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: ESTIMATING FLOWERING TRANSITION DATES FROM STATUS-BASED PHENOLOGICAL OBSERVATIONS: A TEST OF METHODS
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: The author did a very nice job responding to the reviewers' comments and improving the manuscript substantially.  This paper will be a very useful contribution to the field of phenology and help guide researchers in assessing the best approach for selecting methods to calculate transition dates given their dataset limitations and structure.
I only have a few *very* minor edits/suggestions below:
Line 40: change "leafs" to "leaves"
Line 46: clarify you mean repeated observations on the same plant(s).
Line 51; sometimes "First Observed" method is capitalized and other times not, check throughout the manuscript for consistency; also clarify here if you mean these estimators to be for individual or population, or both.
Line 54: I think this statement requires additional citations for context.
Line 81: include that this is for one site/population.
Line 85: could better explain how "population" is defined in the context of these results, some might use these methods to estimate dates across multiple sites/regions. For example, in studies that rely on herbarium data, how are populations defined, and are there often many samples from one "population"?
Line 139: should it be "Last Observed"?
Line 145: by how much was final sample sizes reduced in this study when this restriction was applied?
Line 166: any guidance regarding what a reasonable threshold would be?
Line 209: what is the range of error rates?
I really appreciated the recommendations section of the discussion- very comprehensive and straightforward.


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: VIRALPLAQUE: A FIJI MACRO FOR AUTOMATED ASSESSMENT OF VIRAL PLAQUE STATISTICS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Plaque assay is a classic method for counting viral infectious particles while manual counting of viral plaques is a time-consuming task. The authors aim to develop a software system ViralPlaque to automatically recognize and analyze plaque pictures obtained from a flatbed scanner or a cell phone camera. A machine learning plugin has also been integrated in the analysis algorithm, which is considered to be an application of artificial intelligence (AI).
Experimental design: 1)	In aspect of biological methods, what’s the success rate of your plaque assay method? As far as we know, traditional plaque assays are not easy to conduct and highly depend on the experiences of operators.
2)	How to address the problem of plaque fusion from the aspect of image recognition? You know, sometimes two or more plaques can usually merge to form a larger plaque in disks or plates.
3)	I don’t know how you take pictures by a cell phone. Was the light source from the back of 6-well plates or from the same side of the cell phone?  If the light source is from the back of 6-well plates, the outline of plaques would be clearer than those the light source from reflected light. A flatbed scanner relies on a reflected light source, to our knowledge.
Validity of the findings: The algorithm used in image analysis may be reasonable. The more important things are the repeatability of plaque assays, as well as how you get the raw data of the images. More details regarding these two parts should be described in your manuscript.
Additional comments: The paper is well written and gives an insight into application of technology of digital image recognition, which is similar to face recognition or AI doctor in analyzing CT films. But the latter is based on huge demands while yours is not. For example, there are hundreds of CT film need radiologists to read every day and an AI doctor in image recognition will help finish most of their work. To emphasize the importance of your research, other demands of plaque assay should be mentioned, not just viral titer testing or plaque morphology and dimensions regarding the replication kinetics and the virulence of a particular virus. Especially, when these demands need huge image data, and aim to solve a practical problem.


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: VIRALPLAQUE: A FIJI MACRO FOR AUTOMATED ASSESSMENT OF VIRAL PLAQUE STATISTICS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The plaque assay has been a mainstay of virology research for >50 years and, for some viruses, it remains the best way to measure infectivity. In this generally well written article, Cacciabue et. al. present a new semi-automated image analysis approach for the quantification of viral plaques. Written as a plugin for the commonly used image analysis software Fiji (ImageJ), the method will likely be of use to virology researchers.
Experimental design: The figures outline the design of ViralPlaque and validate it by comparison to traditional manual quantification methods. In this way, the authors provide a reasonable demonstration of ViralPlaque’s ability to make reliable measurements. However, there is focus on quantifying the size of plaques; a reliable completely automated method of counting the number of plaques would also be of use to researchers.
Validity of the findings: I was able to download and run the plugin and some example data provided on the author’s source forge site. Having watched the supplementary videos it was easy to start taking measurements. I also downloaded an assortment of images of different plaque assays from google images and was able to perform some reasonable quantification of these. Therefore, I am satisfied that the approach is working as the authors suggest.
Additional comments: 1. The approach seemed to be very dependent on users manually thresholding the image. This limits its ability to perform automated image analysis. If the macro could also perform some kind of background subtraction to create a homogenous background this may improve the approach.

2. I’m not sure sufficient detail is provided to allow users to use the WEKA method - maybe some notes on training the WEKA segmentation on different data?

3. Whilst running the macro multiple times I seemed to accumulate objects in the ROI manager, the authors might want to include a line of code to clear the ROI manager between runs?

4. Figure 2 multiple incorrect spellings of ‘conversion’.


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: VIRALPLAQUE: A FIJI MACRO FOR AUTOMATED ASSESSMENT OF VIRAL PLAQUE STATISTICS
Review round: 2
Reviewer: 1

Output:
Basic reporting: As image recognition techniques, this research really make sense.
Experimental design: No comments
Validity of the findings: No comments
Additional comments: The authors have responded to almost all of my concerns. They added that they used the classical plaque method firstly described by Dulbecco in 1952. This classical method has been developed for many years since its repeatability is not very high due to its complicated operation. In some experiments, overlay media have been changed into methyl cellulose or Avicel. Yin et al have used the improved method to produce viral plaques in 96-well plates (PMID: 25515071). They also further developed this improved method to screen out viral inhibitors from a compound library (PMID: 30900754). Their ultimate goal is to develop a high throughput antiviral drug system based on plaque inhibitory tests. Candidate inhibitors will also undergo concentration gradient plaque experiments. These attempts will generate large amounts of image data, which really need your ViralPlaque macro. Therefore，in your manuscript, a highly repeatable method as well as huge data production will substantiate the importance of your research. At least, these method developments and their new uses should be mentioned in your discussion section.


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: CHARACTERIZING GENE TREE CONFLICT IN PLASTOME-INFERRED PHYLOGENIES
Review round: 1
Reviewer: 1

Output:
Basic reporting: Walker and Walker-Hale et al. characterized various aspects of gene tree conflicts among plastid coding genes, highlighting the phylogenetic utility of long genes, especially rpoC2. The study is carefully designed, analyses are thorough, and conclusions are reasonable. The figures are informative and easy to understand. The manuscript is well-written in unambiguous language.
In the introduction, the authors summarized biotic and abiotic sources of conflicting phylogenetic signals in molecular datasets, including a paragraph reviewing plastid gene tree conflict due to biparental inheritance and recombination (line 65-83). Yet this study is focused on investigating the abiotic origins of gene tree conflicts. As concluded in the study, analytical error is the primary source of observed gene tree conflicts and the authors do not specifically test for hypothesis of horizontal processes, except that some strongly conflicting genes suggest such possibilities. As a result, these contents should be greatly reduced in the introduction or moved to discussion where it is more appropriate to be presented.
In addition, the authors should also make the reference format consistent throughout. For example, line 680-681: the publication year should follow author names and ‘Algorithms for molecular biology’ should be capitalized. In many cases, the publication year are in parenthesis, which is not consistent with PeerJ standard reference format.
Experimental design: Gene tree conflict among organelle genes is a well-recognized phenomenon. The authors examined how well phylogenetic conflict can be predicted by tree length, sequence length, and data type. Such conflict is measured by comparing phylogenies to two reference trees—tree AT from Soltis et al. (2011) and tree GT from Gitzendanner et al. (2018). The GT tree is built entirely on plastome and eight out of the 17 markers used in the AT tree are also plastid. Using these two trees as reference tree may potentially cause some bias in assessing conflicting phylogenetic signals since we expect plastid phylogenies recovered in this study to match these two trees. The ‘good’ genes identified in this study may be the genes that contain strong phylogenetic signal and drive the AT or GT trees rather than the genes that reflect the true evolutionary history (as discussed in line 544-550). In addition, the AT and GT trees are built using concatenation method without dealing with the issues of ‘strongly conflicting’ signals among plastid genes as identified in this study. I think the authors should at least include in discussion how do the reference trees compared to recent multi-locus nuclear phylogeny based on coalescent methods (e.g., Wickett et al. 2014 and nuclear species tree from the oneKP project deposited at github https://github.com/smirarab/1kp).

