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: HISTORICAL CONNECTIONS AMONG RIVER BASINS AND CLIMATIC CHANGES EXPLAIN THE BIOGEOGRAPHIC HISTORY OF A WATER RAT
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: This work addresses the phylogeographic structure and biogeographic history of the water rat Nectomys squamipes from 50 localities of different river basins, using two mitochondrial genes and six microsatellite loci. Various types of analyses are applied to evaluate evolutionary hypotheses that explain the biogeographic history of the species and test if the genetic structure is influenced by hydrography. This work may become a relevant contribution of an interesting species but I think there are some minor issues as well as important inconsistencies that should be clarified.

Line 89: You may briefly explain how the sampling was performed or you may give the sources from which samples were obtained, for example, in Appendix S1.

Lines 98-99. I do not understand how resequencing can help to detect pseudogenes. Please explain better the measures taken to avoid sequencing pseudogenes, as this is an important issue.

106-107. The thresholds for bootstrap values and posterior probabilities make no much sense here. Explain the values obtained in results.

136-137. Give references for the microsatellites if they were not developed in this work.

150-151. Why using a priori classification by river basins in Structure? If there is no a justification, the results may be more meaningful if you use Structure without any a priori classification.

176-177. It should be obvious that you verified locations with any mapping tool like Google Maps or Google Earth, so you do not need to mention both tools. What it is far from obvious is how you used SpeciesLink and what is the meaning of geoLoc, infoXY, etc. Please explain better if this is relevant.

223-225. The fact that pseudogenes were found for this species reinforces the need to better explain how their sequencing was avoided here.

230 and Figure 2. Several phylogenetic methods are explained in Method. Which specific tree is shown in Figure 2? In any case, this tree does not show branch lengths, only topology. It would be convenient to choose a representation in which branch lengths are shown. It would also be desirable to comment on the differences observed between trees reconstructed with different methods. Same for Figure S3.1.

231-233. You describe the tree with D-loop but then you refer to the tree figure with concatenated sequences. Please clarify.

233-235. It would be easy to test if the outgroups affect the topologies by removing the outgroups from the cytb alignment and comparing the trees with and without outgroups.

250-251. The results obtained microsatellite genetic structure should be given more relevance. They deserve at least being in their own paragraph and a more thorough explanation since microsatellites can reveal more accurately the population structure without the problems of the mitochondrial markers (as the authors comment in Discussion). Take also into account the possibility of using a model without a priori classification, as suggested in a previous point.

262-267. It would be interesting to compare the results of differentiation from mitochondrial data with those inferred from microsatellites using similar tests or, better, Structure with a proper model.

271-275. Since you are not using a coalescent framework, these dates do not reflect the common ancestor of all Nectomys and, particularly, of N. squamipes. These dates only represent the common ancestor of the haplotypes. Care should be taken when using these dates for biogeographical inferences, and possible caveats with these dates should be mentioned.

313-316. In these two consecutive phrases you say that results from mitochondrial markers are "expected" and then that they are "striking". Please clarify the idea.

317-321. I do not find it clear if your microsatellite data is robust or not from this paragraph. Please use additional references (other species, reviews, etc.) to support your data. Otherwise, if the microsatellite results are too weak, it may be better not to discuss them further.

330. Clades with short branches in the phylogenetic trees can only be appreciated in a tree figure with branches (see previous point).

357-365. Again, microsatellite results seem not to fit with the other data but I am not sure if this is simply due to different temporal scales, as the authors argue. As suggested in previous points, the way you pre-classified data by basin may complicate congruence among results.

409-410. The Atlantis forest hypothesis seems to be well supported for this species with the mitochondrial and SDM results. However, I do not understand how nuclear loci (microsatellites) can support the Atlantis forest hypothesis when other paragraphs explain the incongruence between mitochondrial and nuclear data. Unfortunately, the referred criticism that the hypothesis is only based on mitochondrial data may remain. As already stated, a greater effort to analyze microsatellite data should be made and, if the results are relevant, explain better how they support or refute different hypotheses.

426-427. As already stated, your dating analysis cannot be used to date the origin of this and other species.

437. Again, I don't think you can conclude that there is congruence between mitochondrial and nuclear data. This contradicts most of your discussion.

Figure 1: Check the map scale, which seems to be wrong.

Figure 4: I suppose you mean "bar graphs".

Figure 8: Maps should show the same extent, if possible, for easier interpretation.


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: HISTORICAL CONNECTIONS AMONG RIVER BASINS AND CLIMATIC CHANGES EXPLAIN THE BIOGEOGRAPHIC HISTORY OF A WATER RAT
Review round: 1
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: It is not possible to arrive at one of the two main conclusions of the manuscript based on the sampling design used
Validity of the findings: No comment
Additional comments: Review: Historical connections among river basins and climatic changes explain the biogeographic history of a water rat (#25252)

I have read the above mentioned manuscript with attention. This manuscript investigates the phylogeographic structure of N. squamipes using mitochondrial and nuclear markers in order to evaluate evolutionary hypotheses to explain the biogeographic history of the water rat and test if the genetic structure is strongly influenced by hydrography. This is a very interesting manuscript, well written and structured. The introduction clearly presents the background in the subject of study and the results are presented in a clear manner. However, sampling design and the interpretation of some results present certain flaws.
I think that there is scope for this study to yield a paper of interest to a large audience. Unfortunately, I have identified some issues (listed below), which, I believe, need to be dealt with prior to publication. I hope that the authors find my comments below useful.

General comments:

One of the main conclusions of the manuscript is that rivers allow N. squamipes to disperse farther across and inside basins, leading to genetic homogeneity and a shallow genetic structure slightly influenced by river basins. I believe that this conclusion should be reviewed carefully. Firstly, the sampling performed is not representative of all the analyzed basins, since basins such as SeA or EA present between 35 and 60 individuals sampled (Table S 2.4) and basins such as Paraná, SA and SF present only 6 to 13 individuals sampled. This difference in the number of individuals analyzed per basin could be the cause of the lack of genetic differentiation between them only as consequence of a bias in the sampling.
Additionally, AMOVA analyses performed with the mitochondrial control region, a more appropriate marker than cytochrome b for studying genetic structure, showed significant differences between basins (Fct = 0.25, P <0.01, Table 4) and between pairs of basins (Table 3), contradicting the idea of genetic homogeneity between river basins. This result should not be underestimated by the authors, given the importance of maternal lineages in the conformation of genetic structure.
With the aim of solving the bias in basins sample size and trying to make a better use of sequence data with more innovative methodologies, I encourage the authors to add the Bayesian analysis using BAPS or Geneland software in order to estimate the number of genetic cluster present in the study area and then to test for genetic differences between clusters independently of river basin.

Minor comments

Lines 65-66: I think this sentence should be deleted as it is confusing. It is very difficult to imagine that rivers could lead to isolation in this species. Or do the authors refer to high flow rivers that could act as barriers to dispersion? I think that in the case mentioned by the authors, the specialization would be the cause of the isolation and not the rivers.

Line 89: How were the samples taken? Were live individuals captured and sacrificed for liver samples? Or were dead individuals collected? In the case of using live individuals and sacrificing them, had the authors the necessary permits? Please explain in more detail.

Lines 95=96: Only cyt b electropherograms were carefully examined? What about d-loop ones?

Line 103: Which models were selected?

Lines 133-135: Why the BSP was conducted with cyt b sequences only?

Line 143: Why were k values chosen between 1 and 6? How many genetic clusters were expected?

Line 158: Why was MaxEnt chosen among all the available species distribution models? Did you try some other model?

Line 226: If 152 of the 154 individuals sampled were genotyped. Why only 42 of the 50 sampling locations were represented?

Lines 243-244: “Genetic divergences were small within and among Nectomys species, among the N. squamipes clades, and among river basins”. These results must be explained in greater detail. Only the results of intraspecific divergence are displayed

Lines 246-247:  I do not agree that an interclade divergence of 4.0-4.6% for the control region is small. This differentiation corresponds in many cases to that observed between different mammal species.

Lines 262-265: “We found a very large (FST>0.25; Hartl & Clark, 1997) mitochondrial (cyt b and D-loop) differentiation among river basins (Table 3). The largest FST values were found between the Southeast Atlantic and South Atlantic basins (cyt b: 0.74; D-loop: 0.64), indicating little divergence within basins and large divergence between basins”. These results also contradict the idea of genetic homogeneity between basins.

Lines 267-270: It is striking that the AMOVA analysis carried out using cyt b sequences showed non-significant differences and all the paired-Fst comparisons between basins are significant. Please explain.

Lines 315-316: This is not striking at all. This type of patterns is very common in different species of mammals in which males disperse and females are philopatric.


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: HISTORICAL CONNECTIONS AMONG RIVER BASINS AND CLIMATIC CHANGES EXPLAIN THE BIOGEOGRAPHIC HISTORY OF A WATER RAT
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: The authors have addressed the most important concerns and the manuscript has improved with respect to the previous version. However, there are still a few minor issues that the authors should clarify:

Abstract, line 15: "that shows genetic homogeneity across space". It is strange to say in the first phrase of the abstract that the species is homogeneous when the introduction makes clear that this is a controversial issue, with some authors having found a relevant genetic structure (lines 75-77). I also think that "genetic homogeneity" is not properly used in line 30 of the abstract and in several other parts of the manuscript. The authors are in the safe side saying that they found a "shallow genetic structure" because they talk about three mitochondrial clades, etc. but this is not "genetic homogeneity".

