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: RANGE EXPANSION OF A FOULING SPECIES INDIRECTLY IMPACTS LOCAL SPECIES INTERACTIONS
Review round: 1
Reviewer: 2

Output:
Basic reporting: see below
Experimental design: see below
Validity of the findings: see below
Additional comments: July 31, 2017

PeerJ Editor

Re: Manuscript #18935
Review of Speights and McCoy “Range expansion of a fouling species indirectly impacts local species interactions”

Speights and McCoy used a series of laboratory trials to assess the effect of boring sponge infestation on predator-prey interactions between eastern oysters and stone crabs. In general, I thought the ms was clearly written and largely straightforward. The results of potentially longer handling times on non-infested oysters are certainly interesting, and could be of wide interest.

One overarching comment is that the manuscript is extremely succinct – at time perhaps too much so. For instance, the manuscript notes that there may be changes in the distribution of boring sponge, but only introduces stone crabs as an “important native” (e.g., Ln 61). In fact, stone crabs themselves may be experiencing a change in distribution (i.e., poleward range expansion) that is equally important in the context of this 3-member interaction – particularly in the region in which animals were collected for this study (e.g., Rindone and Eggleston 2011). This point could be expanded on in a revised manuscript (e.g., Ln 147).

Similarly, Sara Coleman (UNC-CH) published a thesis in 2014 entitled “The Effects of Boring Sponge on Oyster Soft Tissue, Shell Integrity, and Predator-Related Mortality”. Her results suggested that based on tissue content vs shell integrity, crabs should attack infested oysters over non-infested oysters. However, in feeding trials similar to Speights and McCoy, she found no difference in attack rates on infested vs. non-infested oysters. She concluded that the crushing strength of the crabs she used was so great (based on their size and reported crush force), that no oyster shell was a functional barrier to predation. Perhaps the size of stone crabs used by Speights and McCoy were smaller than those of Coleman – either way, it seems like some broader commentary on how these studies/results fit together could strengthen the discussion.

Other comments:

Ln 12: Could delete “In this study”, and just start “We investigate…”

Ln 37: I wondered how barnacle fouling on their shells increased the proximity of snails to predators. Since this is not intuitive, it would be good to detail the mechanism by which this occurs.

Ln 57 (and elsewhere): I understand I’m being perhaps a bit of a stickler, but I encourage the authors to be careful in how they present “recent changes in the distribution” of sponges. To my knowledge, neither Lindquist 2011 or Dunn et al. 2014 actually tested this. Lindquist mapped the present day distribution of sponges, and Dunn et al. examined sponge infestation in various substrates. Both worked across salinity gradients, and both certainly commented on the potential for sponges to be farther up-estuary (indeed, it’s a logical presumption). However, I’m not sure either had the long-term data to rigorously evaluate a shift in distribution.

It’s also entirely possible that I’ve missed some piece of evidence those researcher did provide, and I am also unfamiliar with the work of Hong and Shen 2012. I suppose I’m just asking the authors to be certain that they are not overstating what researchers may believe is happening, versus what researchers have actually documented.

Ln 86: “Oysters of similar sizes…” is vague. Provide actual sizes (and if sizes changed between trials for an individual crab, or across the four cycles [Ln 103]).

Ln 90-104: I thought the authors did a good job of accounting for the issues related to reusing crabs. I have no problem with interpreting their results keeping in mind that reuse occurred.

Ln 133-139: The increased handling time for non-infested oysters is the crux of this paper. However, I would like to see more detail on how handling time and attack rate (abort rates, etc.) were documented. Were these factors actually observed/recorded to feed Eqs 1&2? Or are they just estimated from the model fits of mortality. If it’s the latter, I have to wonder what sort of confidence intervals have to accompany those estimates. I think this is another place where the manuscript might benefit from further text/explanation.

Ln 151: “inland into estuaries” could be “up-estuary”

Good luck moving forward with this ms, yours,
[this is a signed review]

Joel Fodrie
Institute of Marine Sciences &
Department of Marine Sciences
University of North Carolina at Chapel Hill


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: RANGE EXPANSION OF A FOULING SPECIES INDIRECTLY IMPACTS LOCAL SPECIES INTERACTIONS
Review round: 1
Reviewer: 3

Output:
Basic reporting: Line 65- please add “than” before survival
Experimental design: There are a couple of changes that should be made to ensure this work can be replicated.

Please provide the temperature and salinity of experimental tanks.

Figure 1- please include n=number of trials for each mean/standard error.

Figure 1- Please explain which equation was used for the lines shown here (I believe it was model 1 from your R code).
Validity of the findings: No comment.
Additional comments: This article is well-written. I was able to replicate all results from the code and data provided- nice job. The use of AIC to interpret the finding of this study was compelling. I am choosing minor revisions just because I think the temperature and salinity are necessary to fully interpret your results and compare them to other systems.


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: DECREASED QUALITY OF LIFE AND TREATMENT SATISFACTION IN PATIENTS WITH LATENT AUTOIMMUNE DIABETES OF THE ADULT
Review round: 1
Reviewer: 1

Output:
Basic reporting: The article is written in a clear English.
The introduction and the background are complete and appropriately referenced
to relevant prior literature.
All results relevant to the hypothesis are showed in the text and in the tables.
Experimental design: The research question is clear, relevant and meaningful and the study is rigorous, well detailed and conducted according to technical standard.
Validated tools were used in the language in which the study was conducted.
However, it would be appropriate to enclose questionnaires as supplementary materials, in order to facilitate a better understanding of the items.
Validity of the findings: The study, showing that LADA patients have a poorer QoL profile than patients with T1DM and T2DM,  especially at the expense of DR and insulin treatment, provides new results on a topic that is currently little explored.

However, the study included patients with chronic pathology with a view to assessing the impact on quality of life. Notably, quality of life is dependent on mental well-being. Exclusion criteria include mental illness. Therefore, as the mood of these patients was evaluated?
Would it not be more appropriate to include this variable in statistical analyses to assess its impact on quality of life and Treatment Satisfaction?
Another question.
Another exclusion criterion is the presence of dementia. Who and how did patients evaluate to exclude cognitive impairment?
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: DECREASED QUALITY OF LIFE AND TREATMENT SATISFACTION IN PATIENTS WITH LATENT AUTOIMMUNE DIABETES OF THE ADULT
Review round: 1
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: 1) The most important issue  is the limited number of LADA patients who agreed to participate in the study: 86 contacted patients and only 48 participants, with a refusal frequency equal to 44% .  This high proportion of refusal is an important selection bias that can impact on results validity.
Which factors influenced participation in the study? Can authors exclude that participation was influenced by disease status, socio-demographic factors, quality of life or treatment satisfaction? Which are the differences beetween patient who partecipated and those who refused?
This point is crucial for results validity!

2) Additionally, given the high number of statistical tests, a Bonferroni correction should be applied.
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: DECREASED QUALITY OF LIFE AND TREATMENT SATISFACTION IN PATIENTS WITH LATENT AUTOIMMUNE DIABETES OF THE ADULT
Review round: 1
Reviewer: 3

Output:
Basic reporting: 1. Your MATERIALS AND METHODS needs more details about the inclusion criteria for LADA,T1DM,and T2DM, respectively,especially for the inclusion criteria for the LADA.
2. I suggest that the results you describing in Lines 165-177 shoud be moved forward to the part of MATERIALS AND METHODS.
3.It is noted that your manuscript needs careful editing by someone with expertise in technical English editing paying particular attention to English grammar, spelling, and sentence structure.
Experimental design: no comment
Validity of the findings: Lines 237-239:T2DM patients without insulin treatment showed a significantly lower hypoglycemia frequency perception in comparison with insulin-treated LADA subjects (p=0.006).Please explain why ?
Lines 228-229:Insulin-treated LADA patients had a higher hyperglycemia frequency perception than T1DM (p=0.04)...... Please explain why ?
Additional comments: The number of subjects with LADA is too small so that the conclusion that "LADA patients showed a poorer QoL profile than patients with T1DM and T2DM" in the current study was not convincing.


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: DECREASED QUALITY OF LIFE AND TREATMENT SATISFACTION IN PATIENTS WITH LATENT AUTOIMMUNE DIABETES OF THE ADULT
Review round: 2
Reviewer: 1

Output:
Basic reporting: The manuscript is well structured and adequate regarding literature references.
It contains relevant results to support the hypothesis.
Experimental design: Research question is well defined, relevant and meaningful.
Methods are well described.
Limitations are adequately described in discussion section.
Validity of the findings: Results are significant, but partially inconclusive, suggesting the need for further studies on this issue.
Additional comments: It is worth appreciating the attempt to explore what influences the quality of life of patients with LADA, even if the impact that mood alterations may have on the management and perception of the disease is only partially reported. This is a study limitation. However, this point only partially limits the success of the study which is adequate and complete.