The phylogeny is stratified in order to assess phylogenetic utility at both deep and shallow levels. Yet they seem to be only used for visualization purposes in the figures. When discussing the performance of individual plastid genes (line 383-396), each individual gene was assessed based on overall concordance across the entire tree and all evolutionary depths. Such way of summarization cannot fully reflect the benefit of stratified analysis. Thus, in addition to the current analyses, I suggest the authors assessing the performance of genes as well as different genomic regions (LSC, SSC, and IR) separately in the five time windows to reveal any more detailed patterns. It is also surprising that IR does not perform as well as the other two genomic regions at deep phylogenetic scales. This may be a potential bias because the authors used the number of concordant/discordant genes. This result can be influenced by the length and number of genes in that genomic regions especially for smaller genomic regions as SSC and IR. The utility of genomic regions may be better assessed as proportion of concordant sites ranger than genes.
Validity of the findings: The conclusions are generally well supported by the evidence provided in the study. The authors particularly highlighted the highly informative gene rpoC2 as a marker for phylogenetic reconstruction. In line 119-122, the authors suggested that analyses that relies on branch length should also benefit from rpoC2 for accurate topology estimation. I don’t think the author should make such suggestions because accurate branch length estimation is much harder than getting the topology right. Whether rpoC2 yield a more accurate branch length is not tested in the study, especially compared to a concatenated matrix of various genes.
Additional comments: Minor comments
1.	Line 50-51: Another nice paper to cite here: Li, Hong-Tao, et al. "Origin of angiosperms and the puzzle of the Jurassic gap." Nature plants (2019): 1.
2.	Figure 1: There is a mismatch between what red and green represent in the caption verses in the legend.
3.	Figure 2: The legend should be ‘75%, 50%, 25%’ instead of ‘75%, 50%, 50%’. Same error should also be corrected in Fig S2. Please also adjust the position of label ‘rbcL’ on the upper right circle in Fig 2 to avoid confusion.
4.	Line 374: Appendix S16 only contains performance of LSC, SSC, and IR region without considering bootstrap support? Please revise the table or the title of the table accordingly.


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: CHARACTERIZING GENE TREE CONFLICT IN PLASTOME-INFERRED PHYLOGENIES
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1.	Line 82: ‘vast majority of studies assume no …. (Wolfe and Rand 2004).’ Is this a review of the studies assuming no conflict among gene trees? If not maybe rephrase this sentence or add in more citations
2.	Line 86: Burleigh and Mathews 2007a,b seem to be same citation but slightly different titles?
3.	Line 141: ‘using plastomes have excluding non-coding data.’ I think it should be excluded not excluding.
4.	Line 164: ‘we refer to this is our ‘accepted tree’, …’ Change is to as.
5.	Line 252: ‘the gene was not 1/3rd the length of the amino acid’. I think this could use some rephrasing, it reads like a double negative and a little confusing to understand on the first read.
6.	Line 305: ‘The WAG model was not found to be the optimal amino acid for any gene in the analysis,…’ I think the word model is missing after amino acid?
7.	Line 329: ‘This pattern is similar to that seen in nuclear phylogenomic datasets (Smith et al., 2015)’. I think the use of a single reference here is a little misleading given the general phrasing of the sentence. There are a lot of other studies that assess gene tree conflict in nuclear datasets that could be cited here, and I would suggest adding more in if appropriate. Is this a review paper that goes over several examples of gene tree conflict in nuclear phylogenomic datasets or is this the only paper specific to angiosperm gene tree conflict?
8.	Line 349: ‘We found that a wide range of evolutionary models best fit our data—rather than just a single model for the entire concatenated set of genes’. It is unclear what the authors used to determine best fitting models for their dataset in this instance.
9.	Line 374: ‘with both outperforming the IR whether considering bootstrap or not’ I think the word support is missing after bootstrap
10.	Line 378,389,392: missing commas in base pair lengths
11.	Line 390: ‘In this study, the alignment length of ycf1 was 21,696 bp’ but mentioned in previous sentence that another study found it was ~5,400 bp. Unclear what the discrepancy in length is here.
12.	Line 442: ‘Positive relationships between alignment length, tree length and concordance also suggest that stochastic error is at play, because shorter or slower evolving genes harbor less signal to accurately infer relationships.’ But the authors also find that genes that lead to the most concordant trees with the reference are not always the longest, which might suggest systematic error is at play as well. I think that the discussion weighs heavily on the impact of stochastic error on gene tree conflict but only briefly touches on systematic error (and effectively ruling it out in Line 468-472, and the statement in the conclusion about longer genes being superior (line 654-655)). I don’t think from the results that systematic error is convincingly ruled out (see above comments on model performance in the experimental design section), so I would like to see this argument against systematic error toned down a little/caveated a bit more throughout the discussion.
13.	Line 524: ‘Unlike in that study, there are reasons to believe that biological gene tree conflict could result in the chloroplast genome…’ Given the broader nature of the reader base, I would suggest talking in a little more detail about what some of the expected biological conflicts between chloroplast gene tree could be.
14.	Figure 1: I think it is a little confusing to have green represent two things on one graph (and when zoomed out, green branch looks too similar to black branches on the tree). So I would suggest changing the amino acid alignment color from green to something else.
15.	Figure 3: I think some lines pointing from the histogram bar to the relevant gene would be helpful, since the spacing is tight between some of the gene names/ positioning on the histogram, so it is hard to tell what value is relevant for gene.
16.	Table 1: It is unclear to me why some many decimal place values are needed in the likelihood scores. I would suggest rounding up to make the numbers more visually palatable.
Experimental design: 1.	Model influence assessment
I don’t think that rerunning the analysis with only one other model, and only for the amino acid dataset from what I can tell is a particularly strong assessment of model performance/influence (which is still an admittedly hard thing to assess), particularly given the conclusion later on in the paper that the model choice/systematic error isn’t influential in the gene tree conflict described here. Without directly attempting to assess model performance (e.g. Bayesian posterior predictive assessments, some proposed ones in Brown 2014 Detection of implausible phylogenetic inferences using posterior predictive assessment of model fit, Systematic Biology), it would be difficult to say that because two analyses with different models produced the same tree, there has been little to no influence of systematic error, both models could have common assumptions that are violated by the datasets and produce the same wrong tree. I think it would be interesting to more directly test for model performance, but will acknowledge that it can be a computationally demanding task, so would leave that choice to the authors (could also assess influence of alignment method this way as well, which is less computationally demanding than posterior predictive statistics that require simulating posterior distributions of trees). But if they decide not to assess model performance more directly, I would like to see the arguments of minimal influence of systematic error and model influence on the gene tree conflict caveated more in the discussion than it is currently.
2.	Model choice
Line 139-141: ‘We did not use non-coding regions; as proper orthology at deep time scales can be difficult to assess due to gene re-arrangements and inversions; this is in part why most deep phylogenetic analyses of angiosperms using plastomes have excluding non-coding data.’
So does this mean that non-coding data was excluded from the nucleotide dataset as well in this study? Was there any model choice analyses applied to the data in which GTR+G was selected as the best fitting model from? I think it would be important to carefully discuss the choice of these models for this dataset. I would think that using coding information only will emphasize the heterogeneity in rates of evolution between regions and/or lineages within the dataset and accommodating more heterogeneity (e.g. site-specific partitioning; a CAT model, etc) into modeling might be more suitable/appropriate for phylogenetic inferences, so I think readers concerned about model performance with the dataset would appreciate careful discussion of model choices, as well as the choices to use the same model across all gene regions despite what I think is an acknowledgment that there seems to a range of models that fit across the data set rather than a single one (line 349)?