In general, the authors are advocating too strongly for the "genetic homogeneity" explanation and even give some examples that do not really support it. For example, Galemys pyrenaicus (line 379) shows a particularly strong genetic structure both at large and small scales, so this is not a good example neither for "shallow genetic structure" nor for "genetic homogeneity" and can be removed from here. In general, it will be difficult to find examples of mammals showing genetic homogeneity, but clearly, there are many with shallow genetic structure. In addition, in this part of the discussion the authors sometimes seem to equate small genetic divergence with low genetic structure but these are two different concepts and the authors should be more careful with them.

The issue about pseudogenes is better explained now, at least for cytochrome b. However, given the big problems found with this gene, one could think that they may also be present in the D-loop, at least at some frequency, but nothing is commented on this. The authors could comment in Results if some pseudogene was found for the D-loop or if they were only present in the cytb gene.

In the justification for using the locality prior in Structure in Methods (line 160), you should state that these recommendations are for situations where the amount of available data is very limited (few markers or few individuals), as indicate in the response to reviewers.


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: HISTORICAL CONNECTIONS AMONG RIVER BASINS AND CLIMATIC CHANGES EXPLAIN THE BIOGEOGRAPHIC HISTORY OF A WATER RAT
Review round: 2
Reviewer: 2

Output:
Basic reporting: I would like to congratulate the authors on their hard work, for addressing the comments from a previous review of the manuscript. Authors have done a great job. I consider that there has been a substantial improvement since the first version.  I enjoyed reading the manuscript and recommend considering it to be published in PeerJ.
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: A SARCOPTES SCABIEI SPECIFIC ISOTHERMAL AMPLIFICATION ASSAY FOR DETECTION OF THIS IMPORTANT ECTOPARASITE OF WOMBATS AND OTHER ANIMALS
Review round: 1
Reviewer: 1

Output:
Basic reporting: -
Experimental design: -
Validity of the findings: -
Additional comments: The authors developed LAMP, as a part of their continue efforts in improving diagnosis of S. scabies infestation. The authors proposed that LAMP could serve an important role in a point-of-care or resource-limited setting. I have some comments, below:

> Line 83-85: please provide reference(s).

> Line 173: A total of 87 ITS2 sequences were aligned and shown to have 96.5 - 100% sequence identity. I wonder how much % identity we should expect to consider these sequences were from the organisms with the same species? Should that be 99-100%?

> Line 182, after incubation in a thermal block, the LAMP products were detectable, as visualised by the amplicons on the gel. I wonder what/how interpretation/determination of a LAMP result is relied on? Is it by looking directly at the reaction tube, observing an amplicon on a gel, or checking on a curve/peak (as shown in Fig 2)? A point of care test is expected an convenient way of obtaining and interpretting a result, and that should be described and evaluated in the study.

> The LAMP assay with a heat block (Line 180), a thermal cycler (Line 183), and a fluorometer (Line 186), I wonder which one showed the better detection performance? Comparing an effect of these incubation tools will be useful to readers who may have different devices available, or have to decide which device should be used.

> Figure S2, the marker is not clear, and not labeled.

> Line 194: The authors described the high congruence (95.3%) between LAMP and PCR, and showed that LAMP had higher detection sensitivity than conventional PCR. I feel it is bit confused about the Table 1, where there are the results of 64 clinical samples being compared. However, Line 136-138, the author mentioned that there were a total of 78 clinical samples, consisting of 40 positive samples (from 23 subjects) and 38 negative samples (from 24 subjects) (the authors should briefly described here how these samples were defined positive and negative, although a reference is referred to). Which set of samples  (64 or 78 samples) was used to calculate the sensitivity and specificity? The detection sensitivity and specificity of the conventional PCR should be mentioned.

> Line 246-250, The higher sensitivity of LAMP compared to conventional PCR, how could the authors exclude a possibility of false positive result by LAMP? The authors should also simply perform, compare, and show an analytical sensitivity by using serial dilution samples to investigate which assay (LAMP vs PCR) could detect lower amount of a define DNA sample. In addition, how the authors manage to control an contamination issue of a high sensitivity assay like LAMP?

> The author emphasized an advantage of using LAMP over PCR by that LAMP could be used as a part of a point-of-care (POC) and resource-limited settings. However, it seemed that the results of LAMP were read on a gel, or read by a fluorometer, which may not be considered as a part of POC.

> Line 263-164, the possible targets for LAMP assay can be ITS-2, cox1, 12S rRNA, and 16s rRNA. I suggested that the authors incorporate the initial analysis to indicate that how and why ITS-2 was selected as the target for LAMP, i.e., how much sequence variation, how much %GC, etc.

> Not only detection efficiency, some other factors are important in determining which assay should be selected for use are cost, time, and personal skill. Please describe these factors, for LAMP in comparison with other assays (PCR, microscopy).


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 SARCOPTES SCABIEI SPECIFIC ISOTHERMAL AMPLIFICATION ASSAY FOR DETECTION OF THIS IMPORTANT ECTOPARASITE OF WOMBATS AND OTHER ANIMALS
Review round: 1
Reviewer: 2

Output:
Basic reporting: a.	The introduction provides context, but is lacking information on other assays that are present. It also does not describe limitations to the assay, and why those limitations are tolerable in the context for this assay development
b.	Would like to see Figure S2 and Table S1 included in the main body of the text as opposed to supplementary files
Experimental design: a.	The scope of the research and trials appear to be relevant for the journal.
b.	More information about the materials and methods should be described, as there are some significant portions that are needed to describe the methods. Please see the General Comments below for a line-by-line description.
c.	The objectives and hypotheses for the study were not clearly defined in the introduction. Would like to see them explicitly stated to allow for better flow of research description.
d.	There were really very few samples actually tested in the survey of PCR to LAMP samples. It would be nice to see more samples assessed, but there are of course limitations to the number of samples that one can obtain.
Validity of the findings: a.	Certainly a good body of work that could be a useful diagnostic tool, especially in lower-income countries.
b.	It would be nice to see more discussion on the limitations and benefits of this amplification method over others. There are many new developments that would improve the assay developed here, and there are certainly pitfalls to a LAMP assay that should be discussed.
Additional comments: a.	The abstract could be abbreviated to be more concise and have no separated sections, which would make it easier to read.
b.	Line 85: You should discuss other diagnostic tools. For example, Biomeme has an iPhone qPCR device and Agdia has a portable RP device. It would be nice to see a comparison of methods for this system.
c.	Lines 86-94: There’s several limitations to LAMP as opposed to other methods, especially real-time methods. Turbidity (or lack-thereof) can be misleading in results, whereas a PCR product is pretty distinct. Comment on some of the limitations, and where your methodology accounts for those limitations.
d.	Line 94: It would also be nice to see any comparisons to other LAMP/Nucleic Acid Assays developed for micro-scale arthropods. Are there any limitations to working with those systems (ie PCR/LAMP inhibitors) that may cause issues?
e.	Line 95-101: This seems more appropriate for the abstract. You should list your hypotheses/objectives for the study here as opposed to summarize the work as you’ve already done in the abstract.
f.	Line 104: How many copies of the target region are found in one mite? The LAMP assay can amplify as few as 6, but can the assay detect less than one mite? (ie, should there be broken up mite from the sampling process, could the assay still detect it?)
g.	Line 119: LF and LB are described here but not in the primer design above. Are these loop primers? This should be described in the primer design as well (lines 104-116).
i.	Line 124-125: This should be included in the primer design section.
h.	Line 131: Was sensitivity not assessed during LAMP assay development?
i.	It would probably make more sense to swap the Clinical samples discussion and the DNA extraction methodology since the sample information describes that DNA extraction was conducted.
j.	Line 153: I’m confused as to what you’re testing against for inhibition. Within the skin samples? Likely inhibitors would also be present in the mite only DNA samples as well. Would it not have been better to create a plasmid or an internal control to look at inhibition?
k.	Line 155: Again, I think the DNA extraction should be described before clinic samples. Even if it is described in another paper, a quick overview of the process would be nice in the assay development.
l.	Line 159: Lyse’n’LAMP was not specified in the previous assay development description. Should that not also be mentioned there? Also, why did you switch master mix here? Were there issues with the regular Optigene master mix?
m.	Line 161: “aliquot” instead of “elute”?
n.	Line 164: Please go into more detail as to how this is calculated and why this was chosen as opposed to ROC statistics and/or chi-square/fisher’s exact that are typically used to compare assays. This is not a common statistic, so it should be given more detail.
o.	Line 172: Did you extract DNA from these sources and run the LAMP reaction with those extracts as well? In silico is fine, but an actual assessment with primers under reaction conditions may have differential binding than what is predicted, especially if the reaction temperature is not optimal for the primer sets used.
i.	Found this below (189). Please specify the species assessed in vitro and number of replications. This should be described in the materials and methods as well.
p.	Line 180: Was this detectable by precipitate or using the assay parameters intended for the POC?
q.	Line 184: The sensitivity of the assay wasn’t well described in the materials and methods. I would explicitly describe conditions for this assessment and how many reactions were run with the dilution series.
r.	Line 206: Inhibition of the assay may not be necessarily no-amplification with a LAMP assay given the high affinity for the target region to amplify with the Bst polymerase. It could just delay the amplification significantly to look like non-specific binding. That would be more evident a real-time reaction. There could still be inhibition and amplification taking place. I would rephrase that the potential inhibitors in the system did not prevent amplification from occurring.
s.	Line 214: Please describe this in better detail in the materials and methods
t.	Line 230: qPCR methods and RP methods are capable of this as well, why is this better than those methods?
u.	Line 230: Could you describe the potential minimal amount of DNA present in a typical skin scraping? It may help to describe the need for a lower limit. For example, if there should be less than a mite in a given sample due to poor collection or low colonization of a lesion, would the assay be able to detect ½ a mite or less worth of DNA?
v.	Line 245-250: LAMP can amplify as few as 6 copies of a target region in a given reaction whereas PCR requires more DNA to amplify, so your increased sensitivity is not uncommon and a large appeal for the assay in low-DNA assessments.
w.	Line 253: If you want to discuss the DNA extraction in the discussion, it really should be described in the materials and methods. It seems that it would be part of the novelty of the assay and largely part of the whole process for a limited-technology facility.
x.	There should be discussion about Line 211 findings.  The high variability in the real-time amplification of LAMP products: could this be due to inhibitors present from the crude DNA extraction process? Variability of LAMP product amplification should be discussed since this is not the only occurrence of it in the literature.
y.	Line 277-278: The cost of the equipment is a rapidly changing environment, especially with those using Genie equipment. It’s almost as expensive as some real-time PCR machines. It may be worth elaborating on other diagnostic methods available and why the LAMP assay may be better to pursue in this setting. An actual breakdown of costs does not necessarily show that LAMP is the most inexpensive tool unless crude tools (heat block with visual detection of precipitate) are used.