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: MGST2 AND WNT2 ARE CANDIDATE GENES FOR COMITANT STRABISMUS SUSCEPTIBILITY IN JAPANESE PATIENTS
Review round: 1
Reviewer: 1

Output:
Basic reporting: All information contained in attached review.
Experimental design: All information contained in attached review.
Validity of the findings: All information contained in attached review.
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: MGST2 AND WNT2 ARE CANDIDATE GENES FOR COMITANT STRABISMUS SUSCEPTIBILITY IN JAPANESE PATIENTS
Review round: 1
Reviewer: 2

Output:
Basic reporting: General comments:
The language in this manuscript needs to be improved to a more professional and less conversational style. A review by a native English speaker should be sufficient to raise the manuscript up to an appropriate standard and address the overuse of commas throughout the paper.

The manuscript seems well referenced and the background in the main body of the paper describes the phenotype well but should expand slightly on the previous work by these authors.

The manuscript structure is somewhat inconsistent. The introduction section needs only some language refinement but the method section is very confusing and hard to follow the sequence of work. As a result, interpreting the results and conclusions is impossible.  The tables are poorly formatted and contain confusing information which is not always relevant and appropriate. The raw data was available to this reviewer.

The manuscript appears self-contained, has clear aims and a relevant result reported which is consistent with the hypothesis.

Specific Comments:
Abstract: The Section Background/aim should have a more detailed description of the phenotype since this is a general interest journal, not a ophthalmology journal. A sentence or two should suffice to explain the features to the reader.

Introduction: Lines 73-80. The language here needs to be improved to a more professional and less conversational style.

Figures: The LD figures (1 and 2) are incorrect since no adjustment for kinship has been made and the association analysis they support is entirely inappropriate.

Tables: Table 1 should be completely revised. Table 2 contains a column labeled "Other Methods" which is hard to understand. Table 3 contains an eQTL column which adds nothing to the results.
Experimental design: General Comments:

This is original primary research well within the aims and scope of the journal.

The research question of whether SNPs in two previously identified locations by prior work, is well defined, relevant and meaningful. This is a classic fine-mapping approach which seeks to refine a prior signal in two genomic locations and the family-based study design is appropriate in this context.

The rigor of the investigation was harder to assess. The methods section is confused and poorly structured and raises several questions in this reviewers mind as to the statistical understanding of the authors. They make several assertions that are incorrect, misleading or outdated that significantly undermine their results. Replicating their analyses with the methods section in its current form would not only be quite difficult but would in fact be inappropriate in some parts. I address these failings in the specific comments below.

Specific Comments:

The methods section need major revision in order to meet even basic standards of adequate reporting. The way it is currently structured suggests that certain quality control measures were applied after some analyses were performed. Since this seems unlikely, I suggest the authors use a flowchart to clarify the workflow and use this to restructure the methods section. This flowchart could be added to the paper if figure limits allow or added to a supplementary methods section.

For the description of the study subjects, the table is poorly formatted and should be revised to reduce redundancy. It is typical to include pedigree drawings unless there is a risk of identification and would greatly assist the reader in understanding the cohort. Samples with available DNA could be indicated on these drawings, rather than detailed in the table.

The section describing the genotyping seems straightforward but a table of the primers should be included in the supplementary methods. However, the genotyping rate is rather lower than I would expect and does raise some concerns which the authors should address. Why were there such high missingness rates?

This manuscript contains no discussion of power. Power calculations should be performed and included in the methods and results.

This manuscript contains no discussion of population stratification. Even within apparently homogeneous populations, significant substructure can still exist and can influence the results of association based tests. A discussion of this in the methods would be appropriate. Although linkage analysis is robust to population stratification, it does require good estimation of allele frequencies in the data. This should be included in the methods.

I also have several concerns about how the analyses were performed. The section on association testing uses a completely inappropriate methodology to analyze these data. The authors even seem aware of this themselves, stating that standard chi-square analyses assume independence of the subjects and this standard is not met in this cohort. There is no defending this analysis, it is meaningless and should be removed. A more appropriate association approach would be to use FBAT, EMMAX (with the appropriate caveats about the beta generated) or other methods designed to use or control for kinship between study participants.

The next section discusses Mendelian inconsistencies and this section raises several red flags. The authors mention duplicate subjects but do not adequately explain how duplicate subjects were selected for removal. Equally of concern is the large number of reported Mendelian errors and the methods do not sufficiently detail how these errors were dealt with. Typically, one would remove inconsistent genotypes in a SNP just in a family if only one family had an error. If the same SNP has errors in two or more families, the SNP should be deleted across the entire dataset. It is not clear whether the authors did this in their data.

Although the authors discuss using TDTae, a method to address genotyping errors without deleting data, this is not used widely in the field as it has been superseded by other, better tests. In addition, the authors describe this method as parametric, which is not exactly correct - the error model is parametric but the test for linkage is not.

But no discussion of the other analyses described in the results is included here. These sections should be revised and reordered.
Validity of the findings: The association results are the result of an inappropriate methodology and are meaningless. This section should be removed or the analysis repeated with a more appropriate test as I suggested in my review of the methods section. The results also contain considerable discussion of the methods and these portions should be moved to the methods section. For example, there is no mention of the pseudomarker program until the reader reaches the results section. This is not the right way to report methods or results. The exact models used should be presented and the method used for generating allele frequencies.

As I've noted, methods are poorly described and as a result, it is hard to assess whether any of the linkage results presented here are meaningful, as I cannot ascertain whether the data were appropriately cleaned before analysis. The use of TDTae is discouraged and it is not clear to me if the authors fully understand the implications of using this test in these data.

Finally, the authors make no adjustment for multiple comparisons in these data, despite using several analysis methods and in the parametric linkage analysis, more than one model. It seems likely that such an adjustment would render all results entirely non-significant but should be performed and the manuscript adjusted to reflect the results in the light of this.
Additional comments: Although this manuscript presents an interesting fine-mapping study, I have grave concerns about the statistical analyses presented and as a result, cannot recommend this paper for publication without significant revision.


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

Input:
Title: MGST2 AND WNT2 ARE CANDIDATE GENES FOR COMITANT STRABISMUS SUSCEPTIBILITY IN JAPANESE PATIENTS
Review round: 2
Reviewer: 1

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


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

Input:
Title: BACTERIAL AND FUNGAL COMMUNITIES RESPOND DIFFERENTLY TO VARYING TILLAGE DEPTH IN AGRICULTURAL SOILS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Most of the for the Basic reporting season is acceptable. However, below are some comments about the English language.
Line 50 change Tillage therefore for Tillage, therefore,
Experimental design
Line 62 This however … The use of "this" brings an unclear antecedent. I suggest that the authors rewrite the sentence making a better connection with the previous statement.
Line 83 change Prior to for Before
225 change This for This tillage treatment effect
Line 229 and line 231 the use of this brings an unclear antecedent.
Line 304 change eachother for each other
Experimental design: Some parts of the topic experimental design need to be better explained. Please see the questions and comments below.

The authors proposed the nMDS analysis on their material and methods (see line 194). However, during their results and discussion, they used the term MDS, which is a different kind of multivariate analysis. Thus, I think there is a mistake on the use of MDS term and the authors should do a double check on their manuscript.

For the lines 227-229 and 231 and 232, I have a doubt about which are the statistical analyses that support the both statements. My suggestion is to rewrite these sentences and citing the analysis which endorses it.

The authors used the two-way ANOVA analysis ( e.g. line 233), however, they did not describe in methodology.

I suggest that the author use the envif analysis from Vegan package in R (http://cc.oulu.fi/~jarioksa/opetus/metodi/vegantutor.pdf) to evaluate the correlation between the environmental table (table 1) and the nMDS results. This analysis may contribute to a better visualization of which are the chemical and physical attributes that better explain the nMDS results, being helpful to confirm the hypotheses proposed .

My suggestion for the lines 296-298 is to include a reference for the ARISA methodology statement.
Validity of the findings: no comment
Additional comments: I commend the authors for the manuscript, evaluating the effects of tillage practices and depths on soil microbial community.  The article is written in a good scientific English and text structure. If there is a weakness, it is in the statistical analysis and some better explanation about some topics during the text ( as I have noted above). In my opinion, after the answer of some question and the inclusion of some analysis that could  enrich the discussion, the manuscript will be ready the Acceptance.


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: BACTERIAL AND FUNGAL COMMUNITIES RESPOND DIFFERENTLY TO VARYING TILLAGE DEPTH IN AGRICULTURAL SOILS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The basic reporting meet the criteria, except for:

-The tittles of the figures would benefit from being more descriptive. For example, Figure 2 and three have identical titles.
Experimental design: The experimental aspects appear sound.

Since different tillage techniques are used, it seems relevant to justify the reason for studying them both in relation to depth.
Validity of the findings: I have some comments regarding the validity and discussion of the findings.

The authors could discuss the potential for the fungal community results to have been influenced by methodological factors in the molecular approach. It seems the relative error is greater and thus it appears that the sensitivity for the bacterial and fungal analyses may not be comparable? Thus the conclusion of who is more or less responsive to the management, may need to be revised.

The discussion in lines 284-293 seems too general and not sufficiently insightful and focused.