3.		Gene tree conflict assessment
I think it would be a nice addition to the study to characterize just how much conflict exists among individual genes about the angiosperm relationships directly. There are a lot of sentences that mention conflict is prevalent (e.g. Line 422: ‘We would also expect all plastid genes to show similar patterns of conflict when compared to non-plastid inferred phylogenies’), but I don’t think it is ever directly assessed this in the dataset. One way I think it would be easy to do so would be to do some pairwise comparison of concordance among the gene trees themselves, rather than just to the reference tree. It might also be interesting to see if the longer genes agree with each other, in addition to the comparison of whether they agree with the reference trees that is already presented in this study. It might be able to give some assessment of accuracy that independent of assuming that the reference tree is correct about the relationships.

4.	Reference/plastome tree methodology
I think the authors should explain in a bit more detail about why they chose to use a concatenation method when inferring plastome-wide trees, especially since it seems like in the discussion they warn readers away from doing that. Why not make a ‘plastome’ tree using a species tree method given that there is a lot of gene tree variation going on in the data set, of which the species tree methods attempt to control more for than concatenation methods? Were concatenation methods used to match the current angiosperm phylogenies because the reference trees pulled from other studies were inferred from concatenation methods or as an example of common methods used in studies aiming to determine relationships among angiosperms to highlight the problems with using them when lots of gene tree variation exists in a dataset? I think a little more detail on the choice of phylogenetic inference methodologies used in this study will provide some need clarity.
5.	Choice of Binomial regression
I think the section explaining the generalized linear model is a little confusing and could use some clarity. For example, it is a little unclear to me what format the response variable (concordance) was in, what it actually binomial (concordant vs not over individual nodes) or proportional (percent concordance across entire tree), and was the model run per gene or across all genes? If it was the proportional data, is the quasibinomial the best family to use? Why not negative binomial, which is also used to account for overdispersion but is more traditionally used with proportional data? If it was actually binomial data, was a model for each individual gene and if so, where are these reported? If one model was run over all the genes with binominal data, is gene incorporated as a random effect in the model? I also wonder if the authors used the binomial data and could treat gene as a random effect, would they be able see more subtle patterns by being able to add gene as a random factor to help account for variance that might not be explained by the chosen predictors (e.g. alignment length). I will admit that I have not played around with these particular models with binary data myself all that much, so please take the latter comments about using the binomial data itself as mere suggestion of something to look into and not strong request. But either way, I think the description of this methodology could use some further clarity.
Negative binomial (distributions similar to poison
6.	Line 183: Why the choice of 70% bootstrap support for moderate support nodes? Is not 50% is a more common cutoff (e.g. majority rule consensus trees)? Or wouldn’t we want bootstrap support to be near 100% for nodes that are informative about relationships? I would suggest including a sentence with some justification for bootstrap cut off values for nodes that are informative about relationships vs not informative
7.	Line 205 says the authors calculated a set of summary statistics for each gene, including the number of included species, but it is unclear to why some species would be included or excluded for a particular gene.
8.	Line 234 ‘Investigation of model fits on full datasets revealed….’ It is unclear what is being used to investigate model fit on full datasets in this context, it this assessment significance with logistic regression/quasibinomial regression?
9.	Line 247: ‘We also performed saturation analyses…’ I am not sure the average reader will be familiar with this analysis so I would suggest explaining what it is and what it can tell you in a little more detail.
10.	Line 265: “as described above for each of the ‘plastome’ analyses”. This wording is a little vague and it’s unclear to me what the plastome analyses was. I would suggest rephrasing this sentence for clarity.
11.	Line 314: ‘Quasibinomial regression results for amino acids aligned with FSA and cleaned for 30% minimum occupancy were not significant for all predictors, but this model had an abnormally high estimated dispersion, suggesting this finding to be in error’. I think this sentence could use a little more clarity, it is unclear what the error is here, the alternative alignment method tests having no influence on the results?
Validity of the findings: 1.	Line 106: ‘Our results show that the plastome itself tends to perform well at all levels; however, it contains notable gene tree conflict.’ Is it performing well if there is notable gene tree conflict? I would suggest clarifying what is meant by the plastome and good performance?
2.	Line 107-109: ‘The number of conflicting genes at each node is often comparable to the number of concordant genes; however, the majority of plastid genes are uninformative for most nodes when considering support and thus unlikely to positively mislead studies.’ (Also something similar is mentioned again on lines 641-643). I think that it may be a little more nuanced than this and worthy of stating in more detail in this discussion. While any one gene region that is uninformative on its own about phylogenetic history won’t mislead a study (because it doesn’t have much to say in the first place), it is possible that a study using a large set of genes to infer a phylogeny, one with a lot of less informative regions and only a few high-information regions can be really sensitive to things like poor model performance/systematic error for those few highly informative regions and end up with strong support for an incorrect phylogeny, especially when using methods that don’t try to account for variation in gene tree information content (like concatenation methods). I think that this is a way in which having a dataset with lots of less informative regions in it could mislead studies and a reason why there has recently been more effort to explore how information content is distributed across genes in our phylogenomic datasets. This is explored more in a few studies (e.g. Thomson and Brown 2017 Bayes factors unmask highly variable information content, bias, and extreme influence in phylogenomic analyses, Systematic Biology and Shen et al 2017 Contentious relationships in phylogenetic studies can be driven by a handful of genes, Nature Ecology and Evolution)  and I would suggest maybe discussing this area in more depth when talking about the impact of lots of less informative regions in phylogenomic datasets, perhaps in the discussion.
3.	Lines 112-115: ‘However, some nodes across angiosperm phylogeny show at least several strongly supported conflicting genes, this rare event indicates an area for future exploration, as it provides evidence that the chloroplast is potentially affected by a biological source of gene-tree conflict …’ Could it not also be stemming from systematic error (e.g. poor model fit)?
4.	Line 119: ‘Although as a whole the chloroplast performs well in topology reconstruction,  ...’ Again, I think it would be helpful to clearly lay out what is meant by performing well. Agreeing with known reference trees?
Additional comments: This is a solid initial investigation into the sources of gene tree conflict potentially underlying strong conflict among angiosperm phylogenies.  I think this paper adds to the growing number of phylogenomic studies that are trying to more rigorously investigate methodological choices that can lead to particular inferences about relationships among organisms. I find the results compelling that there is extensive variation and that the paper has useful considerations and suggestions for researchers interested in inferring a robust angiosperm phylogeny.