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 SARCOPTES SCABIEI SPECIFIC ISOTHERMAL AMPLIFICATION ASSAY FOR DETECTION OF THIS IMPORTANT ECTOPARASITE OF WOMBATS AND OTHER ANIMALS
Review round: 2
Reviewer: 1

Output:
Basic reporting: -
Experimental design: -
Validity of the findings: -
Additional comments: The authors have addressed all of my concerns.


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

Input:
Title: A SARCOPTES SCABIEI SPECIFIC ISOTHERMAL AMPLIFICATION ASSAY FOR DETECTION OF THIS IMPORTANT ECTOPARASITE OF WOMBATS AND OTHER ANIMALS
Review round: 2
Reviewer: 2

Output:
Basic reporting: Article follows basic reporting required by journal.
Experimental design: Research questions defined and define research gap. Reviews about experimental design were addressed.
Validity of the findings: Testing was very limited, and few samples were assessed for sensitivity and specificity of assay. The authors describe the limitation of this in the discussion. There is also no sequencing of LAMP products to confirm non-specific binding or primer dimerization. If this work was done, a statement of that work should be included.
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: DATA CONCORDANCE BETWEEN ESRD MEDICAL EVIDENCE REPORT AND MEDICARE CLAIMS: IS THERE ANY IMPROVEMENT?
Review round: 1
Reviewer: 1

Output:
Basic reporting: Line 70, lack of continuous validation of which information?
Line 72-73, this sentence needs to be supported by reference or examples of how this data is being used in policy and practice, and as such why the data accuracy in the USRDS matters?
Line 83, explain what does Kappa statistic represents before providing the values.
Line 92, which three methods? Besides, claims data are not considered complicated data source for health services research compared to other data sources from the EMR, such as lab data.
Line 134, it is ZIP code
Experimental design: Line 96, what is the rational for including environmental-related factors, which factors? The authors need a framework or conceptual model for justifying which variables to include and which hypotheses they will test.
The study is a-theoretical, besides there is no clear objectives and hypotheses that the authors are studying. It seems they included variables that they had access to without clear framework or rational.
The authors need to explain in writing in the text the inclusion/exclusion criteria they implemented. Also, in Figure 1, they must include excluded patients and reasons for exclusion.
Why limit Medicare Claims data for past-year, to the minimum, it should include the current year or two or three years before?
What is the value for including the three methods A, B, C. There are well established methods of calculating co-morbidities from claims data and especially for Medicare beneficiaries.
Validity of the findings: 145, the authors need to explain what each statistic represents?
149, what is the outcome variable, discordance (0-no/1-yes)? Is it a patient level nested model, which patient level variables did you control for? Also, poverty and education are usually highly correlated, how did you handle the correlation in the regression model? Which model A, B or C did you use in the regression analysis?
Claims data is not considered big data in the current health services research (HSR) terminology, claims data is the customary data that has been used over the past decades to conduct HSR.
Table 3 is unreadable, consider revising the table.
Additional comments: What is the link between the ACA implementation and the analysis, there is nothing in the background that motivates the study and why the ACA implementation would have impacted provider reporting behavior in the CMS form.
The writing quality of the manuscript can be substantially improved and the discussion section needs to be strengthened in light of policy implications and results from previous studies, and provider reporting behavior literature.
Line 251, the authors identified by adding more recent claims data you get more complete picture of co-morbidites (which is very evident), is there a reason why they did not include current year in the analysis, for example government reporting requirement timeline for data? These are details that they need to discuss in the discussion section - how is the values for government measures calculated and what are the implications for practitioners and their behavior?
The study could add value to the literature if the authors address the concerns identified in this and each of the previous sections.


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: DATA CONCORDANCE BETWEEN ESRD MEDICAL EVIDENCE REPORT AND MEDICARE CLAIMS: IS THERE ANY IMPROVEMENT?
Review round: 1
Reviewer: 2

Output:
Basic reporting: I thought the articulation of study goals, study design and all other aspects of communication specifically in manuscript were all very clear and concise. I'm almost certain the tables/charts would end up in the manuscript as you approach the final draft but it's worth noting that it was a bit distracting the have to open two different documents (or more) if I'm trying to read and review the charts/tables. Lastly, there might be a small typo in the first paragraph (should "form" be "forms"?)
Experimental design: Overall I thought the experimental design was well thought out, organized and clear. Below are some additional thoughts:

1. It might be wise to shed more light on previous results given that the goal of this work is to investigate potential "improvements" vs. pre ACA. Not much discussion takes place around what those old results are and whether or not the differences are meaningful. I'm left having to take your word for it after a little bit of discussion in the "discussion" section. (ex. how does table 1 here compare with the equivalent table in previous studies and is that important to note?)

2. It might also be good to increase the minimum claims requirements for patients to be in the study cohort. CMS made the burden of proof extremely high for co-morbidities being included on claims data for payments. For that reason, you'll notice that a very sparse sample of patients for which any co-morbidities are recorded. That's because providers have to contact hospitals on patients' behalf to collect any evidence of co-morbidities, signed by doctors. Then they have to run that data through a rigorous QC process before any of it shows up on claims. In summary, it might be the case that the ACA had every intention to improve this data capture but, operationally, collecting this data on claims is a nightmare for providers. I acknowledge here that this might be a separate issue worthy of an entirely different public policy effort. But might be helpful to know and control for those issues in your study (i.e. more claims as minimum over a longer time period).
Validity of the findings: The methods, results and conclusions sound robust and accurate given the underlying data/hypothesis. Not much more to add here.
Additional comments: Thanks for giving me the opportunity to review this great work. I'm hopeful it'll drive the right public policy discussion/actions.


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: DATA CONCORDANCE BETWEEN ESRD MEDICAL EVIDENCE REPORT AND MEDICARE CLAIMS: IS THERE ANY IMPROVEMENT?
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have addressed previous comments which substantially improved the quality of the manuscript in this revision. I have few small additional comments.

In which section of the SEER program they use claims data - cases are identified at the center where patients are diagnosed and/or treated from the medical record. Do you mean in the SEER-Medicare dataset?

Page 6, line 19, you mean county?

Did you exclude patients with Medicare Advantage plans from the study?

Table 1, include footnote how you define healthcare utilization and at which level of analysis - State?
Experimental design: No further comments
Validity of the findings: No further comments
Additional comments: No further comments except those in box #1.


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

Input:
Title: ANALYSIS OF THE LIGHT INTENSITY DEPENDENCE OF THE GROWTH OF SYNECHOCYSTIS AND OF THE LIGHT DISTRIBUTION IN A PHOTOBIOREACTOR ENERGIZED BY 635 NM LIGHT
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The growth rate, cell size and photosynthetic activity were investigated at various orange-red light intensities. The local light intensity and light regime in the PBR were further simulated at different incident light intensity. The work is interesting, and some useful and valuable results were obtained.
Only a little shortage as follows:
In Line 469, as well as other place, the unit of cell size is wrong, it should be μm rather than m.
In Figure 6, what is the detail position of three XZ planes? It should be showed.


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: ANALYSIS OF THE LIGHT INTENSITY DEPENDENCE OF THE GROWTH OF SYNECHOCYSTIS AND OF THE LIGHT DISTRIBUTION IN A PHOTOBIOREACTOR ENERGIZED BY 635 NM LIGHT
Review round: 1
Reviewer: 2

Output:
Basic reporting: In this work, authors describe the investigation of the effect of increasing intensities of orange-red light on the growth of Synechocystis in a laboratory scale photobioreator. The article is clear and well organized.
Regarding references:
Lines: 723, 754, 780, 803, 822, 882 and 958 please add the missing information to these articles. In some cases the Journal title is missing in other the year and the volume/page numbers.
Line 809: article title is repeated for three times, please correct.
Please italicize species name.
Figures 2 and 4. Please replace commas with points.
Table S4. Please correct the gravity force value. Please add unit for incident light intensity.
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The "μ", for micro, letter disappeared in several points in the manuscript. I presume that the symbol is lost with the conversion from .doc to .pdf file. Please check and correct these points.