The conclusion section seems to be too broad, not focused enough of the main findings and their implications.
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: HOW TO NORMALIZE METATRANSCRIPTOMIC COUNT DATA FOR DIFFERENTIAL EXPRESSION ANALYSIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Some text descriptions are really confusing. The mathematical formulas are not clear. Overall, the English writing should be improved.
Experimental design: The simulation steps are not described clearly.
Validity of the findings: Though the simulation results suggest that the proposed taxon-specific scaling outperforms global scaling, the real data results are not enough to support the claim.
Additional comments: The authors claim that calculating scaling factors on the level of taxon instead of on the level of global,  the performance of differential analysis will be improved. I have some comments on the follows,

1. Some formulas are confusing.  For example, formula (3). The estimated scaling factors are only determined by feature i, which is obviously not correct.

2. Some statements are confusing. For example, what does “predictions“ means in line 40? Do the predictions mean DEFs? In line 124, which is “organism-specific abundance”? What is the difference between organism-specific and taxon-specific?

3. The simulation steps are not described clearly. How did the abundance generated, based on what kind of generative model? How did the count matrix of different organism combined together? What is the proportion of each organism?

4. In the real data comparison on page9 13-15,  the gold standard for DEF for both global scaling and taxon-specific scaling. The comparisons of the numbers of DEFs identified of both approaches cannot support the claim one outperforms the other, and vice versa. More identified DEFs could be false positive, and few identified DEFs could be lacking of statistical power. The authors should use other criteria to justify taxon-specific scaling is better than global scaling.


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: HOW TO NORMALIZE METATRANSCRIPTOMIC COUNT DATA FOR DIFFERENTIAL EXPRESSION ANALYSIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: In this manuscript, the authors propose a strategy for normalizing metatranscriptomics data for sequencing depth in the context of differential analysis. They propose the ‘taxon-specific scaling’ to take into account the structure of the metacommunity. This strategy consists in separating the dataset into one dataset per organism, to normalize them independently with standard method such as the method proposed by Anders et Huber in the DESeq2 Bioconductor package and to analyze a combined dataset. They compare this strategy with the ‘global scaling’ one which considers the whole dataset forgetting the notion of species. They study the interest of their strategy, both in simulations and in a real dataset.

The introduction will benefit from a more detailed description of the context of normalization for differential analysis. Are standard strategies for transcriptomic directly applicable for metatranscriptomics ? but also are those for differential abundances from metagenomics data applicable for metatranscriptomics (see Weiss et al. 2017).

The manuscript is not so easy to read for different reasons :
1/ some essential terms essential such as ‘normalization’ or ‘library size’ are not well-defined and need to be explained in the metatranscriptomics context.
2/ it subsists some redundancy (normalization part of the introduction and taxon-specific scaling and global scaling’ paragraph of the materials and methods section).

In order to clarify the manuscript, I suggest to present normalization as between-sample normalization and to define clearly what is the library size. In general, for normalization purpose, the library size is for each sample the total number of sequences which will be analysed. One key point not mentionned here is that normalization depends on the biological question. The main two biases we would like to correct in metatranscriptomics is the composition of metacommunity and the sequencing effort. Both may vary with sample. Depending on the biological question only a correction for sequencing depth or for both is needed. It would be clearer to separate the two and not to speak about ‘organism-specific library size’. I suggest for Simulation II ‘modification in the abundance of species present in the metacommunity’ instead of  ‘with library size variation’. The interpretation of the results of differential analysis will depend on the question and on the normalization step and a feature statistically significant can be result of difference in expression or/and in the abundance of the species it comes from.

I suggest, for my second remark, to merge the normalization paragraph of the introduction with the paragraph in materials and methods. And in this section to distinguish in different paragraphs i) the taxon-specific scaling, ii)the global scaling, and only in a third part to discuss about the way to compute scaling factor (using DESeq2 for example).
Experimental design: The normalization of metatranscriptomics data is a great challenge and the question of when and how to take into account the variations in species abundance is an important question.
The authors explore several interesting scenarios. Nevertheless a real metatranscriptomics study contains generally more than 5 or 12 organisms and most of them are uncultivable. Would the authors discuss this point and the application to metagenomic species ?

I thank the authors for providing the real raw data and a script with R code for simulation and DESeq2 analysis. Nevertheless, I would appreciate to have a minimal script that downloads the data and either simulated data nor script with simulated parameters in order to reproduce the results of  all the analyses.

When using the test.main function or the DESeq2.tax.specific function, it results on a matrix of results with 1000*6 samples and not 5000 features as noted l.316. Could the authors explain why? Is it because they sum over all the organisms?

L156 the authors wrote ‘There are several reasons why the analysis of the recombined metatranscriptome data can be useful ; first of all, the statistical power of organism-specific tests may be low due to decreased counts.’ I do not agree : if the analysis is done organism by organism, the correction for multiple testing will be less stringent (1000 tests instead of 5000).
Validity of the findings: No comment
Additional comments: I regret the absence of discussion on
1/ assumptions for normalization which are i) the great majority of the genes are DE and ii) balance between up and down regulated features. Could the author develop this point and the cases other way to compute scaling factors (ie instead of DESeq2 normalization) is necessary ?
2/ validity of the proposed strategy. Could the authors discuss
i)	on the situations where it is really possible to obtain counts per organisms, and which minimal coverage is needed for each organism, etc. ?
ii)	when it is reasonable to sum over features?
iii)	the case of a great difference in metacommunity diversity, e.g. several organisms absent in one condition?

In the conclusion, the authors write l. 502-506 « Normalization of metatranscriptomic data must have the goal to eliminate the influence of taxonomic variations from functional analysis. (…) We argue that for a correct normalization the metatranscriptome needs
to be decomposed to normalize the organism profiles independently. Then the metatranscriptomic count
data may be recombined from the normalized profiles to look for any global trends in the superimposed count data.’ How to superimpose count data is not well explained . Is it always straightforward ?

Comments for the authors

The reference for DESeq2 is (Love et al. 2014) and in l.186 the authors need to cite Bionconductor and the version they used. One way to obtain references for R packages is to tape « citation(« pkgname ») in the R console.

The reference for R is the following one :
R Core Team (2017). R: A language and environment for statistical computing. R
  Foundation for Statistical Computing, Vienna, Austria. URL
https://www.R-project.org/.

Minor revisions

L50 'transcriptome' is misspelled as 'transciptome'.
L133 ‘separated’ is misspelled as ‘seperated’.
L211 ‘library’ is mispelled as ‘libray’.

l.175 the right name for the normalization method implemented in edgeR is TMM (Trimmed-Mean of M-Values) (Robinson and Oschlack 2010) (l175 , l317,318, 347)

The abbreviation used for differentially expressed features is DEF (l.196) but either DE nor DE is used all along the manuscript. Please choose one and be homogeneous.

Figure 9. the size of the legend and labels is too small. One way to gain place may be to replace « 13d vs 27d » by « 13 vs 27 »

Reference
Weiss,S., Xu,Z.Z., Peddada,S., Amir,A., Bittinger,K., Gonzalez,A., Lozupone,C., Zaneveld,J.R., Va ́zquez-Baeza,Y., Birmingham,A.
et al. (2017) Normalization and microbial differential abundance strategies depend upon data characteristics. Microbiome, 5, 27.


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: HOW TO NORMALIZE METATRANSCRIPTOMIC COUNT DATA FOR DIFFERENTIAL EXPRESSION ANALYSIS
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: In this manuscript, the authors propose a strategy for normalizing metatranscriptomics data for sequencing depth in the context of differential analysis. They propose the ‘taxon-specific scaling’ to take into account the structure of the metacommunity. This strategy consists in separating the dataset into one dataset per organism, to normalize them independently with standard method such as the method proposed by Anders et Huber in the DESeq2 Bioconductor package and to analyze a combined dataset. They compare this strategy with the ‘global scaling’ one which considers the whole dataset forgetting the notion of species. They study the interest of their strategy, both in simulations and in a real dataset.

I thank the authors for the revised version of their manuscript, which considers all my previous comments and suggestions.

Just few typos and one minor comment:

peerj-17882-Additional_File_4:
Fig 4 legend: ‘library’ is mispelled as ‘libray’.

Fig 6 legend : the color box with « white », « black » and « blue » is inexact and not useful.

I tested the code in the peerj-17882-Additional_File_3.R file on the real dataset and I have got the following error message
« Error in `rownames<-`(`*tmp*`, value = colnames(countData)) :
  duplicate rownames not allowed »
I do not use the same versions of DESeq2 and R  (R 3.4.0 and DESeq2_1.16.1). It may be the origin of this error. This error appears using the DESeq2.norm.mat function line 384
YMat <- DESeq2.norm.mat(XMat, cond.vec,type.vec)
A way to avoid it is to check that colnames of XMat are not duplicated before using the function and rename the columns if necessary.


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: MICROBIAL COMMUNITY DIVERSITY PATTERNS ARE RELATED TO PHYSICAL AND CHEMICAL DIFFERENCES AMONG TEMPERATE LAKES NEAR BEAVER ISLAND, MI
Review round: 1
Reviewer: 1

Output:
Basic reporting: In this paper Hengy et al. describe the microbial diversity of four lakes with different physical and chemical characteristics. They describe the environmental differences between and within lakes (different depths) at three different times and correlate them with the microbial community structure. Even though the sample sizes are not large I think the results are clear and concise. This study contributes to the knowledge on the potential effect that environmental variables have on the bacterial community dynamics in aquatic habitats.
Experimental design: Overall, the experimental design is correct. However, in the general comments section you will find some suggestions and aspect that need to be clarified.
Validity of the findings: In the general comments section you will find my main concerns which have to do with rarefying the sequence data and correcting for multiple comparisons when calculating correlations.
Additional comments: Below you will find some general or specific suggestions that are mainly aspects associated to the methods.