The paper presents good evidence that by choosing particular gene regions to minimize the impact of stochastic errors, researchers can produce more robust angiosperm topologies (or at least ones that are more concordant with the available phylogenies used in this study as reference trees). However, I think multiple studies have shown the impact of systematic error in producing strong conflicts that aren’t necessarily related to the level of phylogenetic signal/information content and that the arguments presented in this paper about the minimal influence of systematic error aren’t strongly supported by the analyses/results presented in this study. I realize thorough investigations of model performance are very computationally burdensome and the ability of available test statistics to accurately assessing model performance is not all that clear yet, so a thorough investigation of model performance in this study would be a high bar request for sure, and I am not suggesting that the evidence presented is not any good for leading readers to more robust phylogenetic inference from chloroplast genomes – just that perhaps the strength of claims in some sections of the paper need to be tempered a bit more with caveats.  I would also suggest adding on to the concordance analyses the pairwise comparison of concordance among the gene trees themselves to highlight the extent of gene tree conflict across the plastome and more clarity in the descriptions of several of the methodological choices of this study (listed in more detail in the other 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: CHARACTERIZING GENE TREE CONFLICT IN PLASTOME-INFERRED PHYLOGENIES
Review round: 1
Reviewer: 3

Output:
Basic reporting: Very well done. I find no noticeable issues, so "no comment".
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: Walter et al. have presented a very nice study demonstrating variation in phylogenetic signal across different genes in the plastome. I cannot see any issues with the paper from a technical or presentation standpoint. I very much appreciate that the authors state things very matter-of-the-factly and do not push the capacity of their findings. As a person that does a lot of whole chloroplast sequencing, this will make me consider the different impacts of portions of my alignments on my final phylogeny, especially when doing divergence time estimation.

I found a few minor grammatical/content issues:

Line: 61-62: Plastomes are not always inherited paternally in gymnosperms. It depends on the species.

Line 135: “utilized” should be used”.

Line 263: The literature usually refers to these as Large Single Copy and Small Single Copy, though there are instances of Long and Short.

Line 299: Give RAxML version.

Line 300: “Used” not “utilized”.


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: CHARACTERIZING GENE TREE CONFLICT IN PLASTOME-INFERRED PHYLOGENIES
Review round: 2
Reviewer: 1

Output:
Basic reporting: All comments have been appropriately addressed.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: Good job!


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: CHARACTERIZING GENE TREE CONFLICT IN PLASTOME-INFERRED PHYLOGENIES
Review round: 2
Reviewer: 2

Output:
Basic reporting: I am satisfied with the corrections made by the authors in this revised manuscript!
Experimental design: The added explanations of methodological choices in this revised version improved the clarity in the manuscript.
Validity of the findings: No comment
Additional comments: I think the authors did a good job taking all three reviewers & AE's comments into consideration and the resulting manuscript is much improved.


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

Input:
Title: PAENIBACILLUS STRAINS WITH NITROGEN FIXATION AND MULTIPLE BENEFICIAL PROPERTIES FOR PROMOTING PLANT GROWTH
Review round: 1
Reviewer: 1

Output:
Basic reporting: The English language should be improved throughout the manuscript to improve overall clarity. The current phrasing makes comprehension difficult at many places like line 52, 61-62, 76, 79, 144, 149, 151, 156-159, 172, 174, 212-214, 263 etc.)

Do not repeat the methodology or provide background information in the ‘Results’ section (for example, line 172-174, 227-232). Please revise it extensively.

List names of all bacterial species in Table 1 for better understanding and clarity.

Please see the comments on the annotated copy for further details.
Experimental design: Discuss the main research questions that you wanted to address in this study at the end of the Introduction section.

In my opinion, the Introduction section is too short and enough background and rationale behind this study are not provided.

Details about the sampling sites and plant type are missing in the Materials & Methods section. Plant-type has a direct influence on the rhizospheric bacterial population so brief information about the plants is crucial to provide. The authors can refer to Table 1 here.

Overall in the ‘Materials & Methods’ section authors should thoroughly revise and provide enough details to replicate the procedures. In its current state, the information is ambiguous. Please see the comments on the annotated copy for more details.

Line 194: It is not clear, how all strains can be identified as Paenibacillus? Were the authors specifically targeting Paenibacillus genus in this study? This again links back to the lack of research questions and hypotheses for this study.
Validity of the findings: Some of the information in the Results section is difficult to understand, partly because it cannot be linked to the questions that the authors were trying to answer in this study and partly due to lack of details in the Materials and Methods section or some random new information. Please see the comments on the annotated copy for more details.

Most of the information in the Discussion section is just repetition. Please proof-read it and thoroughly modify it.
Additional comments: Overall, this study highlights the various benefits that strains of Paenibacillus genus can provide to plants. There was significant amount of data generated in this study, however, many pieces of the information are missing in this manuscript in its current state, which reduces its scientific relevance.


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: PAENIBACILLUS STRAINS WITH NITROGEN FIXATION AND MULTIPLE BENEFICIAL PROPERTIES FOR PROMOTING PLANT GROWTH
Review round: 1
Reviewer: 2

Output:
Basic reporting: The Manuscript Number #35859: "Isolation, identification and characterization of Paenibacillus strains with potentials for biofertilizer" is a good research paper for promoting sustainable agriculture by using Paenibacillus strain as potential Nitrogen biofertilizer for vegetable production and an alternative of N2 fertilizer. Authors attempt to screen the nifH gene in isolated strains and also checked its nitrogenase activity along with antagonistic activity with the most common fungal pathogen for selection of efficient biofertilizer. However, the authors should carefully examine the whole manuscript and improve as per the following minor comments:

General comments:

1.	Authors should include some recent references in the introduction part and also discuss the nifH gene mechanism in plant growth promotion section.
2.	Authors should add some information regarding the recent scenario in nitrogen chemical fertilizer usage nationally and globally. And also, how it plays a role in environmental disturbance and loss of soil health.
3.	The authors only tested the effect of biofertilizer on plant growth without any comparative pot trials with nitrogen chemical fertilizer amendment. How the authors justify this when the objective of the study is to provide efficient nitrogen biofertilizer under sustainable agriculture practices.
4.	Authors should briefly explain how the nifH gene plays a role in nitrogen fixation in discussion section with nitrogenase activity in identified strains.
5.	Authors should justify why they selected Wheat, Cucumber and Tomato as test plants during pot trials under this study.
6.	Authors should include pot trials figures/images that showed the effect of biofertilizer.
7.	If possible, author should give the GPS location of soil sample collection.
8.	Authors should mention the significant difference on mean data value of Figure, 2, 3 and table 1 by different alphabet like. a, b, c,d, e………... so this more better for understanding between difference treatment combination.
9.	 Authors should mention the future challenges about microbial consortium or biofertilizers for agricultural production at farmers fields.
In last this can be accepted after minor revision.
Experimental design: ok.
Validity of the findings: good
Additional comments: The Manuscript Number #35859: "Isolation, identification and characterization of Paenibacillus strains with potentials for biofertilizer" is a good research paper for promoting sustainable agriculture by using Paenibacillus strain as potential Nitrogen biofertilizer for vegetable production and an alternative of N2 fertilizer. Authors attempt to screen the nifH gene in isolated strains and also checked its nitrogenase activity along with antagonistic activity with the most common fungal pathogen for selection of efficient biofertilizer. However, the authors should carefully examine the whole manuscript and improve as per the following minor comments:

General comments:

1.	Authors should include some recent references in the introduction part and also discuss the nifH gene mechanism in plant growth promotion section.
2.	Authors should add some information regarding the recent scenario in nitrogen chemical fertilizer usage nationally and globally. And also, how it plays a role in environmental disturbance and loss of soil health.
3.	The authors only tested the effect of biofertilizer on plant growth without any comparative pot trials with nitrogen chemical fertilizer amendment. How the authors justify this when the objective of the study is to provide efficient nitrogen biofertilizer under sustainable agriculture practices.
4.	Authors should briefly explain how the nifH gene plays a role in nitrogen fixation in discussion section with nitrogenase activity in identified strains.
5.	Authors should justify why they selected Wheat, Cucumber and Tomato as test plants during pot trials under this study.
6.	Authors should include pot trials figures/images that showed the effect of biofertilizer.
7.	If possible, author should give the GPS location of soil sample collection.
8.	Authors should mention the significant difference on mean data value of Figure, 2, 3 and table 1 by different alphabet like. a, b, c,d, e………... so this more better for understanding between difference treatment combination.
9.	 Authors should mention the future challenges about microbial consortium or biofertilizers for agricultural production at farmers fields.
In last this can be accepted after minor revision.


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

Input:
Title: PAENIBACILLUS STRAINS WITH NITROGEN FIXATION AND MULTIPLE BENEFICIAL PROPERTIES FOR PROMOTING PLANT GROWTH
Review round: 1
Reviewer: 3

Output:
Basic reporting: Reviewers comments to author
Authors should answer following questions  in the paper
1.	Line no 1, Authors can slightly modify the title of the paper “Isolation, identification and characterization of Paenibacillus strains with potentials for biofertilizer” for making it attractive.
2.	Again in the abstract line no 29 plant hormones production induced systemic resistance or nutrient mobilization. Instead of or it should be induced systemic resistance and  nutrient mobilization
3.	line no 32. Again in the abstract “fourth-fifth of them” word is not correct. This can be replaced with percentage or any number of microbes. Sentence should be reframed.
4.	in the abstract “In this study, twenty-five Paenibacillus bacteria were isolated from plants rhizosphere and identified based on their 16S rRNA”. Sentence is unclear should be reframed
5.	Abstract have many grammatical errors, many sentences are unclear and need to be reframed.
6.	In line no 40 word “provoked” should be replaced.
7.	Line no 68 plant pathogens should be replaced by plant pathogens.
8.	In line no 74 under the heading material and methods, Sample collection isolated, procedures and culture conditions  needs to be reframed.
9.	Line no 197 sentence is unclear
10.	In line no 162 the word reaped should be replaced by harvested.
11.	 In line no 163 Lengths is not correct.
12.	 Paper has many unclear sentences, grammatical errors and spelling mistakes which need to be corrected
Experimental design: Yes It is under scope of the journal.
Yes research question is well defined. although its not entirely novel work.
Methods are sufficiently described.
Validity of the findings: Findings are expected by using the strain, most of the research are going in the same direction.
Data is good and sound.
Conclusions are well stated.
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: PAENIBACILLUS STRAINS WITH NITROGEN FIXATION AND MULTIPLE BENEFICIAL PROPERTIES FOR PROMOTING PLANT GROWTH
Review round: 2
Reviewer: 1

Output:
Basic reporting: Authors has been revised and modified manuscript as per my comments. they have mentioned all red color text in revised manuscript. now this can be accepted for publication.
Experimental design: this is ok.
Validity of the findings: ok.
Additional comments: Authors has been revised and modified manuscript as per my comments. they have mentioned all red color text in revised manuscript. now this 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: EVALUATION OF THE EFFECTS OF THE GREEN NANOPARTICLES ZINC OXIDE ON MONOSODIUM GLUTAMATE-INDUCED TOXICITY IN THE BRAIN OF RATS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The general outline  is satisfactory although the manuscript would benefit from English language editing
Experimental design: 1. Original primary research is within Aim and Scope of the journal
2. The Research question is not clearly defined and would require refining
3. The investigations were performed to a satisfactory technical and ethical standard
4. Methods were not described in sufficient detail to allow for replication
Validity of the findings: There is some novelty in the study although the authors have not clearly defined this.
All underlying data have been provide, the images do not represent region studied
The conclusions are well stated although  the inferences drawn are not supported by the results
Additional comments: Topic:  is it  ……Zinc Oxide Green Nanoparticles….. or …..Zinc Oxide Green Tea Nanoparticles…..?
Abtract:  alteration not alternation. Cortices not cortexes. Duration of administration of msg was not mentioned. methods  and results are being mixed together. What is MSG-HD?
Introduction: Line 35 ‘because they are safe’ safe for what?
Line 35-39 should be revised for clarity of language
Line 46- is this work about evaluating the toxicity of ZnO NPs? if not, then why this line of thought?
Line 51-60- please revise for clarity
Line 68-70- needs English language editing
Line 79-84- the purpose of this paragraph is not clear
Line 85-92- How the contents relate to the present study is not clear
Line 93- This statement is UNTRUE

Animals and ethical considerations
Rats were chosen as the most appropriate animal species for the
experiment……….. Why?
The sample size was chosen based on the lowest number of animals to get actual
results………..What does this statement mean?
What is the basis for the doses of green tea and MSG used in this study?
Estimation of some bioactive compounds as markers for neuropathic pain….. This heading should be changed because what is being measured here is not neuropathic pain
Histopathological slides
There are inconsistencies in the areas of the brain from where the photomicrographs were taken with some reflecting the cortex (slides c, d and g) and a few reflecting the cerebellum (slide a) hippocampus (slide b) or deeper structures (slide f). Also, there is inconsistency of magnification. These problems make the claims regarding morphological changes unverifiable.

Discussion
Line 254….. Microglial actuation produces high amounts of free radicals that can harm proteins, lipids, and nucleic acids at the site of particle deposition ……Please explain the appropriateness of this sentence for this section.

Line 277….. Dopamine and catecholamine are neurotransmitters that occur abundantly in the CNS…….Do the authors consider catecholamine a distinct neurotransmitter?


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: EVALUATION OF THE EFFECTS OF THE GREEN NANOPARTICLES ZINC OXIDE ON MONOSODIUM GLUTAMATE-INDUCED TOXICITY IN THE BRAIN OF RATS
Review round: 1
Reviewer: 2

Output:
Basic reporting: It's good to publish.
Experimental design: It's good to publish.
Validity of the findings: It's good to publish.
Additional comments: It's good to publish.


