HAS A NEUROPROTECTIVE EFFECT IN A MURINE RETINAL ISCHEMIA-REPERFUSION MODEL
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: The study is well defined and relevant. Methods were described with sufficient detail.
Validity of the findings: Data are robust and conclusions well stated.
Additional comments: The authors followed all suggestions of this reviewer. The manuscript has significantly improved and all necessary information has been added.


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 ASSOCIATION BETWEEN SERUM LIPIDS AND RISK OF PREMATURE MORTALITY IN LATIN AMERICA: A SYSTEMATIC REVIEW OF POPULATION-BASED PROSPECTIVE COHORT STUDIES
Review round: 1
Reviewer: 1

Output:
Basic reporting: Thank you for the opportunity to review this paper. It is well written with clear English throughout.
Experimental design: This is a well conceived and designed study. I particularly congratulate the authors  on refraining from performing quantitative analysis on such heterogeneous studies, but instead opt to rigorously appraise the literature and identify a clear need for specific studies to look at the Latin America region.
Validity of the findings: The unique aspect of this systematic review is its population. In this regard, the findings are novel in that there are not enough comparable studies that can be assessed quantitatively. There exists a need for further studies to establish whether or not a link exists between lipid biomarkers and mortality in Latin America.
Additional comments: Thank you for a well-conducted and a well-written study.


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 ASSOCIATION BETWEEN SERUM LIPIDS AND RISK OF PREMATURE MORTALITY IN LATIN AMERICA: A SYSTEMATIC REVIEW OF POPULATION-BASED PROSPECTIVE COHORT STUDIES
Review round: 1
Reviewer: 2

Output:
Basic reporting: The overall reporting structure is generally good.

There are some phrasing/grammatical issues which require attention:
e.g. line 40 (Abstract) "To synthesise the scientific evidence ON OF the association between serum LIPID and premature mortality...". Final English proof reading recommended to tidy up a number of minor issues.

Introduction:
- In describing the "inconsistent findings" of epidemiological data, the authors only really highlight two issues that appear inconsistent:
i. The weaker asssociation between unfavourable lipid profiles and stroke/mortality
ii. The weaker association between unfavourable lipid profiles and older age
- These are not really issues that define the purpose of the manuscript, which aims to examine the association betweeen "serum lipid and premature mortality in Latin America
Experimental design: The methods are well described, with a robust, systematic approach to data retrieval and collection.
Validity of the findings: The major problem with this manuscript is that the results of the literature search yielded only three individual population studies, all of which examined and reported on very different questions.

Crespo et al aimed to examine the relationship between physical activity and obesity with all-cause mortality in Puerto Rican Men, and reported some outcomes according to varied levels of total cholesterol.

Werle et al examined risk factors for the very elderly in a city in Brazil.

Lazo-Porras et al examined the residual dyslipidaemic profile and its impact on mortality.

As the authors have correctly identified, this makes a meta-analysis of the findings impossible. As such, the manuscript "qualitatively" describes the findings of these three studies.

Unfortunately for the researchers, in attempting to uniformly collect and present the data, it makes reading the results more difficult.

I think in such a setting, where the literature search has identified three largely different studies, where meta-analysis is impossible, the authors would be better off writing a narrative review, where discussions regarding issues relating mortality and :
- TC, LDL-C
- residual dyslipidaemic profiles
- elderly lipid profiles
- lipids and coronary vs stroke outcomes

 can all be separately addressed and relevant literature can be presented in each section, combined with relevant literature from Western/other populations, to contextualise it all.
Additional comments: I congratulate the methodical approach to this paper and appreciate the hard work involved, but I feel presenting your findings as is makes it overall confusing to follow, since the three papers analysed produced three very different sets of results.


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

Input:
Title: THE ASSOCIATION BETWEEN SERUM LIPIDS AND RISK OF PREMATURE MORTALITY IN LATIN AMERICA: A SYSTEMATIC REVIEW OF POPULATION-BASED PROSPECTIVE COHORT STUDIES
Review round: 2
Reviewer: 1

Output:
Basic reporting: Thank you for reviewing the manuscript which now reads better and conveys the message in a clearer way.
Experimental design: No concerns. The paper is limited by the number of relevant studies identified but this is acknowledged and discussed adequately by the authors.
Validity of the findings: No concerns.
Additional comments: Thank you for the opportunity to review this paper. I am satisfied with the revised form and have no further 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: IDENTIFICATION OF POTENTIAL BIOMARKERS AND PIVOTAL BIOLOGICAL PATHWAYS FOR PROSTATE CANCER USING BIOINFORMATICS ANALYSIS METHODS
Review round: 1
Reviewer: 1

Output:
Basic reporting: In this work, the authors used bioinformatics tools to analyze the gene expression profile of prostrate cancer from GEO database and identified key genes in the process that may serve as molecular biomarker.

The manuscript lacks on sufficient background information and literature references as further elaborated in general comments.
Experimental design: The aim of the study is well-defined and the methods used are appropriate and sufficiently detailed.
Validity of the findings: Conclusions are well stated but validity of the findings not sufficiently assessed as also noted in general comments.
Additional comments: 1)	Introduction needs improvement and should be rewritten as sufficient background covering previous bioinformatics work is not provided to understand the significance and novelty of the work. For example, lines 57-59, “However, the underlying molecular mechanisms and hub genes or key signaling pathways of PCa were still far from explicit”. There have been several bioinformatics work identifying hub genes in PCa (to mention a few: PMIDs: 29260398, 28800317, 30405806 etc.). Authors should provide information on key genes identified in previous studies, and in the discussion elaborate on the similarities/differences in the hub genes identified in this manuscript to those already reported in literature.
2)	In Table 1, it seems that the top 10 upregulated and downregulated DEGs are identified by their P-values which do not necessarily represent the top regulated genes by fold-change (as can be seen in Figure 1 which identifies genes with fold change values up to ~3). Can authors comment on why fold-change was not used as the criteria here.
3)	Authors should explain in the manuscript why GSE103512 dataset was picked in particular for the analysis since there are other PCa datasets available too (for example: GSE55945). More importantly, authors should validate the hub genes identified using GSE103512 against other PCa datasets.
4)	There are spelling mistakes at several places in the manuscript. For example, “defferentially expressed genes” on line 63.


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

Input:
Title: IDENTIFICATION OF POTENTIAL BIOMARKERS AND PIVOTAL BIOLOGICAL PATHWAYS FOR PROSTATE CANCER USING BIOINFORMATICS ANALYSIS METHODS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Clear and unambiguous, professional English used throughout.
Experimental design: Methods described with sufficient detail & information to replicate.
Validity of the findings: Conclusions are well stated, linked to original research question & limited to supporting results.
Additional comments: I thank the editor for providing an opportunity to review the article “Identification of Potential Biomarkers and Pivotal Biological Pathways for Prostate Cancer Using Bioinformatics Analysis Methods”.

The article is written well and can be considered for publication after addressing these comments:
1.	Fig 1, some of the important genes name should be labeled (especially genes with very high changes).
2.	Figures should be combined together, and a total number of figures should be less (e.g. combine Fig 1 and 2).
3.	Pathway analysis and conclusions are very vague. Cannot be used for therapeutic purpose. Please discuss.
4.	Module analysis showing enriched interaction between many genes, specially KLK3, CDH1, KLK2, FOXA1, and EPCAM. These genes should be annotated and highlighted in Fig 1, 2.
5.	This study is based on one library. The author should validate their findings from other available library and TCGA database.


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

Input:
Title: IDENTIFICATION OF POTENTIAL BIOMARKERS AND PIVOTAL BIOLOGICAL PATHWAYS FOR PROSTATE CANCER USING BIOINFORMATICS ANALYSIS METHODS
Review round: 2
Reviewer: 1

Output:
Basic reporting: NA
Experimental design: NA
Validity of the findings: NA
Additional comments: Authors have addressed all my concerns and the manuscript is now suitable for publication.


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

Input:
Title: IDENTIFICATION OF POTENTIAL BIOMARKERS AND PIVOTAL BIOLOGICAL PATHWAYS FOR PROSTATE CANCER USING BIOINFORMATICS ANALYSIS METHODS
Review round: 2
Reviewer: 2

Output:
Basic reporting: N/A
Experimental design: N/A
Validity of the findings: N/A
Additional comments: Authors have addressed most of the suggestion and comments. This article can be considered for publication after addressing comment:

1.	How come upregulated and downregulated genes are flipped in the revised version? And also, for pathway analysis? If the analysis was done in a similar way, how come results are opposite? Please discuss and clarify.


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

Input:
Title: IDENTIFICATION OF POTENTIAL BIOMARKERS AND PIVOTAL BIOLOGICAL PATHWAYS FOR PROSTATE CANCER USING BIOINFORMATICS ANALYSIS METHODS
Review round: 3
Reviewer: 1

Output:
Basic reporting: N/A
Experimental design: N/A
Validity of the findings: N/A
Additional comments: Authors have addressed all suggestion and comments. I recommend this article for publication.