Line 73: Change “reaction” to “reactions”
Lines 102-103: I assume that the water sample was passed through the larger filter first and then the smaller one? Please explain with more detail.
Line 118: Replace “was measured using and an ion…” by “was measured using an ion…”
Lines 130-131: This sentence is not clear. Was the DNA extracted from both filters at the same time? What is the reason to pool both filters? The 2.2 filter excludes all large particles and cells so I would think the 0.22 is the one that should have all the bacterial cells. Please explain.
L134: the 27F and 1493R primers amplify the whole 16S rRNA gene, not only the V4 region. Please correct this sentence.
Lines 142-151: You need to give more details on the processing of the sequences on this section and some also on the results section:
Include how many sequences were obtained initially.
Which filtering steps did you do in addition to the analysis of chimeras and how many sequences were analyzed after the filtering steps
How many OTUs were obtained?
Did you rarefied the samples? At which depth? If the authors didn’t rarefied the samples I strongly suggest to do it.
Line 154: Change “Statistical analysis (both chemical and biological) was completed” to “Statistical analyses (both chemical and biological) were completed”
Line 153: For this section please clarify why did you retain singletons for alpha diversity and removed them for beta diversity. The same OTU table should be used for both diversity estimates. Otherwise the results may be biased. Also, this section could be organized better.
Lines 155-158: This sentence should be included in the “Microbial Taxonomic Analysis” section of the methods.
Lines 158-160: Move this sentence to line 155
Lines 169-171: How were the samples normalized? Explain the variance transformation.
Lines 185-191. Did the authors correct for multiple comparisons? If not I strongly suggest to do it. Otherwise you might be overestimating the significant correlations.
Line 195: Change “was observable” to “were observed”
Lines 211-212: Move this sentences to methods. Also it is not clear what the authors mean with relative abundance. Do you mean sequencing depth?
Lines 210-224: Is it very important to clarify if the authors rarefied the samples (OTUs) in order to compare diversity values across samples. Otherwise the alpha diversity estimates may be biased.
Lines 281-282: This information should be included at the beginning of the results section. You could include how many OTUs were shared among lakes and how many OTUs were unique to each lake or unique to groups of lakes that are more similar in terms of the environmental variables.

I suggest including a map of the lakes sampled as an additional figure.
Figure 1: You could add a legend for the habitat as is done in the other figures
Figure 2: could be improved, so that the legend can be distinguished clearly. The labels on the X axis could be larger and the bars could be thicker. Also I would suggest to change the colors so that it’s easier to distinguish the different bars


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: MICROBIAL COMMUNITY DIVERSITY PATTERNS ARE RELATED TO PHYSICAL AND CHEMICAL DIFFERENCES AMONG TEMPERATE LAKES NEAR BEAVER ISLAND, MI
Review round: 1
Reviewer: 2

Output:
Basic reporting: Writing should be improved for clarity.
- The emphasis on 'lakes within the Laurentian Great Lakes basin' in the title and abstract is misleading. The Great Lakes basin is massive – 4 lakes is too few lakes to characterize lakes in the Great Lakes basin and it sounds like the study lakes are close together geographically near Beaver Island, MI, making them not especially representative of the extent of the Great Lakes basin. I suggest specifying Beaver Island, MI, not the Great Lakes basin, as the location of the study lakes and being more specific in the abstract concerning the number of lakes sampled.
- You claim to show that conditions in the water column ‘significantly impact’ community structure, yet all of your analyses are correlation based. It is more accurate and clear to emphasize the correlations observed in this study than to imply causation.
- Additional examples where clarity could be improved:
--‘physical, chemical, biological, and temporal processes’ in the first line of the abstract does not make sense to me. Physical, chemical, and biological processes may be temporally variable or dynamic, but I do not think that that there are ‘temporal processes’ that are separate from physical, chemical, and biological processes.
--In the next sentence you talk about ‘similar geographic regions’ – do you mean the same geographic region?
--It would help to be more explicit about the differences between DOC and DOM (and to define them before using the acronyms) and what information you gain from looking at both instead of one or the other (lines 50-51 of the introduction)

Suggestions for improvement on literature references and background/ context:
- You need to include a citation for the R statistical environment (type “citation()’” into R to generate the citation – example from the version of R I use on my computer:
R Core Team (2016). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. URL https://www.R-project.org/.
- The conclusion that ‘despite geographic proximity, each lake harbored a distinct microbial community, suggesting that lake chemistry is a stronger constraint on microbial communities than geographic region’ covers a topic that has previously been explored in freshwater lakes, but it seems like there were a number of relevant studies overlooked from the discussion. I suggest that the authors consider whether previous surveys of lakes provide relevant context to their work, including:
Yannarell and Triplett 2005. Geographic and Environmental Sources of Variation in Lake Bacterial Community Composition. http://aem.asm.org/content/71/1/227
Van der Gucht et al. 2007. The power of species sorting: Local factors drive bacterial community composition over a wide range of spatial scales. http://www.pnas.org/content/104/51/20404.short
- I additionally recommend that the authors consider whether Newton and colleagues (2011) A guide to the natural history of freshwater lake bacteria may provide additional useful context for their sequence data:  https://www.ncbi.nlm.nih.gov/pubmed/21372319. In my opinion, the freshwater database initially curated as part of the Newton review is the best database to use when classifying freshwater sequences (though whether sequence database really matters depends on your question). The database and instructions on how to use it can be found here:
https://github.com/mcmahon-uw/FWMFG

I commend the authors for including accession numbers for their sequence data. However, I am not sure whether there is sufficient metadata available with your deposited sequences. From what I was able to see when I looked up your accession numbers in MG-RAST, I can tell the location but not the depth or time sampled. A key may be needed to connect name to MG-RAST ID to necessary metadata, perhaps this could be included as a supplemental table.

While not necessary for publication, I think it would be useful if the R code, OTU table, and metadata were shared via GitHub.

The article structure is professional, though I do have a few suggestions that might improve the presentation of the study sites and results. I think it would be useful to include a table in the main text with the basic limnological characteristics of each lake (e.g., latitude and longitude, ranges of observed water temperature and chemistry parameters described in supplemental table 2). I am not sure what the value is in including an NMDS plot as well as CCA/ pCCA plots. Since constrained ordinations force samples to correspond to measured environmental parameters, I am not sure how useful it is to include these plots in the main text - perhaps they would be more appropriate as supplemental figures.
Experimental design: The authors present original primary research that appears to be within the aims and scope of PeerJ.

I am unclear on what knowledge gap is specifically being filled. I think the research question could be more clearly defined.

The epilimnon/ hypolimnion contrast is interesting; however, it does not appear that the hypolimnion of Lake Michigan was sampled. It is not unusual for the mixed layer depth in Lake Michigan to exceed the 14.5-18.3 m from which bottom samples were collected. Temperature profile data from supplemental table 1 supports the idea that the Lake Michigan hypolimnion was not sampled.

The sequencing methods are not described in sufficient detail. First, 27F to 1492R primers span almost the entire 16S rRNA gene, encompassing far more than the V4 region. Does this mean that nested PCR was used? What primers were used for sequencing? If the 27F and 1492R primers were the primers used for sequencing, the MiSeq reads could not have merged. If this was the case, did you only analyze forward or reverse reads?

If the number of cycles were varied for each cycle, you should include somewhere, perhaps in a supplemental table how many cycles were used for each sample.

How long were your Illumina MiSeq reads (e.g., 2 x 250)?

You cite Kozich et al. and say that sequences were processed using Mothur, but if you followed the Mothur MiSeq protocol you should specifically state that you followed the Mothur MiSeq SOP and include the date that you accessed the SOP. Any deviations made from the Mothur MiSeq SOP (if this is what you followed) should be specifically noted.

What was the rationale behind removing singletons prior to normalizing samples for beta diversity analyses? I am not aware of this being a standard practice, and it does not seem like a good idea to me.
Validity of the findings: Some of the conclusions may be overstated. For example, stating that the data 'show that differences in phyicochemical conditions in the water column can significantly impact aquatic microbial community structure' seems to imply causation when the study was correlational and involved few lakes.

It is challenging for me to follow the links between the original research question, supporting results, and conclusions.

I find some statements in the results/ discussion to be confusing. For example, the statement starting on line 269 that community composition was indistinguishable between surface water and bottom water communities when oxygen is not stratified seems like a sampling artifact to me. Lake Michigan surface communities from open water stations in the middle of the lake differ significantly from communities sampled from the hypolimnion even though the hypolimnion is oxygenated. A recent paper published in ISME J has also described differences between surface and hypolimnion communities in lakes where the hypolimnion is oxygenated:  http://www.nature.com/ismej/journal/vaop/ncurrent/full/ismej201789a.html?WT.feed_name=subjects_microbiology.
Additional comments: What does it mean to explore microbial communities within lakes on an 'individual level' - one lake at a time (line 38)?