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: EVALUATION OF THE EFFECTS OF THE GREEN NANOPARTICLES ZINC OXIDE ON MONOSODIUM GLUTAMATE-INDUCED TOXICITY IN THE BRAIN OF RATS
Review round: 1
Reviewer: 3

Output:
Basic reporting: -	This article is well written. The level of English is of high quality. Nevertheless, there are some few grammatical and typing mistakes. see lines 21;38
-	The literature cited in this work are relevant to the study and the topic.
The authors should improve in the way some references are written in the references to avoid disparities. See lines 399;406;408 and 430
-	The authors provided raw data of the study in a good format and the figures and tables of the work are of high quality. The figures and tables depict resulst that are relevant to the hypotheses.
Experimental design: -	The work present is an original research article within the aims and scope of Peer J.
-	This research is innovative it gives supplementary information about the pathways involved in monosodium glutamate toxicity. The authors should improve the problem statement. See lines 80-44 and line 93.
-	The methodology use in this study is appropriate, an ethical clearance for the use of the animal in laboratory studies was obtained. The author describes the protocol the used in a manner that can be replicable.
Validity of the findings: -	The author feet this work in the previous works of their laboratory team and also they added additional information about the toxicity of MSG and the possible beneficial effect of natural products against MSG induced neurotoxicity and brain damages.
-	The statistical analysis done hear is appropriate to this type of comparative effect of natural /synthetics products in vivo.
Additional comments: The work presented in this article is of great interest and give additional information about MSG mediated neurotoxicity. Nevertheless, we suggest that the authors your proofread the manuscript in order to reduce the levels of abbreviations, grammatical/typing errors. In the introduction the authors should also clearly state the rationale of the study.


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

Input:
Title: EVALUATION OF THE EFFECTS OF THE GREEN NANOPARTICLES ZINC OXIDE ON MONOSODIUM GLUTAMATE-INDUCED TOXICITY IN THE BRAIN OF RATS
Review round: 2
Reviewer: 1

Output:
Basic reporting: I am now satisfied.
Experimental design: Required corrections are now done
Validity of the findings: Satisfactory
Additional comments: None


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: EVALUATION OF THE EFFECTS OF THE GREEN NANOPARTICLES ZINC OXIDE ON MONOSODIUM GLUTAMATE-INDUCED TOXICITY IN THE BRAIN OF RATS
Review round: 2
Reviewer: 2

Output:
Basic reporting: The article has benefited from English language editing and the final document is well written.
The literature cited in this work are relevant to the study and raw data of the study is in an appropriate format.
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: CALIFORNIA AIR RESOURCES BOARD FOREST CARBON PROTOCOL INVALIDATES OFFSETS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Line 83, "Deforestation continues at a rate..." for this sentence, read this paper: "High-Resolution Global Maps of 21st-Century Forest Cover Change" by Hansen et al 2013

Line 109, was this forest survey the FIA dataset? I thought trees were surveyed more often than six years.

Line 112, is there any studies evaluating the limitation/uncertainty of CARB? Since this is the review paper, it would be good to mention other existing papers addressing the similar questions and what values of this paper adding to it. Or is this paper the only study reporting the uncertainty?

Line 126, yes, NEE is kind the same as NEP, but NEP = -NEE

The reference format (both in-text citation and reference list) is not correct, please check "Instructions to authors"

Figure 3, if there is no negative values, why extending y axis to below zero?
Experimental design: Following the "Instructions to authors" on Literature Review format, The section of "4.0 Survey and Analysis Methods" should be moved up to the second portion.

And there is another section- "Statistical Methods"-appears after "Acknowledgement" in the end. Should this be moved up to the "Survey and Analysis Methods" or be inserted in the Appendix?

Line 667, authors should change the subheading "data sources"to another because below authors have "CARB-CAR Data Sources" and "NEE Data Sources", so this is a summary of all the data sources?

Line 698-699, it might not be necessary to list all the 30 numbers out.

Line 741, were listing those nine columns here necessary? were those all useful for readers to understand the paper?

Line 803, extra indent, not necessary.

Line 812, there are lots of other papers reporting the DBH and biometric equation uncertainty, not just one paper from tropical forest cited here. If authors want to summarize the magnitude of uncertainty, it is better to add more references. Some others are:
Paré, D., G.Z. Gertner, P.Y. Bernier, and R.D. Yanai. 2016. Quantifying uncertainty in forest measurements and models: Approaches and applications.Canadian Journal of Forest Research 46(3): v. 10.1139/cjfr-2016- 0029
Holdaway, Robert J., Stephen J. McNeill, Norman W. H. Mason, and Fiona E. Carswell. 2014. Propagating Uncertainty in Plot-based Estimates of Forest Carbon Stock and Carbon Stock Change. Ecosystems in press. DOI: 10.1007/s10021-014-9749-5
Or if you go to this Ruth Yanai's website "http://quantifyinguncertainty.org/", click "Bibliography-Biomass Uncertainty", there is more.

Line 830-831, this sentence is confusing, "59 sites covering 540 sites..?" Or do authors mean 59 towers?
Validity of the findings: From line 160-192, the first two paragraphs in the "2.Results" are all Methods. I did not see any sentences describing the results here. Just about what methods/steps authors have conducted and in what tables/graphs.

Line 193-197, those sentences are describing the box plot legend, can be inserted in the figure caption, but not here. And same thing for Line 236-239

Line 209, delete extra space

Line 273, why p values being differed among years? were they discussed in the Discussion section?

Line 373, this is an important point. It might be better for authors to add some references reporting estimated soil carbon pool and see how it looks if being added to CARB-CAR.

Line 381, it is also important to discuss some limitations on upscaling eddy fluxes tower data.

Line 429, should part of this section be moved up to the Result section? as a special case for comparing CARB-CAR with NEE1. Because the the limitations discussed above this paragraph-"Protocol Process" and "Soil Carbon Exclusion' are also applied here.

Line 475, any ideas about why the forest survey had larger AGB than CARB-CAR estimation?

Line 580, this is good point. Right now most of the eddy flux tower are run for academic research and supported by NEON. In California, most of the tower are in southern region (dry) but not in northern region (wet), causing a bias if upscaling the results to the whole state.
Additional comments: This paper reviews forest carbon protocols and offsets issued by CARB-CAR, and compared with measurements of annual net ecosystem exchange from eddy flux towers. It reports the large variation of estimated forest carbon within each protocol, and a 5 times differences in annual value comparing the two protocols. This paper adds a great value, connecting economy to academia. It also adds great thoughts on the current application of those protocols.


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: CALIFORNIA AIR RESOURCES BOARD FOREST CARBON PROTOCOL INVALIDATES OFFSETS
Review round: 1
Reviewer: 2

Output:
Basic reporting: see attachment
Experimental design: see attachment
Validity of the findings: see attachment
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: CALIFORNIA AIR RESOURCES BOARD FOREST CARBON PROTOCOL INVALIDATES OFFSETS
Review round: 1
Reviewer: 3

Output:
Basic reporting: Review of ‘’California air resources board forest carbon protocol invalidates offsets”

	Carbon markets remain largely a panacea for contending with climate change, though some nations and states (such as California) are entering into cap and trade agreements. Projects that are designed to defer deforestation or regain C through forest management underlie many of the schemes.