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

Input:
Title: STRUCTURAL, FUNCTIONAL AND MOLECULAR DYNAMICS ANALYSIS OF CATHEPSIN B GENE SNPS ASSOCIATED WITH TROPICAL CALCIFIC PANCREATITIS, A RARE DISEASE OF TROPICS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The English language must be improved to ensure that an international audience can clearly understand your text. To highlight- a few examples in the abstract where language could be improved: - there is no word "destable" in the English language. Consider using "less stable" or unstable etc. In the next sentence "Results from MD-simulation carried for both mutated structures have concluded that mutations lead to structural deviation in protein..." - results are

"..these variants can have deleterious" - the word deleterious should be preceded by an article (a or the).

"Thus cathepsin B gene should be screen in samples" - "Thus cathepsin B gene should be screened in samples...."

"variants can have deleterious effect" - "variants can have a deleterious effect"

"...distribution of variations in patient population" - "distribution of variations in patient the population"

The manuscript is full of such errors, I can't point all of them.
Experimental design: 1.Kindly explain why PSI-BLAST (and not BLASTp) has been used to query PDB sequences? How much % identify in the alignment has been used for homology modeling?
2. It will be useful to show contour maps of the free energies as a function of the backbone angles ψ and φ for residues, AND Energies for binding can be calculated. see: - https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4926353/, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3607329/:
3. Please include results to show secondary structure using do-dssp.
4. MD simulation time should be increased to check longer time-dependent changes.
5. Combined effect of mutation on protein function can also be observed in order to understand their effects.
6. MSA from known model organism procathepsin B proteins can be performed with both wild and mutant.
7. In the section "Materials and methods": - the section "Non-synonymous SNP Analysis" should come before "Homology modeling"
8. Abbreviation should be defined at it's first occurrence, and one time only. Abbreviation for SIFT [Sorting Intolerant From Tolerant] is defined in the line numbers 133 as well as 180- similarly check other abbreviations too.
Validity of the findings: The findings are novel and interesting, however, minor additional analysis, it's integration with other findings combined with language corrections will enhance its impact on readers.
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: STRUCTURAL, FUNCTIONAL AND MOLECULAR DYNAMICS ANALYSIS OF CATHEPSIN B GENE SNPS ASSOCIATED WITH TROPICAL CALCIFIC PANCREATITIS, A RARE DISEASE OF TROPICS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The article would benefit from being checked by a native English speaker. There are many small mistakes throughout the article (eg. missing "the" lines 66, 86, 99).

The introduction contains methods, which are then repeated in the methods section (lines 123-143). Additionally, it contains a large amount of text on the protein structure, I am not sure that this detailed information is relevant for the article's purpose. Many references for the non-synonymous SNP methods section are missing (although in the Intro), and the reference style appears to be inconsistent (e.g. line 202). References are missing for the miR databases in line 371.

Detail on the prevalence of TCP, and how it is increasing mortality rate in tropical countries (line 73) would be beneficial.

Figures and tables: I do not see the need for Fig 2, the numbers are contained in the text and are also in Table 1. Figure 3 could be shortened to only have the sequence for the WT protein, with the two mutations highlighted. Figure 4 and 5 refer to "sticks" - is this the proper terminology for these diagrams? Line 337 refers to Fig 5, is this correct?

Note: Table 1 appears to have the header repeated.
Experimental design: The premise of the article relies on SNPs found to be associated with TCP in a 2006 study. No details of the size of this study were given (here some background information on prevalence of TCP in the general population would also potentially justify using a small study), are there any more recent studies available that could also be used? Is the association with those SNPs replicated in later studies? Which human genome build was used? hg18/19/37?

Are there other SNPs in the CTSB gene? How do they compare cf function/location with respect to those that were found to be associated with TCP?

miRs are often tissue specific. What are the targets of the miRs listed in table 6, and are they known to be expressed in any TCP relevant tissues? There is the potential to analyse this data much more than has been done, noting that it is all in silico prediction only.

The Methods section lists a large number of programs that were used, but some details are missing. For instance, "After these models pass the respective thresholds" (line 173) however no thresholds are given in this paragraph.

Did the authors perform any downstream analysis on the output of any of the programs used?
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: STRUCTURAL, FUNCTIONAL AND MOLECULAR DYNAMICS ANALYSIS OF CATHEPSIN B GENE SNPS ASSOCIATED WITH TROPICAL CALCIFIC PANCREATITIS, A RARE DISEASE OF TROPICS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The article stills need to be checked completely for English and grammar. For example:

line 47: missing full stop
line 50: This lead to activation - "This leads to activation"
line 51: Double full stop; Sentence beginning with "TCP" does not make sense
line 53: Double full stop
line 54: patients majorly - "the majority of patients"

There are numerous other such examples still present in the article.

In the introduction, the authors should group all statements about what they have done into one paragraph, currently its on lines 74-80 and 110-115.

Figures and tables: I still do not see the point of Figure 2 - it is not a complicated figure, and the numbers are in the text. It doesn't add to the paper and could easily be removed.
Experimental design: The additional details regarding the original 2006 study improve the manuscript. However, I think the authors are misleading in their use of language to describe this study. The 2006 paper investigated 23 SNPs in the CTSB gene, but found only 1 SNP to be significantly associated with TCP after adjusting for multiple corrections. And indeed, only attempted to validate 4 of the 23 SNPs. Why then the focus on these 23 SNPs, given that the majority of them are NOT associated with TCP?

The authors state that they couldn't find any other CTSB SNP to be associated with TCP - how did they come to this conclusion? Did they do any association testing themselves, or just a literature search?

My original question of how do these other non-associated CTSB SNPs compare to the TCP-associated SNPs remains unaddressed. This is important, especially now that it appears that many of the TCP-associated SNPs (as defined by the authors) are actually NOT associated with TCP.
Validity of the findings: While the findings might be robust, the premise on which the SNPs were chosen in the first place is not (see my comments above).
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: AN APPLICATION OF PCR-RFLP SPECIES IDENTIFICATION ASSAY FOR ENVIRONMENTAL DNA DETECTION
Review round: 1
Reviewer: 1

Output:
Basic reporting: On the whole, the manuscript is well constructed and presents valuable and interesting results. Some refinements will improve its legibility and the presentation of the data.

Specific suggestions for introduction:

•	Introduction
o	On line 65&66, the authors state that the PCR method has not been applied to other terrestrial species, however an article cited earlier (Olson et al 2013) does so with salamanders and Piaggo et al, 2013 develops a PCR assay to detect pythons in Flordia waters.
	Citation: Piaggio, A. J., R. M. Engeman, M. W. Hopken, J. S. Humphrey, K. L. Keacher, W. E. Bruce, and M. L. Avery. 2013. Detecting an elusive invasive species: a diagnostic PCR to detect Burmese python in Florida waters and an assessment of persistence of environmental DNA. Molecular Ecology Resources 14:374–380.
o	80-90: The section describing the ecological relevance and importance of the target species to conservation may be more effective if presented earlier in the text, before the details about life history and distribution.
o	99-101: The authors describe sampling near the Fukushima power plant, but it’s not entirely clear whether this is relevant to the study beyond it being simply a place they looked for frogs. Is there some connection to the Fukushima disaster?
o	Did they sample in spring/summer for any particular reason?

Specific suggestions for results section:

•	Results
o	For the aid of the reader, it might be helpful to provide examples of the banding patterns expected from different combinations of frog species.
o	Supporting Table 1 appears to be missing from the supporting information
o	Were the sequences generated in this study deposited in GenBank? If so, please provide accession numbers.
o	265-275: It would be very helpful to see a visual summary (figure or table) of what was detected by which methods in both the laboratory and field studies. This is currently presented as +/- on the maps, but I think it would be nice to see presented on its own. That will make it easier for the readier to assimilate the results of the study.
o	276&277: Was Bufo observed visually and in the eDNA results? Was this sequence generated from the eDNA PCR product? I think it was, but it could be described a bit more clearly.