It is not clear to me how relationships between taxa and environmental gradients are being 'deeply' explored (line 84). Is this a reference to sequencing depth?

I am not sure that I agree with the interpretation of Jezbera et al. 2012 lines 317-319; Polynucleobacter comprised 1.1-5% of the bacterial assemblage in most lakes with pH between 8.1 and 8.5.

I am not sure what it means when Polynucleobacter is described as a 'generalist' genus (line 233). It makes it sound like members within the genus are generalists, when I think the reality is that the members of Polynucleobacter are quite diverse.


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: MICROBIAL COMMUNITY DIVERSITY PATTERNS ARE RELATED TO PHYSICAL AND CHEMICAL DIFFERENCES AMONG TEMPERATE LAKES NEAR BEAVER ISLAND, MI
Review round: 1
Reviewer: 3

Output:
Basic reporting: This paper is clearly written and relevant, but there are numerous weaknesses that I think preclude it from being published in its current state.

With respect to literature cites, the authors should consider referencing other comprehensive 16S characterizations of freshwater lakes, particularly ones in the Great Lakes (e.g., Fujimoto, Masanori, et al. "Spatiotemporal distribution of bacterioplankton functional groups along a freshwater estuary to pelagic gradient in Lake Michigan." Journal of Great Lakes Research 42.5 (2016): 1036-1048)

I appreciate the wide array of statistical tests applied, but I don't understand why they are all relevant. Specifically, the NMDS and CCA plots seem redundant and it doesn't seem meaningful for this particular paper to do both, when they are specifically interested in constraining their datasets to the variables they measured. The application of different tests does reads like an after thought, not as something planned with their study. Thus, the data are seemingly made to support their hypothesis, however the experimental design is weak (see below).

With respect to the taxa and comparisons to other studies, the authors should use the freshwater 16S database (https://github.com/mcmahon-uw/FWMFG).
Experimental design: There are no sample replicates, which is, in my mind, a huge weakness. The authors are making vast generalizations about an entire lake based on 120 ml. They also reference "seasonal" differences (line 95 and elsewhere), but all samples were taken in the summer within a few weeks of each other.

The PCR conditions are also problematic; 36-40 cycles is a lot. The Earth Microbiome Project recommends 35 (or fewer) and many papers, past and present, show issues with high cycle numbers (e.g., Qi et al., AEM 2001), especially depending on which Taq was used (which the authors do not report here).
Validity of the findings: I only question the findings because of the experimental limitations (lack of replicates, post hoc decisions of statistical tests, and PCR/database issues. I really think they should tone down their findings should this get published. That said, I think the findings fit the data and tests that they have.
Additional comments: Line 138: "PCR reactions" is redundant
Line 197: So4 is not shown on Fig 1
Fig. 1: "temp" is cut off and abbreviations should be indicated in the figure legend
Lines 202-3: Lake Michigan is also stratified in the summer
Paragraph beginning on line 257: There are numerous contradictory statements here. I think the authors mean to discuss the lack of an oxycline in Lake Michigan, but instead they say that there was no divergence between LM deep/surface communities, which is stratified, but lake stratification is important in structuring communities.
Line 211-12: I don't understand what these numbers mean in terms of relative abundance.
Lines 219-222: These two sentences are contradictory.
Line 274+ What unmeasured environmental differences? This is very vague.


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: MICROBIAL COMMUNITY DIVERSITY PATTERNS ARE RELATED TO PHYSICAL AND CHEMICAL DIFFERENCES AMONG TEMPERATE LAKES NEAR BEAVER ISLAND, MI
Review round: 2
Reviewer: 1

Output:
Basic reporting: The new manuscript has greatly improved in clarity and has gained strength by adding additional references and clarifying several aspect of the methods and results.
Experimental design: I agree with the experimental design but I have made specific suggestions regarding alpha and beta diversity analyses (see general comments below).
Validity of the findings: Data and results are clear. Conclusions are well written.
Additional comments: I agree with all the modifications that the authors made to the manuscript based on the reviewers’ comments. I think that manuscript has greatly improved.

However, I still have one concern regarding alpha and beta diversity analyses. My self (and other reviewers) had previously asked about why the authors retained singletons for alpha diversity and removed them for beta diversity.

The authors gave the following response:
“Singletons were retained for alpha diversity as the chao1 index relies on singleton abundances for proper implementation of the chao1. However, as singletons are often removed to calculate beta diversity estimates and visualization, as many singletons could be sequencing artefacts.”

Based on this response I still think this is not the best approach for two reasons: (1) Chao1 index is presented on table 2 but it’s not even mentioned in the results and discussion and the authors rely only on the Shannon index results. Therefore, I do not see the need of keeping chao1 in the manuscript and therefore I see no need in keeping singletons. (2) the authors stated that singletons could be artifacts so I do not see the relevance of keeping them at all.

The authors should eliminate the singletons and (as I said in my first review) they should use the same OTU table for both diversity estimates.


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: MICROBIAL COMMUNITY DIVERSITY PATTERNS ARE RELATED TO PHYSICAL AND CHEMICAL DIFFERENCES AMONG TEMPERATE LAKES NEAR BEAVER ISLAND, MI
Review round: 2
Reviewer: 2

Output:
Basic reporting: Basic reporting has been improved. I commend the authors for including additional metadata and access to their analysis scripts through github.  There are a few lingering issues needing to be resolved before the manuscript is suitable for publication.

There is still remnant language implying causation, for example:
'driven by' in the title
line 30 - 'can significantly impact'

There are also descriptions obscuring the fact that the hypolimnion of Lake Michigan was not sampled, for example:
line 31-32 - hypolimnion of Lake Michigan was not sampled; most accurate representation would be 'surface' and 'deep'

line 371 - how deep did Fujimoto et al sample? It is likely that you observed differences because you were not sampling analogous deep samples; I do not think your results are directly comparable due to depth differences and the fact that you did not sample the hypolimnion.

line 473-476 - you did not sample the hypolimnion of Lake Michigan and need to be clear about this point

Description of study in the final introductory paragraph needs to be edited for clarity (lines 153-156)
1) 4 lakes, 3 holomictic... does this mean #4 is meromictic? Why would you not say something about lake #4?
2) Lake Michigan did not experience "temperature stratification, but did not stratify"; Lake Michigan was stratified; you did not sample a site that exhibited stratification

line 430 - need more explanation as to why your results differed from Jones et al 2009. I'm guessing they surveyed lakes covering a much broader range of DOC than you did.

Table 1 - There should be separate entries for surface and deep samples; this would improve clarity.

Figure 5 - CCA is most useful for the statistics it generates; plots are typically uninformative. I recommend moving CCA plots to supplemental.

Minor comments related to language and clarity:
Title suggestion:  'near' instead of 'within' Beaver Island, MI

line 37 - temperature is a physical parameter, not chemical

line 39 - constrained analyses demonstrated that DOC did not constrain? (what does that mean?)

lines 115-119 - run-on sentence; break up into more than one sentence

line 410 - 'stratified' instead of 'stratifying'?

line 412 - what about 'Relationship between environmental conditions and microbial beta diversity'? Current phrasing is not clear to me.

line 578 - I don't think 'mitigate' fits here... perhaps 'carried out'?
Experimental design: Necessary details are still missing:
line 250 - explicitly state the primers used for sequencing
line 254 - 'implemented similarly' should be replaced by something to the effect of 'following the Mothur MiSeq SOP with the following modifications' (and then in your brief description, be sure to describe every deviation from the SOP); you need to include the date you accessed the SOP as per the instructions on the website (SOP is not static and has changed over the last few years)
line 257 - what version of Silva?
line 265 - what version of RDP?

Multiple reviewers questioned the removal of singletons and doubletons prior to calculating beta diversity (line 301); rationale and citation provided to justify approach in response to reviewers were not sufficient in my opinion. I recommend re-doing this analysis without removing singletons and doubletons or finding an article that articulates a rationale for this approach, not simply one that went ahead and also removed rarely observed sequences.
Validity of the findings: Validity of the findings is tied to some of the issues described above. I think improving clarity of basic reporting will improve the validity of the findings.
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: MICROBIAL COMMUNITY DIVERSITY PATTERNS ARE RELATED TO PHYSICAL AND CHEMICAL DIFFERENCES AMONG TEMPERATE LAKES NEAR BEAVER ISLAND, MI
Review round: 2
Reviewer: 3

Output:
Basic reporting: The manuscript is improved from the original version. The manuscript is clearly written, uses appropriate citations and background, and the figures and tables are all sufficient.

Line 108: This does not make sense: "...experienced temperature stratification but did not stratify."

Line 124: How deep was Lake Michigan at the site where you sampled? The depth at which you sampled is not even below the thermocline.
Experimental design: I still find some flaw in the design that there is only one sample per lake per depth per time. To extrapolate to whole lake conditions on a couple of liters of water is somewhat problematic, especially since the authors point out that there's lake heterogeneity.

Line 176: Methods are still unclear. Aside from the exceptionally high cycle number used to amplify the full length 16S gene, you then amplified the V4 region specifically from these first amplicons? How many cycles did that add? Do you have references that support using this approach, because it is somewhat different than the standard 16S sequencing protocols?