	This manuscript looks at projects adopted under the California Air Resources Board. I do not confess to understand how these projects are designed or managed to receive C credits, though the authors here outline the forest measurements and simulation models. The authors of this manuscript are concerned that these projections may be flawed or unrealistic, and thus compare C sequestration rates from the CARB protocols to those measured more accurately through net ecosystem CO2  exchange with data from the FluxNet network.

	The major problem with this comparison is that the CARB and FluxNet measurements are conducted at different sites, with the exception of Howland Forest, in the NE. Figure 1b, for example, is quite interesting, but we do not know if some unique management is being conducted at the CARB sites to facilitate the larger negative emissions, or if it is calculation error.

	The authors point out that the CARB C credits at Howland show a unrealistically large rate the first year (2008, I think). I wish they would have clearly identified why that occurred. But, after that, the rates drop down to ~ FluxNet values. Thus, I was not clearly given illumination a comparion of this key site.

	It seems the authors are motivated by the reasonable concern about the validity of C credits on any C market. That is a very fair concern. This manuscript doesn’t quite achieve this:

1.	It is incredibly long, and dense. Shed a lot of the verbiage, and focus on the CARB vs FluxNet at Howland as an excellent comparison. Why does CARB give a huge credit at teh beginning? How do they compare over time after that?
2.	Then, if the authors wish, compare CARB project to FluxNet, but compare projects that have similar site history to FluxNet, so that sites recoverying from disturbance or from forest management enhancements are not skewing rates.
3.	The soil C issue seems out of place here. I can understand why CARB just by passes it. It IS embedded to some degree in NEE, which makes the two metrics somewhat different.
4.	I think Supplement 2 overdoes the summation equations, but under-explains the CARB process.

	To summarize, Fig. 3 underscores the legitimate concern of this paper, but more importantly, the concept that C can be stored and traded in natural ecosystems.  Are the estimates realistic? Does it have climatic significance? With clarification, considerable editorial streamlining and reorganization, this manuscript might offer an important reality check for CARB estimates. However, the manuscript as it now stands is not ready for publication, and needs to be reduced from a dissertation to a digestible article-length analysis.
Experimental design: see above
Validity of the findings: see above
Additional comments: none


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: CALIFORNIA AIR RESOURCES BOARD FOREST CARBON PROTOCOL INVALIDATES OFFSETS
Review round: 2
Reviewer: 1

Output:
Basic reporting: None
Experimental design: None
Validity of the findings: None
Additional comments: I really appreciated authors' hardworking on revising this manuscript. I am satisfied with those responses to my 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: CALIFORNIA AIR RESOURCES BOARD FOREST CARBON PROTOCOL INVALIDATES OFFSETS
Review round: 2
Reviewer: 2

Output:
Basic reporting: The manuscript is now clearly written (though it still could be shorter) for a non-specialist to see what the objectives are, and how the data inform the conclusions. I have to say, the entire premise and framework of the CARB program is remarkably shaky and (if this review is correct) untested.
Experimental design: The premise here is to use publically available NEE data for ecosystems as a constraint on the likely range of NEE under CARB C storage projects, to see if CARB estimates of sequestration are realistic. As I mentioned last time, the CARB sites might start off in such a degraded state they may gain more C than the FLUXNET sites. I can't sort this out, and if the CARB has such information (or other sceintists/foresters), then they can reply. This paper at least opens up the dialogue for scientific evaluation of these schemes.

The paper has the benefit of a direct CARB to FLUXNET comparison the east coast, which at least provides some assurance that the approach here is valid. This site appears to support the contention of the authors.
Validity of the findings: Again, its the history of the CARB sites prior to the start of a project, but even so, there are natural limits to NEE.

The whole arena of C markets and natural storage projects is, to me, a strange and hard to understand space. As a C researcher, I dont have the time to wade through all the regulatory or contractal paperwork, and figure out what is going on. I dont know if ecosystem exchange studies can be conducted for every C sequestration project, but I do think more can be done to remove the black magic aspects.
Additional comments: On Figure S7, both in text and in teh figure caption, there are different statements about what the symbols are, adn what they mean. I dont see any gray circles in the figure, and it is not clear what the gray and black boxes are.


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: CHEMICAL PROFILE AND PANCREATIC LIPASE INHIBITORY ACTIVITY OF SINOBAMBUSA TOOTSIK (SIEB.) MAKINO LEAVES
Review round: 1
Reviewer: 1

Output:
Basic reporting: The editors recommend two separate chapters Results and Discussion. Here they have been combined, which I think is right, because in this type of work, which is actually based on analysis and compounds identification, the discussion can not take up much place if the identification is not in doubt.
Experimental design: The work is in the journal's profile. Separation and identification of compounds have been properly planned and performed.
Validity of the findings: This type of research always expands knowledge in the field of etnomedicine. In this case, it is possible to develop new dietary supplements to reduce obesity. The tested extract has much lower effectiveness compared to orlistat, so it should be considered only in terms of a natural functional dietary supplement. That's why, in my opinion, it would be necessary to add some references to the reviews work, e.g.
Possible anti-obesity therapeutics from nature--a review. Yun JW. Phytochemistry. 2010 Oct;71(14-15):1625-41.
Synthetic and Natural Lipase Inhibitors.Bialecka-Florjanczyk E et al. Mini Rev Med Chem. (2018) 18(8):672-683.
Additional comments: The proper  methods were used and the compounds were correctly identified. Measurements of lipase activity indicate the potential practical use of the extract in the treatment of obesity.
That's why I would like to supplement the references  with some other reviews in this field, e.g.
Possible anti-obesity therapeutics from nature--a review. Yun JW. Phytochemistry. 2010 Oct;71(14-15):1625-41.
Synthetic and Natural Lipase Inhibitors.Bialecka-Florjanczyk E et al. Mini Rev Med Chem. (2018) 18(8):672-683.


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: CHEMICAL PROFILE AND PANCREATIC LIPASE INHIBITORY ACTIVITY OF SINOBAMBUSA TOOTSIK (SIEB.) MAKINO LEAVES
Review round: 1
Reviewer: 2

Output:
Basic reporting: - Some more updated references should be included. There are recent papers (see for example Marrelli et al., 2016 and 2018) dealing with natural phenolics having pancreatic lipase inhibitory effects.

- English should be revised
Experimental design: Experiments are generally well designed and well described.

HPLC farce of the extract seems indeed quite simple, while in Table 1, about 23 compounds are reported.  I would suggest to number the identified ones and add their UV spectra in the table. UV is also very important to confirm identity of flavonoids.
Validity of the findings: Authors should include and compare their data with more recent results from other plants which show inhibition of pancreatic lipase.
Additional comments: Sistematic name of plants (genus species, but not Author or variety) should be written in italic: Authors should check for this in all the text.