•	Language/grammar: overall, the English is well-constructed and legible, but the manuscript might benefit from another read-through and perhaps a review by a native speaker to resolve some awkward sentences and grammatical inconsistencies. Here are a few specific recommendations:
o	42&42: consider something like “the ecology of species and their ecosystems”
o	45&46: instead of “sympatric related species,” try “closely related sympatric species”
o	54&55: I suggest something like “This potentially places limits on the number of institutions able to conduct eDNA studies”
o	55-57: “For ensuring sustainability of monitoring activities and conservation of species diversity, an efficient method should be developed for easy application in many laboratories close to a field site.” This sentence is cumbersome and should be reworded.
o	79: Begin sentences with the full genus & species names
o	82: “Breau” should be “bureau”
o	83: “Depertmet” should be “Department”
o	101: Use “effectiveness” instead of “effectivity”
o	135&136: “For exemplification of applicability in various scales of distribution monitoring studies” is also quite awkward
o	145-147: I suggest something like: “To prevent contamination by the field sampler, we began collecting at downstream sites, and moved upstream as subsequent samples were collected.”
o	148: remove “immediately”
o	149-150: instead of “presence and non-detection,” use “presence or absence”
o	160: instead of “particle retention,” use “mesh size”
Experimental design: The experimental design of this study largely falls within the desired criteria. Here are some specific suggestions regarding the methods:
•	Methods
o	112: the negative control is described as water previously used for other non-target frog species. Was this done (as opposed to using completely clean water) to test for non-specificity of their primers? If so, it may help to add a sentence explaining that.
o	123 & 141: why did the authors choose to filter only 500 mL? Other eDNA studies seem to filter 1L at minimum. Was there any replication? This may be one of the reasons for the lack of detection in running water.
o	123: How were the DNA-free bottles sterilized?
o	124 & 148: How long were the samples kept frozen before they were filtered?
o	133-141: I think it would help to have a broader map of Japan as a whole upon which to place the more specific sampling-location maps. As a non-Japanese reader, it would be helpful to place the study in a geographic context. Similarly, it would be helpful to have the sample locations presented in a table.
o	143: Is it necessary to give the date and time of sampling? If there’s some relevance to the life history or ecology of the target species, please provide that. Otherwise, it’s probably extra detail that can be cut out.
o	149: This section is very short, but it might be helpful to place the visual survey methods under their own sub-heading.
o	159: What was the reasoning behind the choice of 0.7 um mesh size for the filters? There’s nothing explicitly wrong with that but many  other eDNA studies seem to use 0.2-0.45 um filters.
o	160: Please describe the sterilization process in a bit more detail: what was rinsed and for how long?
o	161: Were the filtering controls carried to the sample sites? Or were they just laboratory controls?
o	163: Was the aluminum foil sterilized?
o	164-175: What sort of contamination controls were used during DNA extraction (surface sterilization, laminar flow hood etc.)?
o	197: Is there a citation for the stated expectation of different digestion patterns?
o	203-207: It’s not clear to me exactly what was sequenced? Was it the eDNA PCR products? Specifically, which amplified fragments were sequenced?
o	208-247: It might be helpful to have the qPCR section placed under its own sub-heading
o	248-251: The section on contamination avoidance could be moved to the top of the molecular methods section.
Validity of the findings: The findings of this study are presented clearly. There were some questions about the filter controls used, which I addressed in the experimental design section of this review. For lack of a better section in which to include it, here are some specific suggestions regarding the discussion section:

•	Discussion
o	301: Is the Zemtsova, Montgomery & Levin, 2015 citation relevant to this statement? That study uses qPCR to detect pathogens in blood. Is there a citation that supports this claim for eDNA detections?
o	304-308: Many other studies have been successful at detecting target species in flowing water. It might be helpful to include discussion of what may explain the difference in results between those studies and this one. One possibility that stands out to me is the volume of water that was filtered. Other studies also chose to sample in eddies or pools in flowing watercourses, where genetic material might accumulate. Some also sample below the surface.
Additional comments: This study provides a valuable test of a tool that may simplify rapid ecological survey of an increasingly threatened group of animals. While they failed to detect the presence of frogs in flowing water, the PCR assay is shown to be useful for static water. Further experimentation and refinement may enable the assay to be used for flowing water habitats as well.


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

Input:
Title: AN APPLICATION OF PCR-RFLP SPECIES IDENTIFICATION ASSAY FOR ENVIRONMENTAL DNA DETECTION
Review round: 1
Reviewer: 2

Output:
Basic reporting: The aim of the study was to develop a PCR-RFLP method for eDNA detection of three rana frog species in Japan. The manuscript in general is well written and structured, thought there were several errors in the figure legends and text (see individual comments). The strengths of the study to me were 1) the combination of the lab and field trials and 2) the validation of the PCR-RFLP by field observations, qPCR, and sequencing of some of the amplicons.
Experimental design: 3.	In the methods, the authors had 8 replicates for qPCR but it seems there was only one replicate for conventional PCR. I wonder why and if this may be another reason for false negatives in the field trials. eDNA studies with conventional PCR typically had multiple replicates as far as I know.
4.	There were no lab treatments with all three species. Don’t they coexist in the field at all? If so, does this method still work with all three species?
Validity of the findings: 5.	The conclusion that the PCR-RFLP works for lentic habitats but not lotic habitats is an interesting one. I wonder though if there were any other differences, other than eDNA concentration as discussed, between these two types of habitats that would affect eDNA detection. If the difference is only eDNA concentration as discussed, this means the method cannot detect eDNA even in lentic habitats with low eDNA concentrations or the method should be able to detect eDNA in lotic habitats with high eDNA concentrations.
Additional comments: The aim of the study was to develop a PCR-RFLP method for eDNA detection of three rana frog species in Japan. The manuscript in general is well written and structured, thought there were several errors in the figure legends and text (see individual comments). The results are inconclusive, which is fine according to one of the review criteria. The strengths of the study to me were 1) the combination of the lab and field trials and 2) the validation of the PCR-RFLP by field observations, qPCR, and sequencing of some of the amplicons. That said, I wonder about some issues/questions as follows. With appropriate responses and revisions, the manuscript should be acceptable. It is a neat study. Cost-effective detection of eDNA definitely helps. I hope my comments are helpful.

1.	Although in Intro it is said that the PCR-RFLP method was developed to detect the three common rana frogs in Japan, the results (Fig. 1) seem to show those for only one species (R. tagoi). It would be nice to show the results for all three species.
2.	I wish the quality of the gel images were better. For example, in Fig. 1A it was difficult to see the unique band (~250 bp) of R. japonica in the treatments in which there was only one R. japonica. The tagoi unique band (~300 bp) showed up pretty weak in all tagoi treatments. Then, the same faint bands seem to be present in the R. ornativentris treatments. In Fig 1B, there seem to be faint bands at ~400 bp length. Do they mean anything? I am sure that the authors were able to tell those better by looking at the actual gels. But with the images in the Fig. 2, it is difficult for readers to see them.
3.	In the methods, the authors had 8 replicates for qPCR but it seems there was only one replicate for conventional PCR. I wonder why as eDNA studies with conventional PCR typically had multiple replicates as far as I know. This may be another reason for false negatives in the field trials.
4.	There were no lab treatments with all three species. Don’t they coexist in the field at all? If so, does this method still work with all three species?
5.	The conclusion that the PCR-RFLP works for lentic habitats but not lotic habitats is an interesting one. I wonder though if there were any other differences, other than eDNA concentration as discussed, between these two types of habitats that would affect eDNA detection. If the difference is only eDNA concentration as discussed, this means the method cannot detect eDNA even in lentic habitats with low eDNA concentrations or the method should be able to detect eDNA in lotic habitats with high eDNA concentrations.
6.	In Abstract and Intro, the authors pitched the PCR-RFLP for amphibians in general when the method is only for the three Japanese rana species. Given the scope of the journal, the limited applicability is fine. But it is still misleading.

Individual Comments

Abstract
L 26: Insert “using quantitative PCR or next generation sequencing” after “the cost of the eDNA detection.” There are enough published studies that used traditional PCR for eDNA detection.
L 27-28: “foraying into” sounds odd for the use.
L 28-29: Unlike eDNA barcoding primers for amphibians (Valentini et al. 2016), the PCR-RFLP method discussed in this study is specific for those three Japanese rana species. I am not sure how the specific PCR-RFLP method benefits studies of amphibians in general
L 31: Inclusion of the Fukushima Nuclear Power Plant triggered my interest. Yet, there is no relevant discussion about this in the manuscript.
L 34-35: Please see my major comment on lentic vs. lotic habitats
L 37: “the golden standard of field observation” The point of using eDNA for many is that field observation is not golden for some species. If it is golden, I wonder why the authors became interested in developing this eDNA method. Also a field survey of pond breeding amphibians (calling in the breeding season and counting egg masses) is easy and time efficient.

Introduction
L 42: Change “a species” to “the species”
L 43: Change “conservation of a species” to “their conservation”
L43-46: The sentence can be improved. More importantly, these issues are not limited to field surveys but also relevant to eDNA-based surveys.
L 58: Change “more efficient” to “more cost-effective”
L 66: “to-date this method has not been applied to terrestrial organisms including amphibians” sounds misleading. The authors compared their amphibian study with the Clusa et al. 2017 that developed PCR-RFLP for fish. Some amphibians can be described as “terrestrial” but their larvae are aquatic and this study analyzed eDNA in water samples.
L 66-67: Clusa et al. 2017 developed the method for three trout species commonly stocked in North Hemisphere. Thus, their method has a broad applicability. The PCR-RFLP in this study is specifically designed for the three Japanese frogs and no applicability to other amphibians.
L 86: Delete “which are often”
L 97: It is nice to explain why having various spatial scales in sampling was important.
L 100: Change “regenerating habitats after the disaster” to “habitats recovering after the disaster”

MM
L 113: Please explain why the water that previously contained other frog species was used as negative controls and what those species were.
L 116: I am not familiar with Tahara (1974). I encourage the use of Gosner (1960) staging as it is much more commonly used.
L 120: Was the tap water aged?
L 123: Insert “according to the treatments” after “specific periods”
L 136: I am not sure how having “various scales” has anything to do with checking the performance and sensitivity of PCR-RFLP
L 138-141: A slight mismatch between these site IDs and the ones in Fig. 1
L 141: 500mL is a small volume. Is this an issue for not detecting eDNA?
L 146: Insert “a” before “field sampler”
L 162: Insert “cross” before “contamination”
L 186: Please specify the length of the 16S rRNA fragment amplified
L 208-: Nice to add an explanation as to why R. tagoi was selection for the qPCR assay
L 234-235: Were there any replicates for PCR-RFLP? If not, it would be nice to have replicates. Some studies that used conventional PCR for eDNA detection had replicates.
L 245-247: This explanation about LOD was unclear to me. I would appreciate clarification.