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: ANALYSIS OF THE LIGHT INTENSITY DEPENDENCE OF THE GROWTH OF SYNECHOCYSTIS AND OF THE LIGHT DISTRIBUTION IN A PHOTOBIOREACTOR ENERGIZED BY 635 NM LIGHT
Review round: 1
Reviewer: 3

Output:
Basic reporting: See general comments
Experimental design: See general comments
Validity of the findings: See general comments
Additional comments: Dear editor, dear authors,

I read the manuscript ‘Managing light distribution effects for leveraging photosynthetic activity in photobioreactors’ with great interest. The manuscript is clearly written and easily readable. Figures are of very high quality and advanced modelling tools are used. The experimental dataset generated is valuable as it seems to be applicable for advanced modelling studies. Nevertheless I am not convinced about the novelty of this study because I did not find any results which yield new insights in photoautotrophic microbial growth. For this reason I recommend not to accept this manuscript for publication in PeerJ. Below I will go in more detail on how I came to this conclusion.

The title of the manuscript for me was a bit misleading because in the end there was nothing about ‘managing’ light distribution. The issue of managing light distribution clearly is an important topic for large-scale application of such bioprocesses. I think that this experimental dataset and developed modelling tools can be used in a completely new study really focussed on this topic.

Now I will go further on my conclusion of lack of novelty. The main findings of the experimental work is the finding that growth of Synechocystis is light limited at low light intensities, saturates reaching maximal specific growth rate at increasing light intensities, and decreases again at very high light levels. This is a well-known response for all photoautotrophic microbes (algae and cyanobacteria) grown under nutrient replete conditions and this is not novel. Only the observation that Synechocystis could quickly recover from photoinhibition could be new. But in this context I miss an in-depth discussion on recovery from photoinhibition, for example the relation to the photosystem II damage-repair cycle (see reference below). I think a literature review on this topic probably will show that a rapid recovery to photoinhibition is a much more general feature of microalgae and cyanobacteria.
Theis, J. and Schroda, M. (2016) ‘Revisiting the photosystem II repair cycle’, Plant Signaling and Behavior. Taylor & Francis, 11(9), pp. 1–8. doi: 10.1080/15592324.2016.1218587.

In this manuscript modelling of fluid and gas dynamics and light regime in the photobioreactor is very extensively described. But, also in this part of the study I did not recognize the novelty. In fact, it is not clear to me why modelling fluid and gas flow, and heat transfer was relevant anyway. In the end the only conclusion coming from the modelling study was the observation that light distribution in the culture was heterogeneous. This is widely recognized. The heterogeneity in the y-direction (figure 5) is implicitly present in the Lambert Beer equation. So, there is no need for model calculations because a qualitative evaluation of equation 16 is sufficient to arrive at this conclusion. The heterogeneity of light over the light exposed reactor surface is a direct result of the design of the light source and photobioreactor, and also here complex modelling is not really needed; one could just measure the light distribution over the surface. The lack of use of the models (especially fluid and gas dynamics) contrasts greatly with the extensive description of these models in the manuscript.

At this point I would like to stress that modelling of the liquid flow is relevant in case one wants to apply a dynamic photosynthesis model. For example a model which takes into account photoinhibition in zones with strong illumination and recovery from photoinhibition in dark zones. Please refer to below publication in which such an approach was used. But, such an approach was not taken in the current study so I do not see the use of CFD modelling.
Olivieri, G., Gargiulo, L., Lettieri, P., Mazzei, L., Salatino, P. and Marzocchella, A. (2015) ‘Photobioreactors for microalgal cultures: A Lagrangian model coupling hydrodynamics and kinetics’, Biotechnology Progress, 31(5), pp. 1259–1272. doi: 10.1002/btpr.2138.

In the calculation of the biomass yield on photons I suspect a mistake. A yield of 2.70 g/mol was reported (Table 2). Such a high yield is extremely unlikely as it would mean a minimal quantum requirement of 8 photons per O2 and (!) the production of merely carbohydrates instead of functional biomass, which is not very likely at such low light levels. To my knowledge yields higher than 2 g/mol have not been reported up to date (see for example reference to Schuurmans below). Re-examining the data presented in table 2 allowed me to recalculate the biomass yield on light. Assuming that the optical depth of the PBR is 2.5 cm and assuming that all light is absorbed in the culture I calculated a volumetric light input of 7.2 mol/m3/h (at 50 micromol/m2/s). The volumetric biomass productivity was 8.1 g/m3/h (at 50 micromol/m2/s).  Dividing 8.1 by 7.2 gives a biomass yield of 1.125 g/mol, which corresponds to published data. In this context it is relevant to know the light intensity at the backside of the reactor. Was this measured? (I could not find information in supplementary data).
Schuurmans, R. M., Van Alphen, P., Schuurmans, J. M., Matthijs, H. C. P. and Hellingwerf, K. J. (2015) ‘Comparison of the photosynthetic yield of cyanobacteria and green algae: Different methods give different answers’, PLoS ONE, 10(9), pp. 1–17. doi: 10.1371/journal.pone.0139061.

Minor comments:

In my opinion the discussion of the relation between cell size and specific growth rate should be extended with the possible influence of a persistent circadian rhythm (see reference below). Could such a rhythm have had an impact on the measurements? Was there an change of average cell size over a 24 hours
van Alphen, P. and Hellingwerf, K. J. (2015) ‘Sustained Circadian Rhythms in Continuous Light in Synechocystis sp. PCC6803 Growing in a Well-Controlled Photobioreactor’, Plos One, 10(6), p. e0127715. doi: 10.1371/journal.pone.0127715.period?

With respect to the materials and methods the procedure to measure the specific growth rate could be explained in more detail. As I understand every 24 hours light levels were increased. So within 24 hours the cyanobacteria have to acclimate and reach a new steady state, and then the specific growth rate is determined. Is this possible in only 24 hours? Is it really possible to state that a steady state was reached? If not, what is the possible impact on the data obtained?

Lines 158-159 are not clear to me. How was the turbidostat condition maintained when increasing light levels?

Lines 153-154 are conflicting: pCO2 was adjusted and at the same time CO2 was always at 1% v/v.

Model description is not clear to me with respect to the gas phase, specifically lines 228-229 and lines 241-242. I think this aspect has to be described more extensively.

Assessment of different model parameters is not clearly described. For example gas bubble size in equation 18.

Was the general radiative transfer equation really used? If so, for what purpose? Light regime was modelled with equation 18 I think?

What has been done with heat transfer (equation 20-21)? Is it relevant to model heat transfer since temperature is maintained constant by the FMT150 reactor unit.


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: EPIDEMIOLOGY OF TUBERCULOSIS AND TREATMENT OUTCOMES AMONG CHILDREN IN PAKISTAN: A 5 YEAR RETROSPECTIVE STUDY
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1.	There is noted ambiguity in lines 99 to 114, 175 to 176, and lines 232 to 239.
2.	Language used could be improved in lines 263 to 270 (unsure if personal opinion, which is inappropriate, or if is from references listed in sentences preceding. Language improvement also needed Lines 255 to 260.
3.	Introduction adequate and can appreciate the background intended by author. However, the reference citing need to be improved. These are apparent in line 50 where TB is actually the nineth leading cause of death globally and NOT by an infectious disease. The points of attempting to evaluate the burden of paediatric TB disease in this area within a high burden region is noted but not consistent following intro.
4.	Structure does more or less conform to PeerJ standards but references need to be looked at further i.e.between journal title and volume needs to be amended.
5.	Figures are relevant except the the weight band categories figure is not elaborated upon and confusing at best. I suggest you review the WHO new FDC formulations and weight band categories and try to compare those against standard of available care with regard to treatment regimens available in Pakistan. Good quality explanatory figures explaining trends over fixed period. Figures should have descriptive comments written below, whilst tables should have the title of the table typed above the table.
6.	Raw data seems fine.
Experimental design: 1.	The primary research and evaluation of the district in question is within the scope of the journal and is original, highlighting the need to investigate areas of possible concern within a high burden area.
2.	The research question is well defined and pertinent toward the global context of childhood TB.
3.	There seems to be no IRB ethical approval; no mention of child assent or informed parental/guardian consent; unsure if data available were de-identified to ensure subject confidentiality. Only site approvals from medical superintendents. Investigation tools used seem appropriate but there seems be poor application of paediatric research and clnical definitions.
4.	Noted methods of retrospective analysis of data from TB register. Would you call this a retrospective cohort study?
The inclusion criteria is poorly stated and ambigious as mentioned above in “basic reporting” and suggest you review current up to date or recommended Paediatric TB definitions as well as others eg Graham et al (J infect Dis, 2012, V205, pp 199) or Seddon et al (J of Paed Infect Dis, 2013, V2, pp 100).
Childhood symptomatology are different from adult TB and you aren’t clear about this. The inclusion criteria makes no mention of microscopy for smear status, Xpert availability as a WHO recommended screen for children and phenotypic culture as a gold standard still adhered to for childhood TB diagnosis. Need to be clear about why clinical diagnoses made more.
5.	Personally I feel WHO malnutrition anthropometric defintions might be more useful for a global reader audience i.e. with Z-scores and defining “moderate acute malnutrition” and “severe acute malnutrition” but I do note that you only had TB register data available to evaluate. This is not a must have as is retrospective data analysis.
Validity of the findings: 1.	The incidence of childhood TB globally is mentioned at roughly 5% in the introduction (line 72). No mention of TB high burden region sttaifications by WHO and Pakistan, as part of the Eastern Meditarranean high burden region. Mention of incidence and no mention of prevalence of TB childhood disease in pakistan over a known time period from existing literature and to use that as a backbone for relating current burden of disease, morbidity and mortality in context. Rather your referencing has not kept in mind WHO regions and of a recent Lancet meta-analysis by Jenkins et al reviewing global childhood TB morbidity and mortality to corelate against your findings in discussion.
2.	 Risk factors for Tb mentioned are in keeping with norms in clinical experience and agreed with.
3.	Mention again: for rather looking at recent meta-analysis of TB regions wrt childhood TB.
4.	Line 239 paraphrased states a gold standard “needed to be developed”. This is inaccurate as there is a gold standard with certain limitations which could be elaborated upon – ie phenotypic culture usually MGIT culture.
5.	Line 264 to 270 seems more a personal opinion and needs to be reworded and shown to be of evidence-base.
6.	It is well known that only half of children with TB disease are diagnosed and put on treatment (WHO Global Report, 2016). Those that are inititated on treatment appropriately do very well usually.
7.	Data presented seems statistically reasonable to assess. But if the global burden of childhood TB is roughly 6.9% and we are unsure if over- or under diagnosing, why are your findings across the 3 sites evaluated not improving drastically and high proportionately?
8.	I don’t think the conclusion is well stated and doesnt bring your research question to fruition in terms of local and global context.
Additional comments: 1.	It is a worthy study to have undertaken and glad that data is avaliable to review how these sites contribute to the regional burden of childhood TB in the Eastern Meditarreanean region as well as in the global scene.
2.	Need to mention if IRB ethical approval has been granted.
3.	Reference citing and understanding of current paediatric TB clinical and epidemiological needs work.
4.	Some citations are old evidence and newer, updated revised evidence is available.
5.	Conclusion doesn’t quite coalesce well.