I also do not understand why the authors are so reluctant to classify their sequences using the freshwater 16S database; this would provide more information and better taxonomy than RDP.
Validity of the findings: Paragraph beginning line 315: the observations/discussion seems to have some logical flaws. The authors claim that these lakes diverge over time and compare that to observations during lake mixing; however, these lakes remained stratified and, in fact, became more (oxygen) stratified over time. In fact, of the variables that were measured, oxygen is the only one to change between the the three timepoints, so it is no surprise that this is what can be linked to the change in community structure. Perhaps the authors should discuss what drives the shift to anoxic bottom waters as well.
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: TOLERANCE: THE FORGOTTEN CHILD OF PLANT RESISTANCE
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript addresses a fairly major shortcoming in the use of tolerance resistance in plants to arthropods. The material is presented in a straight forward manner and set up in a very readable format. The figures give good supporting back up to the points made in the text, and the references are thorough equally supportive. Some additional detail in the terminology used in the figures would improve their impact and utility in making the point of the review.
Experimental design: Well addressed, as to the types of normal exp. designs used in plant resistance research, those unique to the study of tolerance and the limitations posed by lack of development of new methods.
Validity of the findings: The logic-based conclusions reached by the authors make use of real-world data to point out the limitations of current knowledge about tolerance plant resistance.
Additional comments: A good and timely review. Nice to know others are thinking of management tactics beyond single-gene, high-dose defenses.


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: TOLERANCE: THE FORGOTTEN CHILD OF PLANT RESISTANCE
Review round: 1
Reviewer: 2

Output:
Basic reporting: All comments are directly on PDF version of ms.
Experimental design: NA
Validity of the findings: All comments are directly on PDF version of ms.
Additional comments: All comments are directly on PDF version of ms.


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: L1 AND L2 READING SKILLS IN DUTCH ADOLESCENTS WITH A FAMILIAL RISK OF DYSLEXIA
Review round: 1
Reviewer: 1

Output:
Basic reporting: The article is well written, covers most of the relevant literature (except see comment on Siegel’s work) sets out the questions clearly.  See also comments on raw data below.
Experimental design: I understand that this journal places a premium on technical adequacy rather than conceptual significance, but at the core of this article lies a pointless analysis, the comparison of the HRDys group with the other two groups.  Whatever the technical adequacy of the analysis, it is pointless because the groups were selected to be distinct on reading in the first place, so finding a “significant” difference is all but a foregone conclusion.  Further, with the well-attested high correlation between reading and spelling, the latter could equally be expected to show a significant difference.  It’s less certain but reasonable guess that they will be poorer in commonly related cognitive processes like PA.  If the article is to be published, the authors should drop that aspect of their analysis.

More worthy of consideration are the questions of how children classified as dyslexic in Dutch fare in English, and how the non-dyslexic family-risk group fare in relation to their non-FR counterparts.  The first of these questions was addressed by the subtraction statistic (Dutch WRF – English WRF), with the finding of a greater difference for the HRDys group than the other two groups.  The only threat to this counterintuitive finding that I can see would be ceiling effects for the LRnonDys and HRnonDys groups (such as many of these participants reading all the words in the allotted time).  Since the authors do not present non-standardized scores, they should address, and hopefully eliminate, this possibility. Supplying raw data would also help readers appreciate is there are any limitations to this finding along the lines that I have speculated.

In their discussion of this particular result they might also refer to a finding by Linda Siegel to the effect that RD children can be superior in the “visual route,” to use dual-route terminology.

The comparison of the two nonDys groups suffers from low power, I believe, leaving uncertainty about whether there is a population difference between them for the variables that were non-significant.  I think the authors would be entitled to use one-tailed tests of significance because they are making directional predictions, which may clarify matters, and in any case their findings are like many others’ and support the continuous nature of dyslexia risk.  It’s just a pity that they did not address the question of power and, if possible, design their study accordingly (or use a Bayesian approach based on prior probabilities of difference).  I also add that it is going well beyond the data to assume that the results support the polygenic account of dyslexia, that “the HRnonDys group are likely to have inherited some but not all genetic risk factors that contribute to the development of a reading impairment” (lines 431-432).  We have no way of knowing this, and it is equally likely that the difference is down to environmental compensation in the nonDYS group, or a range of other possibilities.
Validity of the findings: No comment on top of those in previous section.
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: L1 AND L2 READING SKILLS IN DUTCH ADOLESCENTS WITH A FAMILIAL RISK OF DYSLEXIA
Review round: 1
Reviewer: 2

Output:
Basic reporting: Basic reporting, overall, is of good quality. The following, however,  are recommended improvements:

Ambiguities
(i)  Lines 150 & 176: As these pertain to reading, "sound-to-letter conversions" (or "phoneme-to grapheme") are not relevant. Letter-to-sound (or grapheme-to-phoneme) conversions are relevant.
(ii)  254-257: If the children read aloud, this should be stated. Although labelled as tests of "fluency," they measured the difficulty level of reading accuracy as well as speed of reading. They are tests of "accuracy/fluency."
(iii)  416, 426, 439-441: The text should make it explicit that the Spoonerism measures of PA are for LI (Dutch only).
(iv)  551, 560: It would be helpful to the reader if the "phonological problem" (or "deficit") were specified as either PA or pseudoword reading, and that neither were obtained for L2 (English).

Missing Relevant Background Information
 An obtained result was that high-risk dyslexics, relative to their L1 (Dutch) word reading levels, had a positive advantage in L2 (English) word reading levels (Lines 480-482). In view of this, inclusion of the ages of the participants are recommended for the two previous studies (Miller-Guron & Lundberg,175-178, and van der Leij & Morfidi, 178-181) that gave results of some similarity. In addition, it is recommended that there be information about the method in the van der Leij & Morfidi study by which their subgroup "was shown to have superior orthographic competence" (178-181). If Miller-Guron & Lundberg obtained research evidence about the influence of irregular conversion rules in their study, then it is also recommended that information about their research method be included.

Minor Errors in the Text
See lines 296, 298, 459, and 477
Experimental design: The research design fills a knowledge gap and the method has been well executed. The statistical analyses are of a high standard.

Lines 521-532 should be included in the Participants section (200- ). Although the instructional and remedial history of the participants was "uncontrollable" (526-527), it should be described in summary form for each of the three main groups. This is of some interest in view of lines 558-560 and 563-565 of the Conclusions.
Validity of the findings: (i)  The statistical analyses have been appropriately interpreted to support the findings.
(ii)  However, in 429-432, this "view of" the "polygenetic origin of dyslexia" may be consistent with the results but, without empirical genetic evidence in the study, so also would be a view that there was only familial influence with no significant genetic origin.
(iii)  The expressed limitations and future directions are pertinent to the obtained results.
Additional comments: For testing the "orthographic strategy" speculations (lines 457-461, 492-497, 545-546) the authors should attempt an item analysis of their existing data for the participants' reading of L2 (English) words with only regular grapheme-phoneme correspondences, in comparison with those having one or more non-regular correspondences. If there were sufficient non-regular words in the L1 (Dutch) word reading, the same analysis could be applied to that.


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: L1 AND L2 READING SKILLS IN DUTCH ADOLESCENTS WITH A FAMILIAL RISK OF DYSLEXIA
Review round: 2
Reviewer: 1

Output:
Basic reporting: No further comments
Experimental design: No further comments
Validity of the findings: No further comments
Additional comments: The authors have attended well to the worries that I expressed in my original review.  I now believe that the article meets the requirements of the journal, and support publication.  Congratulations on a nice piece of research.


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: L1 AND L2 READING SKILLS IN DUTCH ADOLESCENTS WITH A FAMILIAL RISK OF DYSLEXIA
Review round: 2
Reviewer: 2

Output:
Basic reporting: The appropriate improvements have been made in this Revision of July 2017.
Experimental design: The changes in this area are also as in 1.
Validity of the findings: In their Rebuttal Letter the authors state that their study "cannot determine whether this familial influence is genetic or environmental." Unfortunately this revision, p. 20, lines 18-22, fails to clearly say this. It is obscured by such terms as "multicausal origin" (their text reads "multiclausal"), "may have," and "support." This reviewer approves the authors clear statement of their Rebuttal Letter, which should replace these obscurities. Anything else would not meet the required standard.
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: L1 AND L2 READING SKILLS IN DUTCH ADOLESCENTS WITH A FAMILIAL RISK OF DYSLEXIA
Review round: 3
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: In this revision, this area now meets the standard required.
Additional comments: No additional comments


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

Input:
Title: INVESTIGATING THE LEVEL OF AGREEMENT OF TWO POSITIONING PROTOCOLS WHEN USING DUAL ENERGY X-RAY ABSORPTIOMETRY IN THE ASSESSMENT OF BODY COMPOSITION
Review round: 1
Reviewer: 1