Results
L 257-258: Please see my major comment.
L 270: Do you have evidence for qPCR being more sensitive than PCR-RFLP?
L 276-280: Nice to better explain how this part about Bufo is related to the development of PCR-RFLP for the three Rana frogs.
L 282-286: Again, the site numbers do not correspond to those in Fig. 1. Fig. 1C says that R. japonica was detected in site 27.

Discussion
L 300-301: I wonder if there is a clear agreement on the sensitivity of qPCR over conventional PCR. For example, Garland et al. (2011, Diseases of Aquatic Organisms) reported that conventional PCR is as sensitive as qPCR. Thus, it may depend on the system.
L 304-306: Please see my major comment

Figure 1
There are no explanations on the panels (A, B, and C) of Figure 1 in the legend. Also it doesn’t say this is about detection of which species out of the three.

Figure 2
I would appreciate better quality of the gel images (please see my major comment). Also (A) is missing from the legend.

Supp Table 1 (L 268) was missing


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 APPLICATION OF PCR-RFLP SPECIES IDENTIFICATION ASSAY FOR ENVIRONMENTAL DNA DETECTION
Review round: 2
Reviewer: 1

Output:
Basic reporting: Upon review of the revised and resubmitted manuscript, the authors appear to have satisfied the original comments and concerns of both myself and the second referee and I believe it warrants publication as it is.
Experimental design: See section 1.
Validity of the findings: See section 1.
Additional comments: See section 1.


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

Input:
Title: EFFECTS OF TRAPPING EFFORT AND TRAP PLACEMENT ON ESTIMATING ABUNDANCE OF HUMBOLDT’S FLYING SQUIRRELS
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: This is a nice short paper that looks at trap placement, and how many trap nights are required to get reasonable population parameters for Humboldt’s flying squirrel: the answer probably being placing traps on tree trunks for 8 nights. The authors estimate abundance, capture probability, and recapture probability over 4, 8, and 12 trap nights using two datasets spanning 6 and 4 years from several sites in HJA and SIU forests. Trapping method was mark-recapture using Tomahawk live traps, and conducted in accordance to proper animal care and handling procedures. Data was analyzed using Huggins robust design modeled in RMARK, which is an appropriate and well accepted method for deriving population parameters.

In short, this is a well written paper, with clear methods, rigorous analysis and results reporting, and sound description of conclusions. The trapping methodology was carried out according to proper animal care procedures. The study was granted appropriate permits, and no undue stress was issued to the animals.

I do not have many suggested edits, but I would like to see an addition in the Results and Discussion exploring how trapping for 4-, 8-, and 12-nights affected animal condition or mortality. The authors mention that reducing trap nights from 8 to 12 reduces stress on animals, but the authors do not explore it (line 332-344).  This addition would add valuable and pertinent information to their article, and should not be particularly onerous. Adding animal condition information would be especially important considering the authors describe cases where researchers have increased trapping duration from 8 to 12 nights to improve capture numbers in low density populations. What is the actual cost for animals trapped over 12 nights as opposed to 8? Is there an increase in mortality, or a decline in apparent condition of the animals?

Line 114: I would like to see a small map of the area added in

Line 117-120: SIU elevations are reported twice, and are different

Line 332-344: There is some speculation here. I would like to see this section expanded with actual data to further explore how animal condition changes over 4-, 8-, and 12-trap nights.

Line 348-350: I agree with the authors’ conclusion that prebaiting does not seem to impact trapping periods of 8 or 12 days. However, the authors do not mention the effect of prebaiting on the 4-night period, where prebaiting would arguably have the greatest impact, and which does appear to be different. I would like to see this paragraph expanded to include the impact of prebaiting on the 4-night trapping session.


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

Input:
Title: EFFECTS OF TRAPPING EFFORT AND TRAP PLACEMENT ON ESTIMATING ABUNDANCE OF HUMBOLDT’S FLYING SQUIRRELS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The basic reporting in this manuscript is well done and conforms to Peer J standards and raw data were provided in supplemental files
Experimental design: The manuscript appears to represent original primary research and is within scope of the journal. The basic research questions are well defined, relevant and meaningful and It is stated how the research fills an identified knowledge gap.
It appears that rigorous investigation was performed to a high technical and ethical standard.  The field and analytical methods are described in sufficient detail to replicate.
Validity of the findings: Based on my examination, the data appear to be robust and statistically sound.
Additional comments: In this paper, the authors examine the efficiency of various live trapping strategies (ie, trap placement and successive nights trapped) on a small mammal, Humboldt’s flying squirrel. They evaluated how bias and precision of estimates for apparent annual survival, abundance, capture probability, and recapture probability of Humboldt’s flying squirrels varied with the number of secondary trapping occasions.

The authors used a sequential modeling procedure to analyze their trapping data and found that reducing the length of primary trapping occasions from 12 to 8 nights had very little impact on parameter estimates, but further reducing the length of primary trapping occasions to 4 nights caused substantial bias in parameter estimates and decreased precision. Thus, this study suggests that 8 nights of trapping is sufficient to effectively sample Humboldt’s flying squirrels, reducing the need for extra nights which would likely cause extra expense and may cause unnecessary stress to animals. The authors also found that attaching Tomahawk traps to trees increased the site- and year-specific capture frequency of flying squirrels. This is consistent with my own personal experience trapping for these squirrels.

Overall, this is a solid, well done and well written paper.  It provides useful information for field biologists working on Humboldt’s flying squirrels, and provides a road map for evaluating optimal trapping strategies for other species.  The figures are clear and useful and the methods and results are well described.

This is an interesting, well done paper that provides useful information on study design for live trapping Humboldt’s flying squirrels, and is a good example of how to determine an efficient live trapping regime for small mammals in general.  I liked the sequential modeling approach the authors used to analyze their data; it is logical and seems statistically sound to me.  The authors also confirmed that placing some traps on tree trunks increased trapping efficiency.  This is a common practice, but it takes extra time in the field to attach live traps to trees, so it is nice to know that the extra work seems to have a positive return.

One suggestion I have is to add a citation for Humboldt’s flying squirrel in the introduction around line 70.  This species was just described in 2017, and many biologists might still not be familiar with it and it’s Latin name, Glaucomys oregonensis.  Something like “Two species of flying squirrels, the recently described Glaucomys oregonensis (Arbogast et al 2017) and G. sabrinus, have been of interest because they serve as prey for the federally threatened northern spotted owl...”

I also think the Figure Legends could be tightened up a little. For example, for Fig 1, consider “Recapture probabilities of Humboldt’s flying squirrels in the H. J. Andrews Experimental Forest and the Siuslaw National Forest in western Oregon. Estimates from 3 nested subsets of data are shown for each study area with vertical bars indicated the 95% confidence intervals.”


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

Input:
Title: EFFECTS OF TRAPPING EFFORT AND TRAP PLACEMENT ON ESTIMATING ABUNDANCE OF HUMBOLDT’S FLYING SQUIRRELS
Review round: 1
Reviewer: 3

Output:
Basic reporting: Basic reporting is reasonable.
Experimental design: Design is sound and fundamental questions are well defined.
Validity of the findings: Findings are well-supported and reasonable.
Additional comments: The authors use capture-recapture data on flying squirrels collected during a series of 12-occasion surveys from two areas to explore the consequences of reducing effort to 8 and 4 days on parameter estimates and to evaluate the relative effectiveness of traps placed in two different locations.  Although I offer a number of comments and suggestions below, I think the paper is solid and well written.  My main criticism is that the focus is quite narrow for a paper exploring design alternatives.  Consequently, my principal suggestion focuses on broadening the scope of the work to increase the relevance of the paper to other species and to a wider range of sampling designs.

One issue with using only field data to explore design options is that we do not know the true value of parameters, which eliminates the possibility of exploring bias (see below) and makes exploring precision complex for several reasons, one of which it becomes conditional on the choice of model for analysis.  Although I appreciate the realism we get from field data (and I am an empiricist), simulations provide clearer insights on the effects of altering design options.  Therefore, my primary suggestion is to add some simulations to the paper.  That will require a modest amount of effort, but what you learn through that exercise will complement the results from field data and strengthen the paper in multiple ways, one of which is allowing you to delineate more carefully the influence of effort on bias and precision.