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

Input:
Title: EPIDEMIOLOGY OF TUBERCULOSIS AND TREATMENT OUTCOMES AMONG CHILDREN IN PAKISTAN: A 5 YEAR RETROSPECTIVE STUDY
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript “Epidemiology of tuberculosis and treatment outcomes among children in Pakistan: A 5 year retrospective study (#24857)” by Laghari et al presents interesting data and conclusions on the epidemiology of TB in children in Pakistan.

 It is the opinion of the reviewer that the manuscript can be accepted for publication with minor corrections.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: - Did the authors observed if there was any difference in the treatment success rate between patients infected with MDR strains of TB and patients infected with strains sensitive to drugs? This could be an interesting data.

- Line 82-83:  Since the conclusions of the manuscript indicates that there is a significant difference in the success rate of the treatment between children living in rural and urban areas, can the authors include a short comment indicating the major differences in living style and condition between rural and urban areas?
- Line 123: Please clarify the treatment regimens reported in Table 1. It is not clear
- The authors should indicate s the antibiotic treatment regime advised in Pakistan.

- Table 1: Please indicate the meaning of the abbreviations “R”, “H”, “Z” and “E”
- Figure 2: please indicate what the abbreviations stand for.


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: EPIDEMIOLOGY OF TUBERCULOSIS AND TREATMENT OUTCOMES AMONG CHILDREN IN PAKISTAN: A 5 YEAR RETROSPECTIVE STUDY
Review round: 2
Reviewer: 1

Output:
Basic reporting: 1.	Must commend the authors on a much improved manuscript.
2.	Introduction good. Minor comment: Language line 62 “This is for the reason” can be improved for an international reader audience.
3.	Minor Language comment in Line 263 “Like” could be changed.
4.	Figures and tables much improved. Unsure if PeerJ would like Figure captions to be below figure and have Table headers above table as shown.
Experimental design: 1.	Recommendations addressed and am happy with design.
2.	Re: “suggest you review current up to date or recommended Paediatric TB definitions as well as others eg Graham et al (J infect Dis, 2012, V205, pp 199) or Seddon et al (J of Paed Infect Dis, 2013, V2, pp 100).”
i.	Most paediatric Tb publications used recommended definitiions as part of academic consensus for terms labelled “Definite Tb, Probable TB, Possible TB” or “definite, probable or not TB.”
ii.	I’m happy with the authors rebuttal but a comment to authors for reader audience interested in their findings.

3.	Wrt my previous comment re: “Personally I feel WHO malnutrition anthropometric defintions might be more useful for a global reader audience i.e. with Z-scores,” I must apologise to the authors.
(i)	WHO anthrometric gradings with centile and z scores (as used in South African paediatric clinical settings) theoretically are not as appropriate to be used in the developing world setting as the WHO growth charts are more targeted to a healthy index population.
(ii)	The CDC definitions are more appropriate.
Validity of the findings: 1.	 Re: "Rather your referencing has not kept in mind WHO regions and of a recent Lancet meta-analysis by Jenkins et al reviewing global childhood TB morbidity and mortality to corelate against your findings in discussion." – I still would have liked the authors to have read an article and refer them to:
i.	Stark et al (Lancet Infect Dis, 2016, V 17, pp 239) for commentary on Helen Jenkins article on Global childhood TB mortality.
ii.	This gives context to their article and think the authors need to realize that the article targets a paediatric academic community also, esp the paediatric TB academic community.
2.	Mention again: for rather looking at recent meta-analysis of TB regions wrt childhood TB. – see comment 1.
3.	With reference to known paediatric TB epidemiological trends I am unhappy with the explanation in lines 245 to 250 (not explained well and seems not appreciative of disease spectrum reasoning behind it).
i.	Trends show that with adolescence there an increased trend of TB disease in this age group presenting with more adult type of disease spectrum. Younger children have paucibacillary pathogen burden while older children much higher burden
ii.	There is also hypotheses that adult sex hormones via the HPG axis might account for these.
iii.	I think these lines should be looked at carefully and look at epidemiological age curves demonstrated from various publications incl WHO. Perhaps can look at articles by Peter Donald too.
4.	Line 278 to 285 seems more a personal opinion still. Is there no qualitative research evidence to support this to make your point more poignant and contextual? Noted comment on being a hypothesis so perhaps state it clearer that its specultive in terms of personal opinion; it is quite a long explanation describing the hypothesis.
5.	Data presented seems statistically reasonable to assess. I am happy with the authors rebuttal.
6.	Conclusion sounds good.
Additional comments: 1.	I am very happy with the revisions made and commend the authors for the hard work put in.
2.	My comments arise from being a paediatrician and a childhood TB researcher. Comments were only geared to help you target an interested audience.


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: EPIDEMIOLOGY OF TUBERCULOSIS AND TREATMENT OUTCOMES AMONG CHILDREN IN PAKISTAN: A 5 YEAR RETROSPECTIVE STUDY
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors have replied to all the comments from the reviewers and the article can now be accepted for publication
Experimental design: no additional comments
Validity of the findings: no additional comments
Additional comments: As I mention in my previous review, the article present interesting results about epidemiology of TB in children in Pakistan. The authors replied to the comments of the reviewers and the article now can be accepted for publication.


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

Input:
Title: AGE DIFFERENCES IN HEAD MOTION AND ESTIMATES OF CORTICAL MORPHOLOGY
Review round: 1
Reviewer: 1

Output:
Basic reporting: •	Major points.

1. Generally clear, professorial language.

2. Clear progression, structure and background.

3. Well formatted and clear.

•	Minor points

1. Slight typo on line 16 – perhaps should be 'also differs'

2. Line 16 ‘Here I…’ is a slightly awkward sentence and could be clearer, e.g. ‘within…’ to ‘with a larger dataset than has been demonstrated previously’.

3. Consider referencing the Hitchcock film used fully (e.g. https://learn.solent.ac.uk/mod/book/view.php?id=3381&chapterid=3455).

4. Consider referencing previous literature regarding ‘Models with differences in BIC>2 were considered…’ (line 106-107).
Experimental design: •	Major points

1. Well defined and a meaningful question.

2. Conducted to a generally high technical and moral standard.

3. Generally, sufficiently detailed.

4. Is there a male vs. female effect? Some basic demographic details like sex frequencies, average age, stroke frequency etc. for the sample would be helpful. If these are stated in other papers (for CamCAN), please explicate that.

•	Minor points

1. Perhaps IQ or personality metrics predict head motion at rest/movie-condition, or the amount of improvement from rest->film, independent of older age. You could consider this analytically or note for future research (if you think this might be a valid factor; only a suggestion, and ditto this for any possible sex effect as suggested above).
Validity of the findings: 1. Very important, easily-applied amendments for human brain imaging research (i.e. that films should be shown during structural scans). I believe the data are robust, statistically sound and this cautious conclusion is reasonable in that context.