Output:
Basic reporting: The article was written well for the most part. However, there were some instances where phrasing and spelling could be improved (see below under General comments). Existing literature was discussed effectively, with limited previous investigations performed in this area. Raw data were mostly made available, however the findings related to participant comfort were not presented effectively and the clarity of graphs contained in Figures could be improved.
Experimental design: While the question seems novel it is not well developed in the Introduction section leading from discussion on previous research. Also, there is some confusion over the actual research question at times given “reliability” is referred to consistently and is not actually measured in the submitted article. The methodological approach regarding the scanning procedures and protocols was presented at a high level.
Validity of the findings: The findings are mostly presented in sufficient detail; however, some data analyses were not explained in the Methods and surprisingly appeared in the Results section. Further, the conclusion is unclear given a definitive recommendation based on the provided data was not provided and some findings were not discussed regarding differences between approaches. Some adequate explanations for various findings was presented in the Discussion.
Additional comments: Introduction
Line 86: “on regular” should be “on a regular”.
Lines 95-99: This is a one-sentence paragraph that doesn’t really develop an idea fully. I would suggest combining it with the following paragraph or elaborating on the studies presented to present some actual reliability data for supportive evidence.
Line 111: “moderate level” of what specifically? Are you referring to “evidence” here? Please specify.
Line 113: “is the only study” should be “completed the only study” or similar. Also, why is Shiel et al. not given with a year – is this the unpublished work? If so, probably no need to reference.
Lines 120-123: There needs to be a stronger link to this paragraph. You finish the previous paragraph by highlighting that research demonstrates Nana’s protocol is more reliable and comfortable, but then don’t set up the importance of your investigation. Further development to show the need for your study is needed here in 1-2 sentences I feel.

Methods
Line 131: “the” should be inserted before “scanning bed”.
Line 132: Was block randomisation followed whereby the same number of participants completed each scan first?
Line 137: “and” should be between males and females without a comma.
Line 139: This is confusing as your study doesn’t seem to be a reliability study but rather a comparative study between scanning protocols. Please make sure your terminology and direction match the actual research question throughout.
Line 152: “jewelry” is incorrectly spelled.
Line 155: Has this device been shown to be reliable and valid?
Line 182: “data was” should be “data were” given it is the plural form.

Results
Line 207: There is no mention of calculating the CV previously in the manuscript?
Table 2: Can “Whole body” please be presented horizontally rather than vertically?
Figure 3: The numbers of the axes are very hard to see in this figure – can it be reworked to be larger?
Overall: There was no detail regarding the participant rating for comfort – this is a key outcome that should be presented in your results somewhere in this section.

Discussion
Line 238: “This studies primary aim” should be “The primary aim of this study”.
Line 254: Please delete “studies regional analysis” and replace with “study” here.
Line 264: Please delete the second “as” in this line.
Line 268: How does the feet distance apart affect movement artefact? Some elaboration on this suggestion would strengthen your argument here.
Line 271-274: This paragraph doesn’t offer anything that is not already known in the manuscript. Further, reliability of the approaches were not assessed but rather the level of agreement between the two was. This needs to be made clear throughout. I would recommend elaborating on these results further to strengthen this paragraph and transmit an important outcome with explanation to the reader.
Line 301: “minuet” is misspelled.
Lines 300-304: This is not really a measured outcome of the study though and is a little off topic. I would consider removing this paragraph.
Line 308: What is meant by “the real difference”? Were your findings not “real” or are you referring to a more valid approach?
Line 311: But size was not factored into your approach so I don’t see how it should shape your clinical outcomes.
Line 312: So what approach is recommended? From your descriptions here it appears that you are recommending either can be used, however your Bland-Altman data showed the tissue data where consistently higher in the Nana than the NHANES. This is not discussed anywhere in this section. Also, in the Introduction you mention that the Nana has been shown to possess greater reliability, and given reliability was not measured in your study, shouldn’t existing reliability data be factored into your conclusions?

References
There are inconsistencies throughout the reference list that require addressing – e.g. some journals do not have capitals assigned to words, some article titles are given capitals for each word, & is used in some journal titles, etc.


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: INVESTIGATING THE LEVEL OF AGREEMENT OF TWO POSITIONING PROTOCOLS WHEN USING DUAL ENERGY X-RAY ABSORPTIOMETRY IN THE ASSESSMENT OF BODY COMPOSITION
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript is generally well written and clearly describes the study (although see comments below).

Fig 3 is unclear, the Y scale should be compressed and the axis titles and legends are uninformative (what does Mmeanlean actually mean?). The 2 SD limits should be stated. What are the units?  Please redraw.

Tabulate LOA data for all body regions.

How widely is the Nana protocol used? The paper has been cited 52 times on Web of Science but it appears that citing papers are concerned with the main aim of the Nana pape, the effects of daily activities on DXA" rather than the use of the protocol. If it is not widely used then this point should be made. This should not be seen, however, to question the worth of the present paper.
Experimental design: The authors have generally described the methods adequately. However a few points require attention.

Was the survey of participant's protocol preference undertaken in a systematic manner? was a specific survey form used or simply anecdotal comment? Provide more details.

The authors used data automatically provided by the equipment software. This is OK but does need clarification, for example, what is exactly defined as "tissue". This is a generic term that is presumably being used in specific way by Lunar. Please explain.

The statistical analysis is generally acceptable but I question the correctness of the use of ICC for method comparison. I believe that Lin's concordance correlation is more appropriate. I suspect that you will find that Lin's r and ICC are very similar but statistical rigour should be applied. The authors also refer to Bland Altman plots. This is colloquial, it would be more appropriate to refer to limits of agreement analysis. Paired t tests can also be used to assess the significance of position differences (or combined for all body regions in an ANOVA to see if there is segment-position interaction).

Single scans for each position only were performed. This begs the question as to whether the inter-position differences are similar to intra-position differences and hence may not be "true" inter-position differences. This needs to be addressed.
Validity of the findings: The authors present limits of agreement analysis but fail to discuss adequately these findings. For example, are the positioning interchangeable? The mean difference in lean is small (Fig 3) but the LOA are about =/- 2 (kg?) for a mean lean mass of around 50 kg. This equates to a 4% difference. Is this clinically acceptable? Can you justify the final sentence of the Discussion in the Abstract? This implies exchangeability of methods.

The LOA plots are for whole body only. I suspect that the LOA may be wider for body segment data. This should be provided. Rather than additional plots a table of bias and LOA for all body regions could be supplied.

Discussion requires a strengths and weakness paragraph.
Additional comments: Minor points to note

Line 77 It would be more correct to refer to different degrees of attenuation rather than "rates".
Line 109. It would be better to phrase as " Shiel et al (unpublished data) have systematically...."
Line 181. The website has a number of spreadsheets - which one was used. Why was this used rather than SPSS?
Line 262,. It is unclear how the studies have accounted for biological error? Please explain.
Line 182 and elsewhere. "Data" is a plural, i.e. data were...


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: INVESTIGATING THE LEVEL OF AGREEMENT OF TWO POSITIONING PROTOCOLS WHEN USING DUAL ENERGY X-RAY ABSORPTIOMETRY IN THE ASSESSMENT OF BODY COMPOSITION
Review round: 2
Reviewer: 1

Output:
Basic reporting: Elements of basic reporting in the submitted manuscript meet the journal standards.
Experimental design: Elements of experimental in the submitted manuscript meet the journal standards.
Validity of the findings: Elements of validity in the submitted manuscript meet the journal standards.
Additional comments: The authors should be commended on addressing the author comments thoroughly, and as a result producing an improved manuscript. I do however have some minor points that require consideration:

INTRODUCTION
Lines 107-109: This seems like a bit of a stretch regarding development of a need for your study. I think speculating that bias existed in previous work should not be included and that an alternative reason/explanation should be presented here to show more work is needed in this area. Perhaps some of the positioning issues raised in this study might be useful.

DISCUSSION/CONCLUSION
Can you make a recommendation using the collective evidence from past research and your findings, considering aspects such as reliability, validity, comfort, etc.? You conclude that the protocols should not be used interchangeably, but which is ultimately recommended?


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: INVESTIGATING THE LEVEL OF AGREEMENT OF TWO POSITIONING PROTOCOLS WHEN USING DUAL ENERGY X-RAY ABSORPTIOMETRY IN THE ASSESSMENT OF BODY COMPOSITION
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors have satisfactorily amended the manuscript in response to queries raised in my original review.
Experimental design: The authors have satisfactorily amended the manuscript in response to queries raised in my original review. I accept the author's arguments for the use of ICC although I still believe that concordance correlation rather than ICC is preferable for the type of analysis described here.
Validity of the findings: The authors have satisfactorily amended the manuscript in response to queries raised in my original review.
Additional comments: The authors have satisfactorily amended the manuscript in response to queries raised in my original review.


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

Input:
Title: INVESTIGATING THE LEVEL OF AGREEMENT OF TWO POSITIONING PROTOCOLS WHEN USING DUAL ENERGY X-RAY ABSORPTIOMETRY IN THE ASSESSMENT OF BODY COMPOSITION
Review round: 3
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The authors have sufficiently addressed all suggested revisions. Thanks for the opportunity to review this revised version.