Specifically, exploring a range of plausible values for the key parameters under a series of scenarios would allow those interested in other species to benefit from your work and allow you to view flying squirrels as a case study.  Because capture and recapture probabilities are quite low for flying squirrels (especially at one of your sites), your work only has relevance to a narrow subset of similar species and similar sampling designs.  I appreciate the practicality of 4, 8, and 12 occasions in your specific study, but exploring a wider range (e.g., 4-12) would help to illustrate and inform a range of other possible design alternatives.  Same holds for exploring a range of different values for the target demographic parameters and detection-related parameters.  You would not need to explore all that many values for each parameter to inform choices relevant to a wider range of species and sampling designs.  In fact, in addition to the values you report for flying squirrels, I’d inform the ranges of values to explore based on empirical work from other small mammals.

Below, I’ve provide a number of line-by-line comments.  Although some are substantive, I see them as relatively minor compared to broadening the scope.

Line 20.  I’d be cautious about using the word bias to represent deviation from estimates based on 12-occasion surveys.  Strictly, bias is deviation between the expected value of an estimate and the true value of the parameter, which you don’t know (but would with simulations).  I don’t think it’s unreasonable to assume the 12-occasion estimates are ‘better’ than those based on fewer sampling occasions, but you have no real way to gauge that.  So I think the quantity is useful to report, but I would call it something different (maybe apparent bias?).

Line 41 and elsewhere. You use abundance and population size interchangeably; probably best to be consistent (I’d vote for abundance).

Line 97.  Smaller than needed to achieve what objective?  These sorts of statements only make sense for specific objectives.

Line 160.  Sticking with one approach eliminates model bias, so I don’t mind that you only considered only Huggins’ models.  But I think you should explain why you made that choice and whether you explored other approaches.  To be clear, I don’t think you need to include information on that initial effort (assuming there was one), but I was curious how and why you chose the one you did.

Lines 179-210.  Unless I missed it, you do not discuss the criteria you used to choose among models (I assume you established an arbitrary value of AICc) or how you dealt with competing models.

Line 197.  Replace ‘if the data supported the modelling of’ with ‘if there was evidence for’

Line 198.  Considering a behavioral response to trapping seems fine, but I wondered why you didn’t also pursue heterogeneity (and perhaps b + h) as it emerges as a reasonable model for many species.

Line 215.  If site and year were modeled as random effects (which I think you’re implying), I’d mention that explicitly.

Line 216.  It’s no big deal, but I don’t understand the value of R-squared when the question revolves around a single parameter.  In the results (lines 290-296), R-squared gets the lion’s share of attention and the actual result is relegated to the end.  Therefore, I’d suggest moving the result to the front of the paragraph and offering R-squared parenthetically (if at all).

Evaluating the question of model fit is a lot more valuable when the goal is parameter estimation, but you did not include it there and probably should (i.e., goodness-of-fit tests for your top models).

Line 239 and throughout this section. You should include units for each estimate you report otherwise readers might think they are direct probabilities, when each beta is on the scale of the link function.

Line 245.  Something seems off here – perhaps an aberrant ‘to 0.53’?  More importantly, this entire section is challenging to work through, so you might consider eliminating contrasts such as this when they are based solely on point estimates (i.e., the CIs largely overlap, so I am not sure claims of systematic patterns are especially compelling).

Line 246-7.  Seems awkward to use both a model-selection approach (I suspect by ‘some support’ you mean that one or more of the competing models included a site effect) and a frequentist approach (because they are based on an alpha level, CIs are frequentist measures).  Seems cleanest to me to simply go with whether the CI for beta overlaps zero as you do elsewhere.

Line 249 and throughout this section.  You sometimes report probabilities as percentages; probably best to stick to probabilities.

Lines 262-266.  I think it would be better to convert these to a per hectare basis rather than on the scale of the plot.

Line 273.  Delete the word ‘parameter’

Lines 290-296.  See my comment offered for line 216.

Lines 307-309.  I’m not sure I see the contrast implied by the ‘but’ in the middle of the sentence.  We don’t expect bias to change systematically with sample size, just precision (as you note), so CIs should always overlap.

Lines 311-314.  This all seems likely to be an artifact of low capture probabilities, which is true for many of the patterns you report.  Here, based on Figure 2, CIs around the point estimates for the 4-occasion surveys seem to include a neutral or even negative beta, so it doesn’t seem that the change in sign is especially meaningful.  The issue is likely an artifact of so few recaptures for a species with low detection rates and even lower recapture rates.  That seems to be the important point – you need sufficient data to get good estimates.

Lines 332-344.  Of course, your goal is to explore the consequences of reducing effort, but as I mentioned earlier, whether than is a reasonable trade-off will depend entirely on the overarching objective.  For long-term work, optimal sampling designs will always be different than those for short-term work.  I think it would be worthwhile to readers to explain how design is always a series of trade-offs and that the ideal design depends largely on the features of the target population and the target objective.

Figure 1.  Unlike Figure 2, each set of related values illustrated here are model-based prediction based on a constant beta, so it could be illustrated more simply by plotting the betas and their CIs.  More importantly, as the paper now stands, the focus is effectively on how precision of estimates changes based on different subsets of data.  Therefore, I’d think it would be more effective to illustrate directly how precision (say SE or CI width) of parameter estimates changes as the number of sampling occasions changes, rather than illustrating model effects, which is not the primary focus (at least according to the title).

Also, you should add the word ‘Estimated' to the start of the legend of this and related figures and indicate the model you’re using to estimate the values displayed.

Table 1.  Given that temporary immigration and emigration are model parameters, you should at least mention that you held them constant in the table heading.  More importantly, I am curious as to why you chose to include site as a covariate rather than a few habitat covariates (e.g., tree density, stand age, etc.).  Sites is okay, but requires many more parameters (unless fit as a random effect) and carries no ecological information, so a few key habitat might be more interesting and more parsimonious.

Table 2.  I don’t follow the information in the footnote – specifically, I am not sure what you mean by ‘held to site.’  If you mean identifying sites as structural units in the analysis, wouldn’t adding a simple site effect allow for additive differences among sites (i.e., there’s no need for interactions)?  Perhaps I have that wrong, but either way, it would be good to clarify this section and even include a section in the methods if it’s complicated. Lastly, I don’t think the AICc column is useful.

I hope my comments are of some value to you.

Best wishes,
Bob Steidl


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

Input:
Title: EXPRESSION OF LEPTIN AND LEPTIN RECEPTORS IN COLORECTAL CANCER—AN IMMUNOHISTOCHEMICAL STUDY
Review round: 1
Reviewer: 1

Output:
Basic reporting: Professional English was used throughout the manuscript.
The literature references were detailed.
The Tables were of professional quality. The Figure quality was good.
Note: I was unable to find any Figure legend in the manuscript.
The results were relevant and supported the hypothesis.
Experimental design: The experimental design was relevant to the study area. The statistical analysis was detailed.
Validity of the findings: The results were elaborate and the discussion was well written.
Additional comments: Manuscript title:  Expression of leptin and leptin receptors in colorectal cancer – an immuno-histochemical study
Shibli, SM et al. have performed immuno-histochemical analysis on 44 colorectal cancer patients. There results demonstrated that both Leptin and Leptin receptor are over-expressed in surgically resected colon cancer tissues when compared to normal adjacent colon tissue.
The manuscript is well written. The results and conclusions are crisp and to the point. The discussion section was compiled in a very professional manner. I would like to compliment the authors for comparing their findings with previously reported literature in great details. In my opinion, the manuscript is suitable for publication.
Some minor comments:
1)	Figure legend for Figure 1 was missing.
2)	A comparison of LEP and LEPR levels in cancerous tissues versus inflamed colon tissues (from non-cancerous patients) would have made this manuscript even more interesting. This would have helped in understanding if LEP and LEPR can be used as a potential marker to differentiate between colorectal cancer and non-cancerous colorectal inflammation related diseases.
3)	Please consider removing lines 31 and 32 where the authors mention that they are the 1st to report such a high presence of LEP and LEPR in colorectal cancer in any part of the world. Reason: IHC results are based on the techniques and reagents used across the samples. Moreover, grading in IHC results (eg. +1. +2 or +3), until quantified by specific digital methods, is not comparable among multiple studies, unless the same pathologist grades the tumors at the same time.
4)	Please reconsider modifying lines 280 and 281. Based on this manuscript, the increase in LEP and/or LEPR expression has not yet been characterized to be a cause or an effect of neoplastic progression. Moreover, the role of LEP and LEPR signaling pathway has not been characterized in this manuscript. Thus, the authors may reconsider their conclusion of considering the LEPR pathway as a potential candidate for anti-cancer therapy. Alternatively, the authors can include that their findings combined with research from some other research groups (as mentioned in the introduction part of this manuscript) make the LEPR pathway an interesting candidate for potential anti-cancer therapy.