2. A generally commendable paper which conducts detailed analysis of a well-outlined problem, and links conclusions to robust data and questions.
Additional comments: A good, clear 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: AGE DIFFERENCES IN HEAD MOTION AND ESTIMATES OF CORTICAL MORPHOLOGY
Review round: 1
Reviewer: 2

Output:
Basic reporting: For the most part the paper is well written in proper English.  However, I did not find the background information presented in the introduction sufficient to fully justify the work and to understand the importance of the findings.  Without further elaboration, it is not clear what problem or issue this work addresses.  Although the work serves as a replication of some of the authors prior findings, the results are completely predictable and there is no clear indication why such a replication is either necessary or helpful.

I think this overall weakness is reflected in the discussion which highlights only that this work found the same correlations previously reported by the author.

In my view, the introduction needs substantial elaboration to understand how this work contributes to our understanding of how head motion influences estimates of cortical thickness in aging.

More minor potential edits:
In the opening paragraph, it appears that the author means to say that head motion leads to lower estimated cortical thickness – but the language of “decreased estimates of cortical thickness” on line 34 is ambiguous and should be edited.

On line 36 the author uses the term “Taken together” to summarize studies that have not been discussed (or at least adequately discussed).
Experimental design: The methods lack detail about the data and how it was analyzed.  Although the data come from a database and the techniques have been used and reported elsewhere, it is necessary that this paper stand on its own without the needs to reference a handful of other works to grasp the implementation.

Please provide information about the MRI machine, fMRI pulse sequence, number of slices, preprocessing on fMRI data, etc.

A more detailed description of FD should be presented in the paper rather than referring readers to other papers, particularly because it appears to be an approach developed by the author and is not widely known or adopted.

Describe the measure of frame-wise displacement and why this measure was used instead of other measures of head motion.

Section 2.4 Model comparison approach is under-described and contains no citations for referencing the explanatory tools or their application.  In particular, describe your hierarchical regression procedure including what software was used.  Also provide more detail on model construction implementation.  It appears that age and head motion may be at different levels of the model, but the description is somewhat vague. Describe Bayesian Information Criterion (including writing it out in words before using the abbreviation) in more detail including how it was applied to the data in the study.  The use of this measure implies the evaluation of different models.  Be explicit about how many models were tested, how each was constructed, and which model comparisons were made.
Validity of the findings: The author makes an argument that the fMRI sessions are an appropriate predictor of motion during structural scans based on a correlation between the two fMRI sessions. I think it would be useful to discuss possible limitations of this assumption in the discussion section.

Without further analysis and evidence, there is no justification for the observation beginning on line 121 for systematic stimuli evoked increases in head motion.

It seems that with the available data there is an opportunity to provide more detailed and potential more interesting data about head motions effect on thickness estimates in different regions of the cortex, particularly because thickness estimates are not influences uniformly across the brain.

The delta BIC in table 1 is difficult to interpret because it is not clear what models are being compared.  Moreover, if a delta BIC of greater than 2 is considered significant, why is only the Age + Motion(Movie) condition considered as containing additional explanatory power when 8 of the reported 12 delta BIC measures exceed a value of 2?

It would be helpful to see the BIC for each model in addition to their differences.
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: AGE DIFFERENCES IN HEAD MOTION AND ESTIMATES OF CORTICAL MORPHOLOGY
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The author has addressed my concerns, particularly with respect to the methods section of the paper.  Although I would still like to see a more thoughtful discussion of the findings and at least some consideration of the source for the relationships (or lack thereof) reported in this work, I also recognize that PeerJ is concerned more with the findings than the scholarly impact.  As such I feel the paper provides enough analysis and novel material to move forward to publication.

Note that there are some small typographical errors in the paper that I recommend the author correct before publication.


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

Input:
Title: ‘ROLLING’ STONEFLIES (INSECTA: PLECOPTERA) FROM MID-CRETACEOUS BURMESE AMBER
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The only point that may raise doubts is the naming of a new Acroneurinae genus on the basis of larva alone, since larvae are unknown for several extant Acroneuriinae genera. But of course, it is very improbable that Electroneuria ronwoodi would belongs to a recent genus, and I agree that it is probably not a Largusoperla larva. However, it should be corrected that correlation between the length of cerci in larvae and adults regards only to Systellognatha and not all the extant stoneflies.


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: ‘ROLLING’ STONEFLIES (INSECTA: PLECOPTERA) FROM MID-CRETACEOUS BURMESE AMBER
Review round: 1
Reviewer: 2

Output:
Basic reporting: line 24, you should add the reference to Wang et al too (Fossil record of stem groups
employed in evaluating the
chronogram of insects (Arthropoda:
Hexapoda)

line 25 or 34, there are more recent papers on the phylgeny inside the order
(chen et al 2018
Molecular phylogeny of Systellognatha (Plecoptera: Arctoperlaria) inferred from mitochondrial genome sequences
By: Chen, Zhi-Teng; Zhao, Meng-Yuan; Xu, Cheng; et al.
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES  Volume: 111   Pages: 542-547

line 37-38, what do you think about this taxon, there are few supporting synapomorphies, even none is indicated, if I am not wrong

line 82: the nomenclature should have a reference, please precise
line 105, no it is not, the oldest resins are Carboniferous !!

line 128: please precise in the diagnosis which characters are autapomorphies of the new family, it is very important for the readers

line 690, remove one 'is'

line 699: if it is a an ancient trait, it is a plesiomorphy?, so it supports nothing at all
furthermore the basal position of RP is quite frequent in polyneoptera

line 711: no, a plesiomorphy supports nothing at all, if you want to exclude from the crowngroup, maybe find autapomorphies

maybe a key to species could be welcome ?
Experimental design: methods, investigations, research question are ok
Validity of the findings: of interest because it is a new family and oldest taxa in recent families, from a crucial period of changes in aquatic ecosystems
Additional comments: a very nice paper, well written,
maybe just to add some precisions on the apomorphic characters supporting the taxa (in diagnoses) and some small questions to answer


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: ‘ROLLING’ STONEFLIES (INSECTA: PLECOPTERA) FROM MID-CRETACEOUS BURMESE AMBER
Review round: 1
Reviewer: 3

Output:
Basic reporting: "“Rolling” Stoneflies (Insecta: Plecoptera) from Mid-Cretaceous Burmese amber" (#27907) by Sroka et al., corresponding author Arnold Staniczek;
The paper deals with new species of fossile stoneflies and describes seven new species from four genera. Additionally one new family and three new genera are established. The ms is clearly structured and well written in appropriate English language and fits within the scope of the journal. Descriptions are detailed, although sometimes – due to general conditions of types – quite vague. Figures are of high quality and of high relevance.
As I am not a specialist in fossile stoneflies, I can only suspect that this is state of the art within this specific and sometimes speculative discipline. On the other hand the discussion is deep and pros and cons are carefully debated in a scientific way including morphology, temporal and spatial scales. The systematic position of the newly described taxa are transparently deduced, including some hardly testable hypotheses.
The ms follows a charming approach, naming fossils after „fossile“ rock-band members, which, although a general trend nowadays, dusts the scientific community a little bit.
Summarizing, not being a specialist I can only annotate few misspellings. From my general understanding I support publishing the ms with minor revisions and hope the other reviewers have more substantial comments.
Introduction, page 1: line 20, Stoneflies have aquatic larvae, reformulate: Stoneflies have in general aquatic larvae (there are several semiterrestrial species);
Line 25: within the order was analysed by (Zwick, 1973, 1980): change into: was analysed by Zwick (1973, 1980);
Petroperla mickjaggeri sp. nov. Description, line 185: of femora and on dorsal surface of tibiae: insert a dot after sentence.
After the description I miss a short paragraph dealing with affinities of the family/genus which is given in the discussion but is added in other examples directly below descriptions, please harmonise
Line 374: skip underscore after abdomen
Line 638: start with capital letter after the dot.
Line 690: skip one „is“ within th sentence
Line 701: after e.g. recent instead Recent
Line 710: change prescnce into presence
Line 757: space between Figs and 5D, harmonise Figs and figs within the entire document
Line 785: change Stark and Lenz 1992 in Stark & Lenz, 1992
The abreviation approx. should be changed in approximately within the entire document
Experimental design: there are no experiments
Validity of the findings: New species are a hypotheses which will be validated in future
Additional comments: "“Rolling” Stoneflies (Insecta: Plecoptera) from Mid-Cretaceous Burmese amber" (#27907) by Sroka et al., corresponding author Arnold Staniczek;
The paper deals with new species of fossile stoneflies and describes seven new species from four genera. Additionally one new family and three new genera are established. The ms is clearly structured and well written in appropriate English language and fits within the scope of the journal. Descriptions are detailed, although sometimes – due to general conditions of types – quite vague. Figures are of high quality and of high relevance.
As I am not a specialist in fossile stoneflies, I can only suspect that this is state of the art within this specific and sometimes speculative discipline. On the other hand the discussion is deep and pros and cons are carefully debated in a scientific way including morphology, temporal and spatial scales. The systematic position of the newly described taxa are transparently deduced, including some hardly testable hypotheses.
The ms follows a charming approach, naming fossils after „fossile“ rock-band members, which, although a general trend nowadays, dusts the scientific community a little bit.
Summarizing, not being a specialist I can only annotate few misspellings. From my general understanding I support publishing the ms with minor revisions and hope the other reviewers have more substantial 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: CLIMATE-INDUCED PHENOLOGICAL SHIFT OF APPLE TREES HAS DIVERSE EFFECTS ON POLLINATORS, HERBIVORES AND NATURAL ENEMIES
Review round: 1
Reviewer: 1

Output:
Basic reporting: The present study aimed to evaluate potential effects of climate change on the ecological interaction between apple and different animal groups: pollinators, pests and natural enemies. This is an interesting topic and research is welcomed.