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: TUMOR-INFILTRATING CD8+ LYMPHOCYTES PREDICT DIFFERENT CLINICAL OUTCOMES IN ORGAN- AND NON-ORGAN-CONFINED UROTHELIAL CARCINOMA OF THE BLADDER FOLLOWING RADICAL CYSTECTOMY
Review round: 1
Reviewer: 1

Output:
Basic reporting: Language used:
The language should be edited for clarity.
Sentences that are unclear:
Lines 64-65: please edit for clarity/make more concise
Lines 152-154: Unclear if “the authors” defining high CD8 density are the authors of the present study or if this sentence refers to the most recently referenced papers; please edit for clarity and insert reference(s) as appropriate.
Lines 163-165: This sentence invokes possible (and likely very important) immunosuppressive effects of the local tumor microenvironment (TME), but is unclear; please edit and insert reference(s) as appropriate.
Lines 174: As is, this is a long sentence; suggest refining to minimize redundant statements.

Sentences that could be improved for style and readability:
Line 148-150: awkward construction of final clauses
Lines 158-160: awkward construction
Line 175-176: wordy

Literature references, etc:
No comments.

Article Structure:
Lines 132-135: The statement describing the results of the multivariate Cox proportional hazards regression model is closer to a conclusion than a simple statement of findings; please focus on describing the correlation identified without straying into interpretation (value as prognostic/predictive factor) in the results section.

Self-contained with relevant results to hypothesis:
See comments below (Experimental Design section) for recommended additional analyses to include in the manuscript; several may have already been performed but should be explicitly stated.
Experimental design: Aim & scope:
Appropriate.

Research Question:
The study adds to the literature by demonstrating differential correlation between overall survival (OS) and infiltrating CD8+ T-cells in organ-confined and non-organ confined disease.  OS is a strong, important end-point while the semi-quantitative method for scoring CD8 positive cells infiltrating into neoplasm is likely reproducible in the clinical setting.

Rigorous Investigation:
It would be interesting if the authors examined correlation of CD8+ TILs and OS for pT2-4 lesions as a comparison to Sharma et al (PMID: 17360461); this would also test the hypothesis that the negative correlation between CD8+ TILs and OS in organ confined disease is driven primarily by pT1a lesions.

Method description:
1) The authors should clarify their scoring methods – is the percentage CD8+ cells out of total cells or total lymphocytes?  Was this an estimate or was this counted?  An estimate is acceptable, but should be stated explicitly.

2) The authors should explicitly discuss in the manuscript why/how they selected bins for CD8+ TILs in their analysis (I.e., CD8+ vs CD8-negative, as opposed to other score combinations such as CD8+ score = 0 & 1 vs CD+ score = 3 & 4)

3) If known/recorded, it would be nice to include how many cases required a consensus discussion between the reviewing pathologists for scoring.
Validity of the findings: Data
Robust; details provided.  Concerns are expressed above about method description and other possible analyses that might make the results more impactful and demonstrate both consistency with prior literature and highlight new findings (i.e., differential correlation of CD8 TILs with OS in low-stage vs high-stage disease).

Conclusions
Conclusions would benefit from increased clarity in writing and more concise statements.
A minor point: the authors have not demonstrated activity (pro or anti-tumor) of the TILs.  They have demonstrated presence of CD8+ cells alone; activity would require markers of T-cell activation.  In general this distinction is clear, but grows slightly fuzzy in lines 162-5.  Afterall, as the authors note in Lines 164-5, the local tumor microenvironment very likely has an important role.

Speculation
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: TUMOR-INFILTRATING CD8+ LYMPHOCYTES PREDICT DIFFERENT CLINICAL OUTCOMES IN ORGAN- AND NON-ORGAN-CONFINED UROTHELIAL CARCINOMA OF THE BLADDER FOLLOWING RADICAL CYSTECTOMY
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript meets standards. Minor points and comments are as follows:
1.	Row 48: These 2017 numbers are projected and not actual numbers. Please revise.
2.	Did any of the patients receive any immunotherapy. A comment on the existing knowledge on immunotherapy of TCCs could be useful.
3.	Rows 142-143: The authors write “…however, which was a favorable…”. Please rephrase for syntax.
Experimental design: The manuscript meets standards.
Validity of the findings: The authors investigate the presence of CD8-positive tumor infiltrating lymphocytes (TILs) in organ-confined and non-organ-confined excision specimens of bladder transitional cell carcinomas. Based on their findings, the authors conclude that the presence of TILs is an independent prognostic factor in both organ-confined (unfavorable) and non-organ-confined (favorable) disease. No other clinicopathological correlations are identified based on the findings of the present study. While technically statistically significant in both cohorts (p<0.005), the statistical correlation is not compelling, as the reported p values are approaching the 0.005 cutoff. To further weaken the findings, the results are contrasting between the two cohorts. The authors should state this as a limitation of this study and should also try to offer possible explanations to the contrasting results between organ-confined and non-organ-confined disease. Finally, some information should be offered, with regard to the current state of immunotherapy in bladder transitional cell carcinoma, as this closely relates to the presence of TILs. Although unlikely, information (or lack thereof) should be included on whether patients have had immunotherapy as part of their treatment, and report any correlations with presence of CD8+ TILs.
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: TUMOR-INFILTRATING CD8+ LYMPHOCYTES PREDICT DIFFERENT CLINICAL OUTCOMES IN ORGAN- AND NON-ORGAN-CONFINED UROTHELIAL CARCINOMA OF THE BLADDER FOLLOWING RADICAL CYSTECTOMY
Review round: 2
Reviewer: 1

Output:
Basic reporting: This is a re-review of the manuscript submitting following minor requested revisions.  I have four truly minor, albeit important, requested modifications.
1) The authors have now twice addressed concerns expressed from multiple reviewers about the selection of bins of CD8+ scores and explained their rationale for using CD8+ TIL negative vs positive (grouping 0 & 1 scores vs 3 & 4 scores).  They have adequately explained in the rebuttal letters that there was no statistically significant association for other combinations of scores.  However, unless I missed something, the authors have still not included this explanation in the primary text.  I strongly believe this rationale should be explicitly discussed in the text (the results section would be appropriate).  Without such a clear, concise discussion in the results section: a) the rationale behind Figure 2's construction is unclear; and b) the readership is left with outstanding methodological questions.  The single sentence provided in the rebuttal letter is sufficient explanation and an important result.
2) Similarly, the authors explain in the rebuttal letter that percentage of CD8+ TILs was estimated as a percentage of total cells.  That methodology is definitely acceptable (to me, at least).  However, this has not been made explicit in the manuscript text.  I suggest modifying Lines 89-90 as follows: "based on the [estimated] percentage of positively stained cells out of total cells."  (Adding "estimated" to existing text; brackets indicate my modification/suggestion).
3) Figure 2 still uses TCC (transitional cell carcinoma) in lieu of urothelial carcinoma (UC) which has replaced all other uses of TCC in the text.
4) A minor point: there is inconsistent use of "Figure X" vs "Fig X"; please compare lines 109 and 123 and choose one for consistent style.
Experimental design: No concerns.
Validity of the findings: No concerns.
Additional comments: I believe this paper is deserving of publication and congratulate the authors on their efforts.  However, I believe the four really minor corrections in Part 1 (above), particularly the first two points, really are necessary corrections prior to publication and will better enable colleagues' interpretation of the data and results, and the TIL community's ability to replicate the methods in the paper.


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

Input:
Title: TUMOR-INFILTRATING CD8+ LYMPHOCYTES PREDICT DIFFERENT CLINICAL OUTCOMES IN ORGAN- AND NON-ORGAN-CONFINED UROTHELIAL CARCINOMA OF THE BLADDER FOLLOWING RADICAL CYSTECTOMY
Review round: 2
Reviewer: 2

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: The authors have adequately addressed the reviewers' comments and have presented an improved version of this manuscript


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

Input:
Title: A RETROSPECTIVE STUDY: DOES UPPER AIRWAY MORPHOLOGY DIFFER BETWEEN NON-POSITIONAL AND POSITIONAL OBSTRUCTIVE SLEEP APNEA?
Review round: 1
Reviewer: 1

Output:
Basic reporting: I think the article can be published with corrections. Given the limitations of a retrospective study, the reported data and the conclusions still make sense. The authors used routine polysomnography and CT scanning in the appropriate manner and  the data were unsurprising. The fact that NPOSA , compared to POSA, is worse and due to anatomical abnormalities is not at all a surprise. And the reasons appear totally logical in that there was, on the average,  a lengthened soft palate and a reduced or narrower  glossopharynx.

My concern is with the clinical significance with respect to treatment regarding these observations. If one has OSA it requires treatment regardless of whether one has POSA or NPOSA due to the well-known debilitating consequences of untreated OSA. One possible conclusion is that NPOSA would probably almost always require PAP therapy whereas POSA might be more susceptible to a mandibular repositioning device. Some discussion along these lines might be helpful.

Wrt data reporting, there is no need for 2 decimal points. For example, patient age should be 30.2 +/- 9.4 years not 39.23 +/- 9.41 years. And the same applies to BMI, AHI, and so on.
Experimental design: They authors used routine Procedures to assess OSA and to measure the patients' anatomical characteristics. And the statistical evaluation followed standard and correct procedures.
Validity of the findings: The findings are valid while not being very surprising. However, nice to have meaningful statistical data.
Additional comments: The comments above should suffice but happy to make further comment if the review is unclear in any way.