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

Input:
Title: EXPRESSION OF LEPTIN AND LEPTIN RECEPTORS IN COLORECTAL CANCER—AN IMMUNOHISTOCHEMICAL STUDY
Review round: 1
Reviewer: 2

Output:
Basic reporting: Well written English and literature citation.
Experimental design: Methods and research question well defined.
Validity of the findings: Conclusions need revision to bridge with old and future findings.
Additional comments: Comments on manuscript # 37549 "Expression of leptin and leptin receptors in colorectal cancer - an immunohistochemical study. " by Al-Shibli et. al.

The author studied the expression status of hormones and hormone receptor (Leptin and Leptin receptor) in a bunch of colorectal cancer tissues as well as the adjacent normal tissues but the role is still missing as per author’s findings.

Following things need to be addressed in the MS.
1.	Line 93-95 and line 264-266: Normal adjacent tissues showed low expression of LEP and LEPR, but CRC tissues showed high expression of LEP and LEPR and author claimed in accordance with other authors findings that high expression is correlated with high prognosis, how? That explanation is missing in MS, which is the main finding of this MS.
2.	Line 80 and 97: LEP (ob/ob) animals showed less bone growth/mass but increase on LEP administration and LEP acts as a growth factor in-vitro and not in-vivo-these are itself controversial statements. Should be explained in MS?
3.	Line 235: Author stated that patch distribution of LEP was found in CRC tissues in accordance with reference 80. Is that the purity reason of the tissue, if author claimed that it is not differentiation specific or grade specific problem? Please explain in MS.
4.	Figure 1: H&E staining should be included to see the proliferation/tissue integrity of the tissues used for IHC staining?
5.	Validation of IHC results with other technique, like Western blotting or qPCR-would confirm the handling/analysis of IHC results.
6.	Conclusion of the findings of this MS w.r.t. novelty or addition to the already known findings is specifically lacking.
The paper is well written in nice English. Comments above- will make the MS better.


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

Input:
Title: EXPRESSION OF LEPTIN AND LEPTIN RECEPTORS IN COLORECTAL CANCER—AN IMMUNOHISTOCHEMICAL STUDY
Review round: 2
Reviewer: 1

Output:
Basic reporting: Professional English was used throughout the manuscript.
The literature references were detailed.
The Tables were of professional quality. The Figure quality was good.
The results were relevant and supported the hypothesis.
Experimental design: The experimental design was relevant to the study area. The statistical analysis was detailed.
Validity of the findings: The results were elaborate and the discussion was well written.
Additional comments: Manuscript title: Expression of leptin and leptin receptors in colorectal cancer – an immuno-histochemical study
Shibli, SM et al. have performed immuno-histochemical analysis on 44 colorectal cancer patients. There results demonstrated that both Leptin and Leptin receptor are over-expressed in surgically resected colon cancer tissues when compared to normal adjacent colon tissue.
The manuscript is well written. The results and conclusions are crisp and to the point. The discussion section was compiled in a very professional manner. I would like to compliment the authors for comparing their findings with previously reported literature in great details.
The authors have made the necessary modifications to the manuscript as suggested in my 1st review.
In my opinion, the manuscript is now suitable for publication in PeerJ.


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

Input:
Title: EXPRESSION OF LEPTIN AND LEPTIN RECEPTORS IN COLORECTAL CANCER—AN IMMUNOHISTOCHEMICAL STUDY
Review round: 2
Reviewer: 2

Output:
Basic reporting: Well written in Professional English and literature citation.
Experimental design: Research question well defined and performed.
Validity of the findings: Results nicely concluded as per data.
Additional comments: Author revised MS as per all reviewer's comments, including mine and now the revised MS looks clear and understandable as per scientific point of view.


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: EXAMINING THE INFLUENCE OF PICTURE FORMAT ON CHILDREN’S NAMING RESPONSES
Review round: 1
Reviewer: 1

Output:
Basic reporting: see General comments for the author
Experimental design: see General comments for the author
Validity of the findings: see General comments for the author
Additional comments: Review of ms (#35015) entitled "Line drawing or photograph? Examining differences of picture format in children’s naming" by Naroa Martínez and Helena Matute for PeerJ

The manuscript reports "norms" that were collected from line-drawings and photographs on thirty-eight and thirty-four Spanish-speaking children aged from 8 to 10 years. The findings indicate that the format of pictures plays a role in the naming performance. Pictures of objects represented by photographs lead to higher name agreement scores (and less alternative incorrect names), higher visual complexity scores compared to the corresponding pictures of objects represented by line-drawings. The only exception is familiarity for which no significant difference was found between the two picture formats.
Evaluation
I found the study reported in the ms interesting. It addressed an important issue, namely whether certain psycholinguistic norms collected from line-drawings and from photographs of the same objects are different. In particular, different psycholinguistic "norms"  were collected on children which is not often the case in the literature. In effect, most normative studies that have used pictures of objects have been conducted in adults. Therefore, the findings have important implications for the investigation of picture naming speed and accuracy in children. The methodology used is generally sound even though the choice of a complete within-design (Picture format x Norms) in the two studies is questionable because of carry-over effects. The analyses were correctly done. The overall message and the conclusions are clear. However, there are major concerns that need to be addressed before publication is concerned.

(1) The Introduction needs more details. Indeed, the review of the literature is clearly incomplete. There are a lot of normative studies that have been done on line-drawings or on photographs that are not reported in the ms but that are worth citing. Therefore, some good efforts should be made to include more of these studies in a revision. It is important to mention that a lot of studies have used the same sets of pictures (e.g., Snodgrass and Vanderwart, 1989 and the corresponding colorized version by Rossion and Pourtois, 2004) to collect norms in different languages and cultures. To give a few examples of studies that should be included in the ms:
*Navarrete, E., Arcara, G., Mondini, S., & Penolazzi, B. (2019). Italian norms and naming latencies for 357 high quality color images. PLoS ONE 14: e0209524.
*Bonin, P., Peereman, R., Malardier, N., Méot, A., & Chalard, M. (2003). A new set of 299 pictures for psycholinguistic studies: French norms for name agreement, image agreement, conceptual familiarity, visual complexity, image variability, age of acquisition, and naming latencies. Behavior Research Methods, Instruments, & Computers, 35, 158–167.
*Pind, J., Jónsdóttir, H., Tryggvadóttir, H.B., & Jónsson, F. (2000). Icelandic norms for the Snodgrass and Vanderwart (1980) pictures: Name and image agreement, familiarity, and age of acquisition. Scandinavian Journal of Psychology, 41, 41–48.
*Raman, I., Raman, E., & Mertan, B. (2014). A standardized set of 260 pictures for Turkish: norms of name and image agreement, age of acquisition, visual complexity, and conceptual familiarity. Behavior Research Methods, 46, 588–595.
*Russo, N., Hagmann, C. E., Andrews, R., Black, C., Silberman, M., & Shea, N. (2018). Validation of the C.A.R.E. stimulus set of 640 animal pictures: Name agreement and quality ratings. PLoS ONE, 13: e0192906.
*Tsaparina, D., Bonin, P., & Méot, A. (2011). Russian norms for name agreement, image agreement for the colorized version of the Snodgrass and Vanderwart pictures and age of acquisition, conceptual familiarity, and imageability scores for modal object names. Behavior Research Methods, 43, 1085–1099.

(2) Some references must be updated. I was very surprised to see that the authors were referring to one review paper published in 1996 (Johnson, Paivio, & Clark, 1996). Indeed, there are more recent reviews that the authors should be aware of and consider reporting in the revised version of their ms. Again, I provide the following two references that should be included in the revised ms, but there are certainly others worth mentioning, for instance certain chapters in Brenda Rapp’s (2000) book entitled "Handbook of Cognitive Neuropsychology: What Deficits Reveal About the Human Mind" (Psychology Press).
*Levelt, W. J. M., Roelofs, A., & Meyer, A. S. (1999). A theory of lexical access in speech production. Behavioral and Brain Sciences, 22, 1–75.
*Roelofs, A., & Ferreira, V. S. (in press). The architecture of speaking. In P. Hagoort (Ed.), Human language: From genes and brains to behavior. MIT Press

(3) There are two studies which are directly relevant to the issues addressed in the current study but that were surprisingly insufficiently described.
*The study by Bonin et al. (2019, Reading & Writing) is described only very slightly. Indeed, more space should be given to this study. In effect, the authors compared three different formats in written naming and investigated whether and how image characteristics influence written naming performance in adults. There were black-and-white pictures (Snodgrass & Vanderwart’s 1980 drawings), grayscale and colored pictures of the SV drawings as provided by Rossion and Pourtois (2004). Among the main findings, the authors found that colorized pictures yielded shorter written naming latencies than line drawings with the grayscale pictures being situated between the two. The addition of gray texture did not have an effect on the ease of recognition of the objects. The latter finding should be added more explicitly, for example by stating that the greyscale does not improve recognition as indexed by name-agreement scores. In any case, this study should be better described because it is fully relevant for the issues addressed in the ms.
* The methodology used by Saryazdi et al. et al. (2018) is interesting and should also be reported since it is share some features with the current study.