The background helps to fix the aims of the MS and shows relevant literature. (L90. The correct reference is Rosa García & Miñarro 2014; the same in the reference list).

Bar graphs were used to show the results in Figures 1 to 4. However, bar graphs do not provide information about the distribution of the data and are not the most appropriated for continuous data (Weissgerber et al 2015 PLOS Biology 13 (4)). Boxplots could be a better choice. The authors used grey colors when treatments differed from the control. It is very visual but the common method of using different letters for different values could be more informative at showing differences among treatments.

The y-axis legend of Figure 2, ‘Abundance’ could be also more informative (e.g. number/1000 flowers/min), for an easier comparison of visit rate with other studies.

Legend of Figure 3. Is it necessary to provide: ‘a = 0: log (…) Simpson index’?

Supplemental Figures are provided as .eps files which could be difficult to open. When I finally managed to open them I could not find figure legends. I suggest to provide them in a more common way.

Raw data are supplied.
Experimental design: The authors use a clever method to manipulate bud burst: a greenhouse to advance phenology and a cold store to delay it. However, whereas the greenhouse advanced the bloom peak a reasonably period (6-9 days), the cold store retarded it from 16 up to 38 days, which is a huge delay in any current scenarios of climate change. Thus, this fact partially conditions the results, in particular those of treatment Delayed2. A much lower difference in days would be a more realistic scenario.

In addition, and as authors pointed out and tried to explore (they recorded lower flower number, leaf size and fruit-set in delayed trees), such methodology might have had physiological consequences for trees that had affected the plant-animal relationships.

These two points should be more deeply reflected in the interpretation of the results.

I think it is not clear enough how pollinators were sampled, if all the tree was observed, how many trees, or if transects were done… I understand that on each treatment pollinators were sampled two days, 15 min each day (i.e. 30 minutes in total) (I have a concern related with that and with the results and their interpretation. Wild bees were much more abundant in treatment A1 than in the rest, and the authors argued that it is because earlier species occurred at that moment (‘due to the lack of alternative floral resources’) and that later (control) they diminished due to a dilution effect of having many more flowers. However, three days later (A2) those wild bees were less than 8 times lower (but conditions (e.g alternative resources) should not have changed so much), and then increased again in the delayed treatments (similar results in diversity; Fig 3A). I wonder if this result could be the result of a sampling effect because pollinator assemblages may change considerably according to the time of the day as well as with the weather conditions, and two observations could not be enough to reflect the pollinator assemblage if conditions are not exactly the same. Detailed information on pollinator sampling and on time of the day and weather conditions should be provided, and a potential sampling effect should be also considered at interpreting the results.

L115. What is the phenology of cv Resi, in comparison with reference cvs (e.g. Golden Delicious)?

L117-118. I do not understand that of eight blocks and three orchards.

L121. ‘Similar numbers’ were used but see Table 1 (from 31 to 42 trees).

L143-146. Beating started before trees in the delayed treatment had flowered. How could this have affected the results?

L164-165. I do not understand the reasoning.
Validity of the findings: Results are in general valid but comments above should be considered.

Is the Conclusions section too long?
Additional comments: L285-286. And likely of blossom weevils.

L330. Could the higher number of flowers in the control partially explain the higher abundance of weevils?

L332-333. Do you mean more adverse effects than in other herbivores? Then, please rewrite.

L335. Is A. pomi a low-specialized oligophagous aphid and not a specialized one that spend all the cycle in this host? Are you sure that all they were Aphis pomi and not the similar species A. spiraecola (also frequent in Hungarian orchards: Andreev 2007. Journal of Plant Protection Research, 47, 109–112.)?

Regarding green aphids and its relation with its host, apple, it could be also discussed that delayed phenology could hamper aphids to develop from winter eggs as those hatch before bud burst and fundatrices would die by lack of food, as happens with the rosy apple aphid (Miñarro and Dapena 2007. Environmental Entomology 36(5): 1206-1211). Despite that could not be reflected in the present study it is likely to happen in natural conditions. Although it is also true that secondary colonization is possible in green aphids, unlike rosy aphid.


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: CLIMATE-INDUCED PHENOLOGICAL SHIFT OF APPLE TREES HAS DIVERSE EFFECTS ON POLLINATORS, HERBIVORES AND NATURAL ENEMIES
Review round: 1
Reviewer: 2

Output:
Basic reporting: The authors studied the possible effects of climate change, here through shift in the phenology of apple cultivars, on pollinisation and abundance of some pests and their natural enemies. This kind of approach is novel for agricultural conditions. The general context, and the associated concepts, are well and clearly presented in the Introduction section making it easy to read and very interesting.
Experimental design: The experimental design is well introduced (worst-case scenario) and is scientifically sound (and the number of replicates impressive). I have only minor comments on that point : (i) the date of outdoor planting are not ranked according to the different treatments (is it normal?) and (ii) the effects of the orchard where the experiment was carried out (and of its phenology) is only marginally presented and will appreciate to find a more in-depth discussion about the possible effects (bias?) of this on the main results.
Moreover, it was strange not to find more information on the apple cultivars used (for the experiment (indoor previously) and for the orchard site).
Validity of the findings: The Discussion is balanced (between clear findings and others requiring some speculation) and well constructed.
Additional comments: Other comments :
L76: the word selection here is too vague (selction of what)
L118 n=8 for each block, right?
L115: provide information on the apple cultivars
L137: something unclear: how to monitor the pollinators for 15 mn if an insect is sometimes used (it is a perturbation)
L139: no information on the rosy apple aphids (presence and infestation rate)?
L158: « thus » sounds strange here (a sharp decline do not prevent the use of raw data in a mixed model)
L180: it is very surprising that the orchard was not a random factor in this model!!!
L189-191: better explain (I do not understand)
L221: if only one orchard was considered, this important information should be provided in the figure legend
L233: hard to follow (especially because visually the mean values are close but then significantly different). Why not dividing the mean values by the number of flowers?
L391-394: it could mean that evenness would be modified. Then the absence of this index (due to Renyi) is strange and disappointing
Figure 1: not sure ‘fitness’ is the right word (fitness is the number of descendants per generation) and for me leaf size is not directly associated to fitness.
Figure 1- panel D: the SD (or SE) is sharply decreasing between advanced 1 and Control. Can this be discussed?
Figure 3: eveness is missing
Figure4: why not separating spider into families, those than can be known to be natural enemies in apple orchards and those with unknown or even negative effects (for example those known to predate syrphids)?
Table 2: (last row) does it mean that spiders are very sensitive to phenological shifts? and then why?


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: METAL COMPLEXATION BY HISTIDINE-RICH PEPTIDES CONFERS PROTECTIVE ROLES AGAINST CADMIUM STRESS IN ESCHERICHIA COLI AS REVEALED BY PROTEOMICS ANALYSIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The authors of this manuscript have clearly stated the biological significance of this study. The authors proposed a sound hypothesis of the molecular mechanism that governs the adaption of E. coli to toxic cadmium stress. The manuscript provided sufficient background information with appropriate literature references. The whole information provided in this manuscript was well structured; the figures and tables were properly displayed for supporting.

This reviewer suggests adding another figure to better describe the authors' conclusion. The authors described a network of protein that are regulated under the stress of cadmium and the expression of PolyHistidine. A picture of this network would be a nice summary for the proposed mechanism of cadmium adaptation.
Experimental design: This manuscript represents an article for an original primary research within the aims and scope of PeerJ. Understanding the mechanism for bacterial in response to toxic ion strength is of significance in Microbiology, Ecology and other scientific fields. E. coli, used in this report represents an excellent target at lease because it is a convenient and relevant organism to deal with in a laboratory setting. The authors have conducted proper experimental design. The experimental data are sufficient and unambiguous to support the authors' hypothesis and to facilitate further studies in this topic.
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: METAL COMPLEXATION BY HISTIDINE-RICH PEPTIDES CONFERS PROTECTIVE ROLES AGAINST CADMIUM STRESS IN ESCHERICHIA COLI AS REVEALED BY PROTEOMICS ANALYSIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Work is a labor intensive effort. The presentation and content are nicely lined up.
Experimental design: Experimental design is presented very well.
Validity of the findings: The results of the work are well presented. However, since the results of E. coli in different expression properties are close to each other, what is the effect of the measurement uncertainty of the study on these results? It is unknown. This should be well explained.

***I strongly recommend evaluating these results by a scientist working in proteomic field.
Additional comments: Work is a labor intensive effort. The presentation and content are nicely lined up.
The results of the work are well presented. However, since the results of E. coli in different expression properties are close to each other, what is the effect of the measurement uncertainty of the study on these results? It is unknown. This should be well explained.


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: METAL COMPLEXATION BY HISTIDINE-RICH PEPTIDES CONFERS PROTECTIVE ROLES AGAINST CADMIUM STRESS IN ESCHERICHIA COLI AS REVEALED BY PROTEOMICS ANALYSIS
Review round: 1
Reviewer: 3

Output:
Basic reporting: This manuscript describes the use of cytoplasmic and membrane bound polyhistidine derivatives for bacterial adaptation to toxic cadmium ions. Using susceptibility assays and proteomics, this study compares effects on bacterial growth and protein synthesis.