(4) There are also certain claims in the ms which are incorrect and should be corrected (see line-by-line comments).

Other line-by-line comments
Lines 48-51. I was really surprised to read that "Snodgrass and Vanderwart in their 1980 paper showed that pictures were named more readily when a particular word…" because in this publication the naming performance in adults was not measured online. The sentence must be reframed because this does not reflect was SV did exactly in their study.
Lines 52-53. Martein (1995) did not use the SV set of pictures.
Line 60. As far as MultiPic is concerned, please indicate some of the languages in which norms were collected in adults.
Lines 81-83. The authors mention "name agreement" and then "response latencies" when discussing the influence of picture format. This is not the same DV as they know. This must be corrected and clarified.
Lines 90-92. I do not understand what is the meaning of what is written here, please clarify.
Lines 94-96. The description of Johnson’s (1992) study is very confusing.
Lines 101-107. Please indicate explicitly which studies used black-and-white vs. colored drawings vs. black-and-white (or colored) photographs and compared performance in memory tasks and which compared performance in picture naming.
Lines 107-110. The description of Bramão et al. (2011) is very allusive. How was "recognition" measured in the 35 experiments?
Line 131. "name agreement, alternative names, unknown responses". When name agreement is measure using the H statistics, alternative names are taken into account. Please be explicit when describing the different measures.
Lines 151-187. Materials
More information concerning the stimuli set should be reported: the different semantic categories (and how many items per category). Instead of reporting in the main text the statistical characteristics of the items (Mean, SD, range) for the variables corresponding to frequency, length, etc., perhaps a table could be used instead and inserted in the Materials section.
Please indicate here whether the full set of pictures can be downloaded freely. This is an important information.
Lines 353-372. Same remark as for Experiment 1 (lines 151-187).
Discussion
Line 454-457. It should be noted that in adults the %NA agreement has been found to be higher with line drawings than with photographs. This should be mentioned somewhere in the ms and the conclusion about the influence of picture format on the naming performance should be more nuanced (lines 460-476). Indeed, photographs do not always facilitate the picture naming performance in adults even though in the present studies it was clearly observed that in children photographs yielded more accurate naming performance than line-drawings. For example, in adults the overall name agreement scores have been found to be higher in both the SV database (88%) and the Bonin et al. (2003) database (77%) than in the BOSS database (64% in the Brodeur et al. study [2010] and 59.45% with the latter pictures normed in Thai by Clarke, & Ludington, 2018).


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

Input:
Title: EXAMINING THE INFLUENCE OF PICTURE FORMAT ON CHILDREN’S NAMING RESPONSES
Review round: 1
Reviewer: 2

Output:
Basic reporting: In the article entitled "Line drawing or photograph? Examining differences of picture format in children's naming", authors proposed to develop a photography database for picture naming experiments in children. They argued the choice of photographs rather than line drawing pictures because of the fact that several studies have reported an influence of format on both norms (e.g., Name Agreement) and spoken latencies. In general, the rational of the study is of good quality, the literature being well-cited and well-used. The subject treated is of great importance both for pscyhoeducation research (as indicated by authors) and also for fundamental researches. There are, however, certain points which prevent me from accepting this manuscript for publication in its present form. I do, however, show great interest in this study, and think that with modifications it could be a publication of importance.
Experimental design: A total of 38 children participated in the study. They are divided into two groups: one of 17 (3rd grade) and one of 21 (4th grade).  A set of 53 concepts was used in both image and photographic format (106 stimuli in total). The material seems to me to correspond to what is classically used in this type of study.The authors asked the children to perform three tasks: one of written image naming, one of conceptual familiarity assessment and one of visual complexity. In this way, the authors obtained norms for the three factors: Name Agreement, Familiarity and Visual Complexity.

It is the same with regard to the calculation of different scores. Research question is weel defined and it is stated clearly how authors' research fills an identified knowledge gap. The study was conducted under the appropriate ethical conditions.The methodology seems to me quite suitable. There are enough details to allow replication.
Validity of the findings: There are two points in this part that ask me questions.
First, I'm not sure I understand the reason for doing two experiments. If the authors have (as I think) the goal of providing a standardized material for children's photography, there is no reason not to share the material  to make one study. More generally, I'm not sure I understand the logic of using an experimental approach. Why make a standard collection study an experimental study?
Then, the authors use the analysis of variance to compare the normes obtained with the two image formats. I think that may be a problem. I am not sure that the reported data fit with a normal distribution. A test of Shapiro-Wilk or Kolmogorov-smirnoff can be used to test this point. If these tests are significant (as I think) a non-parametric tests (in this case, Mann-Withney rank tests) can be much more adequate and much less biais. Without this verification, it is hard to discuss results and conclusions.
Additional comments: Finally, I will have some general questions.
First, the authors worked on three classic norms: Name Agreement, Familiarity, and Visual Complexity. However, studies in adults indicate at least two other factors that have a direct impact on picture processing (for a recent synthesis see Perret & Bonin, 2018 Behav Res (2018) [url]Https://doi.org/[url] 10.3758 / s13428-018-1100-1): Image Agreement and Image Variability. Is there any particular reason why these factors (in direct relation to the visual characteristics of the image) have not been addressed?
Next, the authors used a relatively small sample of images, compared to other studies. Is there a particular reason for this small sample size?
In the same vein, are there criteria other than the linguistic ones that led to the selection of the data?
Finally, I am not sure what I mean by the phrase "No significant age differences were found ...".

I would like to reiterate the importance of this work to provide standardized photography material for children. It seems important, however, that authors address previous concerns before accepting the manuscript to publication.


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

Input:
Title: EXAMINING THE INFLUENCE OF PICTURE FORMAT ON CHILDREN’S NAMING RESPONSES
Review round: 1
Reviewer: 3

Output:
Basic reporting: As a preamble, please note that details concerning the different issues, that is to say basic reporting, experimental design, … are given in the attached file.
The submitted text generally complies with scientific articles standards. General structure is well organized. Bibliographical references are nearly sufficient, but are sometimes provided with erroneous or insufficiently precise and balanced arguments. Although the English is comprehensible, it seems sometimes uncomfortable and probably needs to be improved (as our proper English is sometimes questionable, we however do not try to give suggestions for this aspect). Results are however questionable essentially given the experimental design in use.
Experimental design: The issue addressed by this work pertains to the vast open debate concerning which type of 2-D images are better suited for such or such population and/or how and when cognitive processes could vary with the format. As pictures are sometimes the unique possibility to conduct experiments with children, for which the preceding questions were rarely addressed, it is a timely topic, which could be of interest for PeerJ. Despite the experimental design is in our opinion not well-suited and the materials choices are questionable, they are generally well described.
Validity of the findings: Participants and material choices are questionable given the literature and already existing databases : Arguments should be given to convince that the selected age band for participants is a good choice to speak about "children". Existing results indeed suggest that 8-10 years children productions are close to adults ones. The choice to build new sets of pictures while large normed databases already exist for adults should also be developed, particularly in view of name agreement measures, for which adults computations have been shown to be not well-suited for children.
The use of a repeated design leave uncertainty about the results that can be drawn from the data. We highly recommend to employ independent groups of participants, both for the norms and for the formats.
The way computations were done for one of the measured variables (NA%) should also be argued.
Bibliographical arguments given for supporting results have to be carefully checked in order to be more matched with the restricted measures that were done. They should be also more balanced.
These points are developed in details in the attach file.
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: EXAMINING THE INFLUENCE OF PICTURE FORMAT ON CHILDREN’S NAMING RESPONSES
Review round: 2
Reviewer: 1

Output:
Basic reporting: see general comments
Experimental design: see general comments
Validity of the findings: see general comments
Additional comments: This is my second review of this manuscript. The authors have taken seriously the concerns that I raised and the changes have resulted in a stronger paper. I have no remaining concern.


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: EXAMINING THE INFLUENCE OF PICTURE FORMAT ON CHILDREN’S NAMING RESPONSES
Review round: 2
Reviewer: 2

Output:
Basic reporting: OK All my concerns have been taken into account
Experimental design: OK. All my concerns have been taken into account
Validity of the findings: OK. All my concerns have been taken into account
Additional comments: OK. All my concerns have been taken into account. I think this manuscript is reaching for publication


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

Input:
Title: VARIATION OF 13C AND 15N ENRICHMENTS IN DIFFERENT PLANT COMPONENTS OF LABELED WINTER WHEAT (TRITICUM AESTIVUM L.)
Review round: 1
Reviewer: 1

Output:
Basic reporting: The article is well written in an understandable English

In my opinion there is more literature that should be commented in regard to d13C and d15N labeling and natural abundance. There is a lot of publications in wheat and other crops that studied C and N remobilization and measure natural abundance d13C and d15N, and use labeling with these two isotopes. See Zhou et al., 2014,2015, 2016 and papers published by Jose Luis Araus group, and Iker Aranjuelo (Aranjuelo et al., 2011; Plant Biology ISSN 1435-8603).