The manuscript has a lot of scope for improvement with regard to grammar, sentence structure and scientific accuracy. Some examples of areas to improve are listed below:

Abstract

Lines 39-42: the main point is not clear. Please rewrite these lines to better describe the results related to expression of key proteins.
Line 43: “minute expression of MnSOD…”, relative to what?

Introduction

Line 55 – Opening sentence is missing references.
Line 56 – More specific descriptors of the effects of Cadmium ions. For e.g., “breakage” of DNA, state the nature of alteration of cell division (slower, faster, dysregulated, etc).
Line 63 – The knowledge gap is not clear. The authors describe several mechanisms of metal toleration, e.g. reduction of uptake, sequestration, efflux pumps, yet state that “the underlying mechanism of metal toleration in bacteria remains unclear…”/
Line 69 – The meaning of “accumulation” is not clear.
Lines 71-73 – please revise so as to improve sentence structure.
Line 87 – describe the specific mechanism being referred to. Do all these proteins adopt the same mechanism?
Line 94 – replace ‘prove’ with a more appropriate term, such as ‘test’ or ‘determine’.

The Introduction is missing a description of the current understanding of the toxic effects of cadmium ions on E coli, mechanisms of cellular response and adaptation. I urge the authors to conduct a thorough literature survey. (A simple Pubmed search for the keywords ‘cadmium toxicity bacteria’ led to more than 900 hits.) Moreover, the authors should provide a strong rationale for studying protein expression in their model strains. How does cadmium ion treatment affect protein expression profiles?

Materials and Methods

Line 110 – host ‘cells’ should be singular.
Line 126 – Comment on the choice of Cadmium dose. Did the authors perform a dose response assay?
Experimental design: Results

Line 221 – Title includes ‘growth pattern’ but the following text does not discuss it.
Line 229 – For this experiment and Figure 2, report the doubling time, i.e. time required by cells to double their optical density, for all strains and conditions. Presenting this analysis in a table format might be more informative to readers.
Line 233 – Please explain the choice of Cadmium ion dose (0.2 mM). Did the authors perform a kill assay to determine susceptibility/survival of all relevant E coli strains used in this work, as a function of Cadmium dose? This is essential to rationalize the choice of dose for all the experiments described in this manuscript.
Line 235-236 – The assertion that the presence of hexahistidine-GFP promotes cell growth is merely correlative at this point. Please rewrite this sentence to better describe the observation. For example, ‘Cells expressing the hexahistidine-GFP fusion protein displayed similar growth rates in the presence and absence of Cadmium ions.’
Lines 246-263 – These lines describe changes in protein expression profile in the three strains as a function of cadmium ion stress. I urge the authors to report on the total number of proteins identified in each strain and condition, including common proteins identified in more than 1 strain and condition. For each protein, state the fold-change values while comparing expression profile changes in the various strains. Some of this information is provided in Table 2, but including the numbers in the results section will help readers.
Line 256 – Please be specific in reporting changes observed in expression profiles. For example, ‘H+ transporting ATPase was found to be expressed in an opposite manner.’ Explain what the ‘opposite manner’ is.
Line 263 – ‘… while OmpF was found in all cases in response to cadmium ions.’ The point of this line is not clear. Do the authors mean that OmpF was identified in all cases? Or was the trend of fold-change the same in all cases?
Lines 273-274 and Figure 5 – Please state the number of replicate experiments performed to generate this data. Figure 5 is lacking error bars. Please represent OD600 data as mean +/- standard error of at least 3 biological replicates.
Overexpression of OmpA results in similar growth rates in the presence and absence of cadmium ions. The increase in growth levels observed in the case of polyhistidine-OmpA is small. It remains to be seen whether the difference in growth rate/levels between OmpA and hexahistidine-OmpA are within the error of the experiment. I urge the authors to be cautious while interpreting any differences in the growth rate of the two strains. Based on the data, the contribution of the hexahistidine tag to Cadmium resistance is unclear to me. Also, it is important to provide proper context to these results relative to a wild-type control carrying the empty vector.
Line 284 – needs appropriate referencing.
Line 290 – ‘was disappeared’ may be replaced with a more appropriate term, such as ‘was missing/not detected’.
Line 303 – ‘expression’ should be replaced with ‘overexpressing’
Lines 288-312 – for the fold-change trends reported in this section, I urge the authors to explain their use of descriptors such as ‘a remarkable’ expression… (line 301), MnSOD became ‘the most important’ target… (line 303). Discuss fold changes observed in cells expressing native GFP – what is the explanation for differences in expression between cells expressing native GFP and cells carrying the empty vector (for example, MnSOD expression, line 330)?
Line 387 – OppA should be OmpA?

Line 318 – ‘great discrimination’ – consider alternative terminology

Line 432-434 – I disagree with the authors on this conclusion. Figure 5 shows that cells overexpressing the polyhistidine OmpA exhibit slightly elevated growth rate/level relative to cells overexpressing OmpA. As mentioned in a previous comment, this growth curve should be replotted to include average data from at least three independent biological replicates (mean +/- standard error). Moreover, a growth curve alone provides little information on the mechanism of cytoprotection, if any. It is preliminary and inaccurate to suggest that this experiment proves that inhibition of metal uptake by the polyhistidine residues confers resistance to cadmium toxicity.

To further investigate the mechanism of cytoprotection mediated by the polyhistidine moiety, I suggest the authors measure intracellular levels of Cadmium ions in all the strains, under all the treatments, described in this work. Similar studies have been performed by Sousa et al (Nature Biotechnology 1996), who used hexa-histidine conjugated LamB, an outer membrane protein in E coli. The authors should explain their choice of OmpA for tagging with hexahistidine, as opposed to LamB (as in Sousa et al).

2D-DIGE – Was reciprocal labeling of the samples performed, to eliminate artifactual spots due to dye-dependent interactions?
Validity of the findings: Lack of relevant controls

The susceptibility and proteomics experiments are missing appropriate positive controls, i.e. strains that have been demonstrated previously to affect Cadmium ion toxicity (for e.g. hexa-histidine LamB in Sousa et al). Use of a positive control is essential to provide context to the effect of unknown alterations, such as poly histidine GFP and OmpA. Moreover, the OmpA experiments are missing a control strain that does not overexpress OmpA. This must be included in any repeat experiments because OmpA itself facilitates transport of ions and solutes, therefore, its overexpression might alter transport of cadmium ions across the membrane. I suggest the authors repeat their experiments to incorporate relevant positive controls.

The fold-change in expression of OmpA in the strains described in the text should be measure relative to native expression in the appropriate parent strain. Plasmid based overexpression is often accompanied by misfolded or aggregated proteins, such as inclusion bodies. The correlation between expression and activity of OmpA needs to be validated.

It will be important to compare the susceptibility and proteomics results obtained in hexahistidine-expressing strains with previous regulators of cadmium toxicity such as OxyR, SoxRS, RpoH, etc, which are already outlines in the Introduction (Line 86). The proteomic signature, in particular, might reveal important mechanisms of response based on these previously known models.
Additional comments: This study provides new information on the effects of cadmium ion treatment in bacteria. The findings are undoubtedly relevant to several areas of microbiology and biotechnology. I urge the authors to incorporate control strains to provide proper context to their findings. Specific suggestions in this regard are provided in my comments. Additionally, significant improvements can be made to all sections of the manuscript, this will surely elevate the impact of this work.


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

Input:
Title: METAL COMPLEXATION BY HISTIDINE-RICH PEPTIDES CONFERS PROTECTIVE ROLES AGAINST CADMIUM STRESS IN ESCHERICHIA COLI AS REVEALED BY PROTEOMICS ANALYSIS
Review round: 2
Reviewer: 1

Output:
Basic reporting: Work is a labor intensive effort. The presentation and content are nicely lined up.
Experimental design: Experimental design is presented very well.
Validity of the findings: Explanations about the experimental design of the article by the researchers regarding the criticisms are sufficient.
Additional comments: I recommend the paper for publication. In my opinion, the paper should be published in PeerJ.


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

Input:
Title: INTEGRATIVE MACHINE LEARNING ANALYSIS OF MULTIPLE GENE EXPRESSION PROFILES IN CERVICAL CANCER
Review round: 1
Reviewer: 1

Output:
Basic reporting: "Machine Learning" is a very broad term. I suggest the authors to be more specific than the current form on the Abstract section. Whether it is supervised or unsupervised learning? Is it predictive or exploratory?
Figure 1 is nicely drawn for a bird-eye view on the manuscript.
Experimental design: Sufficient experimental design for "PeerJ".
Validity of the findings: The last sentence in the abstract looks troublesome: "... Nevertheless, further validations are needed in order to validate the significance of selected 9-potential-gene expression signature in cervical cancer." It significantly weakens the scientific soundness. In addition, such a sentence is also repeated in the conclusion.
Additional comments: In general, the authors have written a reasonable manuscript for PeerJ. It has the basic components in computational genome studies. I have the following suggestions:

Since the study is a data integration study. Special care has to be taken for data normalization among those input datasets. The authors should carefully write down most of the parameter setting for readers. Otherwise, readers may not be convinced.

From line 118 to 200, the authors have described the off-the-book-shelf concepts in bioinformatics. I doubt if it is necessary for the authors to repeat those basic knowledge on this scientific research manuscript?

It would be interesting if the authors can describe their works in the context of pathway network analysis on top of GSEA.