The article has a professional structure in figs, tables etc.. My only concern is that in the discussion there is a part in regarding to CV of d13C that seemed like a extension of the results. There is not real discussion with other publications. Please compare your results with other publications and try to find a sense to the different variation between high and low N treatments, and between the different organs. For example why the authors think that there is higher CV in the lower leaves than in the upper leaves? supported with other references.

In my opinion, the hypothesis of the manuscript is not clear enough or too descriptive, I will explain myself. Isotope labeling is a very powerful tool, to study C and N cycles, and has been used to study N2 fixation (Sanz-Saez et al., 2015), measure sink strength between organs and different cultivars (Zhou et al., 2014, 2015, 2016), N use efficiency (Felini et al., 2007), etc. However, in this study the author´s only objective is to describe the variation of 13C and 15N, not linking the labeling to a physiological process. There is no hypothesis or the intention understand a physiological process. In fact, there is no a reason why th authors are interested on describing the isotope labeling at different stages or organs. I think this part needs to be improved so the manuscript has a focus or a clear objective with physiological meaning.
Experimental design: The manuscript fulfill the scope of the journal

The research question is not well defined. See coments in point 1.

Comments to the Methods:
Please describe more in depth the 15N labeling procedure. Which was the N source for the labeling. How much was the richness of the 15N portion. How much 15N enrich fertilizer was added? please clarify.

The 15N labeling experiments need to be clarified. For what I understand (and I might have understood it wrong) the 15N treatments is only applied in the +N experiment, there is not +N that is not labeled control. I think that by only adding the 15N in the +N treatment, the authors may be mixing effects. To avoid this problem, the authors should have added an extra treatment in which they should have added a +N treatment but without labelling. I think that is the way to calculate the excess 15N value. In my opinion the N excess value as it is calculated now mix 15N and +N effects, so it is not completely correct. The authors should show the total N content (usually it is analyzed in the same 15N analysis) and in that way they will see how the N in the +N is much more concentrated. In that way authors would be able to calculate the difference in the 15N labeling between -N and +N to calculate the excess 15N in a more accurate way.

Did the authors take soil samples to analyze the 15N in the soil at the beginning of the experiment and at the end? That would help to see how much different the two N treatments were in the d15 signature.

The sample after the d13C labeling is at 28 days after labeling (DAL), can the authors explain why they choose those days. It seems to me that 28 DAL is too long to wait to analyze the labeling, the C13 can be respired in the majority and only a small proportion is still stored in the plant as starch or as structural sugars (see Aranjuelo et al., 2011 and Nogues et al., 2006).
In addition, a labeling of 7 hours for only one day seems a little insufficient to sample after 28 days, please support information that justify that methodology and maybe a reason, like the researcher wanted to study to which storage or structural molecules the C goes after respiration etc.

Can the author provide data about the 13C content in the chamber during the measurements? This  is important because if there is not controlled and stable d13C signal that could explain the very high CV.

Justify why the authors  separated the plant (stems) in three lengths. Is there any previous publication or justification for this? Use that in the introduction
Validity of the findings: In the discussion the authors speak of increase in biomass of 35% at high N but they don´t provide any data besides the d13C excess. Please provide the data. It would be useful to provide the data of leaves, stems, spike etc.to have more of a broad view of the data.

pp233-238. The authors suggest that the higher allocation of C to ears can be caused due to allocation from roots too ears. However the authors don´t continue the hypothesis or give any additional and original data to support the hypothesis, so I suggest to eliminate this part or say that is a speculation only based in data from other publications.

The part of the conclusion regarding to d13C does not clarify the source of variation in d13C. Please state all possible sources of variation like differences in the labeling, different leaf areas, photosynthesis.
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: VARIATION OF 13C AND 15N ENRICHMENTS IN DIFFERENT PLANT COMPONENTS OF LABELED WINTER WHEAT (TRITICUM AESTIVUM L.)
Review round: 1
Reviewer: 2

Output:
Basic reporting: The research titled as “Variation of 13C and 15N enrichments in different plant components of labeled winter wheat (Triticum aestivum L.)” tried to figure out the impact of labelling with 13C and 15N on two differenced developmental stages. The main innovation in this work is evaluate the labeling times to achieve a uniform labelled material. However, the results are not innovative. In addition, the text should be improved in English language as in some points that I describe below.
I suggest that do not use “trend” in a research work, and less when there are significant differences among your treatments.
Experimental design: The section Material and methods requires a higher explanation about the experimental set up.
I suggest explain the experimental design at the beginning of the “Treatments” section (i.e. total number of plots, number of plots per treatment and number of plants per plot).
L81. Replace CK by –N.
L82. You indicate that each treatment included four replications but until the line 97 do not indicate the number of plants per plot. I suggest group that.
L83. Did you apply 15N urea since the beginning of the experiment? Did you replace the 100% of N-fertilizer by 15N? Did you have wheat plants fertilized with N but non-labelled?
L102. You splitted the N fertilizer for the grain filling harvest. Plants that only received 1 doses of fertilizer also received 90 mg N hg-1 air-dried soil?
L117-122. I suggest extend this part for a clearly understanding. You indicate that for 13C labelling you chose 4 –N and 4 +N pots and 8 C-unlabeled controls. Were these controls 4 –N and 4 +N?
L145. Here you indicated that 3 plants per pot were assessed for the 13C and 15N enrichment and in line 82 you indicate that each treatment had 4 replications. So, you had 4 biological replicates with 3 technical replicates in each pot. Therefore, you had n=4 or 3 and you made 3 technical replicates for each treatment. Tables 1 and 3 and figures 1 and 2 indicate that the values are the means of 9 replicates. What do you consider as biological replicate and what as technical replicate?
L156. Did you used non-labelled material to calculate the 15N excess?
L178. I suggest indicate with words better than with symbols (next appearances as well)
L182. N fertilization decreased 13C values….
L184. N fertilization did not change the 13C values at grain filing stage (no significant differences).
L190-192. Upper stem did not present higher 13C values than middle stem. Here, the relevant result is that ear showed the highest 13C contents followed by upper leaves, which indicate the strong remobilization process.
L192-194. Are you meaning at grain filling stage? At elongation stage, there were not significant differences between upper and middle portions.
Validity of the findings: Your discussion needs more detail. I suggest that you discuss more the importance of ears at grain filling stage due to its importance as sink. Your results showed a high labelling of this component that could affect the other components by a strong remobilization process.
Results must be revised carefully and write that statistic indicates.
Conclusions are well stated and limited to supporting results.
The question of what do you consider biological replicate must be answered.
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: VARIATION OF 13C AND 15N ENRICHMENTS IN DIFFERENT PLANT COMPONENTS OF LABELED WINTER WHEAT (TRITICUM AESTIVUM L.)
Review round: 1
Reviewer: 3

Output:
Basic reporting: This manuscript describes the methods of artificially labeling outdoor-grown plants with 13C and 15N, and presents data on the resulting 13C and 15N enrichment in various plant parts.
Stable isotopic techniques may provide powerful insight to coupled soil-plant processes, and future advances in our understanding of the fate of plant C and N entering the soil will depend on efficient techniques which help to elucidate rather than obfuscate C and N cycling through the soil system. The authors seem to be fully aware of the importance of a uniform isotope distribution among the plant parts and chemical fractions being labeled to prevent misinterpretations.

The analysis of isotope homogeneity is a worthwhile contribution, but the results indicate that there was considerable inhomogeneity.

More serious is the failure to discuss several critical variables, including labeling frequency and chamber environmental conditions (temperature, humidity).
Experimental design: The experimental design is fine and clear.

Report the source and cost (in US dollars or Euros) of the NaHCO3 at 98 atom%
Validity of the findings: The results are clear and comprehensive however the proposed labeling method was marginally adequate, and produced residues that were marginally acceptable for subsequent decay studies, because  13C was not uniformly distributed among the plant parts. There is much room for improvement, and ideas to improve the labeling procedure should be shared openly.

The labeling intensity (duration of each pulse and frequency of pulses) may have a more important influence on above-ground residue quality and on isotopic homogeneity.

A more transparent discussion of strengths and weaknesses would add credibility and utility. Perhaps the authors might identify some mistakes, and advise readers on how they might be avoided. Actually this study provides very little support for conducting labeling in the field, the main benefits being ample PAR and a natural root environment and distribution.

Please see Tahir et al (2018) Field 13C Pulse Labeling of Pea, Wheat, and Vetch Plants for Subsequent Root and Shoot Decomposition Studies. Rev. Bras. Ciênc. Solo vol.42
Additional comments: The work has clear objectives and was carried out with rigor. However, the originality is limited, being a reapplication of the technique, as quoted in the manuscript.