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: CHEMICAL PROFILE AND PANCREATIC LIPASE INHIBITORY ACTIVITY OF SINOBAMBUSA TOOTSIK (SIEB.) MAKINO LEAVES
Review round: 2
Reviewer: 1

Output:
Basic reporting: Authors have answered to previous comments and manuscript is now acceptable for publication. I have annotated some minor corrections to be done.
Experimental design: See above. Authors have improved the manuscript
Validity of the findings: See above
Additional comments: Manuscropt has been improved and is now acceptable 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: A DRAFT GENOME AND TRANSCRIPTOME OF COMMON MILKWEED (ASCLEPIAS SYRIACA) AS RESOURCES FOR EVOLUTIONARY, ECOLOGICAL, AND MOLECULAR STUDIES IN MILKWEEDS AND APOCYNACEAE
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript presented by the authors reports on a draft genome and transcriptome assembly of the common milkweed (Asclepias syriaca). They also discussed the structure of the genome in comparison to other related species, such as Coffea. I think this is a well performed analysis with clear implications as to why these data should be published and accessible. Overall, I found the paper to be very well written and the need for this work well articulated. I particularly appreciated the comparison of the Asclepias transcriptome with other Apocynacae transcriptomes.
Experimental design: The experimental design detailed by the authors was appropriate for this study. For the most part, the methods and statistical analyses were clear and a well-defined. I think the authors could be a bit more clear regarding the number of individuals sampled and sequenced, as it became a bit confusing at times in their sequencing details (see specific comments in the methods section below). However, in my opinion, this is something that can be easily addressed.
Validity of the findings: Based upon the methods used, the results of the study appear to be valid. The methods and analyses were appropriate. Their conclusions were well thought out and could be directly link to the results presented.
Additional comments: Specific comments:
Line 130: It would be better to give these in terms of final concentrations rather than number of microliters. Alternatively please indicate what total volume was used so that the audience can determine final concentration.

Line 141: “Frozen tissue was sent to GlobalBiologics..”
Is this from the same individual that was collected McDonough Co.?

Line 144: “Purified DNA was provided to the CGRB….”
Again, were all of the libraries of different sizes prepared for the same individual? I am assuming so, but I would just reiterate that because otherwise it is somewhat unclear.

Line 146: “… one of 15 samples pooled on a lane..”
I’m confused as to what these other 15 samples are from.

Line 148: ‘’ one of 3 samples pooled on a lane…’
See comment above.

RNA-seq methods: In my experience plants also contain a large number of ribosomal RNAs. Were these removed prior to library preparation?

Line 180-181: “…. To yield 101 bp single end reads…”
In my experience de novo transcriptome assembly is more efficient when using paired end reads. Why were single-end reads used?

Lines 323: I would suggest writing out Progesterone 5-beta reductase when first introducing it in the methods.

Line 387: Are the RNA oligos used to capture the SNPs listed somewhere?

Line 489: I’m not sure what the authors are referring to when they state…. “This represents a total of 865 BUSCO genes, or 93.6%.”

Line 483: Perhaps it would be a good idea to describe the transcriptome assembly in a bit more detail, such as providing N50 data, etc. I feel as though the information about the transcriptome gets a bit lost in this paragraph.

Lines 505-521: While interesting, it is unclear clear as to how gene death rates, birth rates, and innovation rates were calculated.

Line 580: Change ‘cosely’ to closely.


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 DRAFT GENOME AND TRANSCRIPTOME OF COMMON MILKWEED (ASCLEPIAS SYRIACA) AS RESOURCES FOR EVOLUTIONARY, ECOLOGICAL, AND MOLECULAR STUDIES IN MILKWEEDS AND APOCYNACEAE
Review round: 1
Reviewer: 2

Output:
Basic reporting: The authors have presented nice, well written manuscript presenting a reference genome for Asclepias syriaca.

The introduction is well written overall, cited the relevant literature, and was concise. I felt that the first three paragraphs that were sometimes redundant in their structure and wording, particularly the listings of the multiple studies that use Asclepias as a model found at the end of each paragraph. A rewrite should address these issues.

In some places, I was confused about which set of reads were used for a particular analysis. An example being on line 392 where I wasn't sure which set of reads were used for the linkage analysis.

Images of Asclepias syriaca would be a great addition to this manuscript to showcase this beautiful and unique plant, particularly its unique floral structures. I suggest swapping images of the plant for Figure 1. I don’t think Figure 1 adds much to the main manuscript and might be better suited as a supplemental figure.

Minor points and other comments have been added under the General Comments section.
Experimental design: The materials and methods are largely well-written. There were several places where some revision would help with clarity and flow, particularly when describing how and where each library was constructed and sequenced. I particularly found the paragraph describing the mate-pair library (lines 141-151) construction to need revision. Other comments and questions have been included in the General Comments.

I was very pleased to see the section on contaminant removal. This is often an ignored, but very important step in genome projects.

Using only short read data to construct a reference genome is challenging and it appears that the authors undertook the appropriate methods for assembly and annotation.

If the authors are planning on future sequencing efforts to expand this genome or sequence additional Asclepias, it might make a nice addition to talk about these points in the Discussion.
Validity of the findings: I was initially concerned by the relatively large number of scaffolds and small N50 size, but I feel that the authors acknowledge this throughout. Their discussion of the challenges in assembling highly heterozygous genomes from only short read data and comparing this assembly to other similarly fragmented assemblies was a good inclusion to relieve these some of these concerns. Creating a reference genome for a non-model plant can be expensive given the large size of many plant genomes. It would be beneficial to eventually add long-read data to this project and examine what parts of the genome are missing from the current assembly.

Despite a very fragmented assembly, I found the linkage and synteny analyses quite interesting. The development of SNP markers using the assembly graph bubbles from Platanus was an approach I had not encountered before and seems to work as intended for creating a targeted enrichment probe set. On line 399, the authors state that they called SNPs using bcftools call. In my experience, SNPs are called using the mpileup and view commands. A clarification here about the approach and specific commands used would be helpful.
Analyses identified 11 linkage groups from the full set of individuals and 22 groups from the full-sib set. Asclepias and Coffee both have a chromosome count of n=11, 2n=22. It would be very interesting for the authors to expand more on the connection between 11 linkage groups and 11 chromosomes in their discussion.

Overall, I found the Discussion to be well-written and thoughtful.
Additional comments: Some minor points and areas for clarification:

L131: What were the settings for sonication on the BioRuptor and what was the desired fragment size?

L132: What was the library kit used to prepare these two Illumina libraries?

L153: Were the raw read files assessed for quality, overrepresented sequences, etc. using FastQC or similar tool?

L156-157: Please specify what commands/parameters were specified for Trimmomatic, including which set of Illumina adapter sequences were used for search and removal.

L176: Was the RNA checked for quality and integrity prior to library preparation? If so, please specify here.

L182: Similar to above, please specify the commands used for Trimmomatic.

L186-188: It was mentioned on line 178 that strand-specific RNA-seq libraries were prepared and I see no mention of any read orientation specified to Trinity (--SS_lib_type). If you didn’t provide read orientation, Trinity treats the reads as non-strand specific. I’m curious if you re-ran your Trinity assembly specifying the proper read orientation for strand-specific libraries using the dUTP method (--SS_lib_type RF) if you would find any differences or additional transcripts.

L192-193: Which databases did Mercator and TRAPID search against for functional annotation? The choice of database, such as the NCBI Nr or UniProt, provide different levels of information. Usually doing both, with higher priority given to the curated UniProt database provides enough information to confidently assign putative function. Additionally, were any specific options used for Mercator or TRAPID, or the default settings? It would be good to specify.

L198-200: Please provide the NCBI accession numbers here for the downloaded Rhayza and Calotropis transcriptomes.

L286 and L483: What do you mean by “best” ORF?

L468: I am curious to know how well the scaffolding performed. How many contigs were initially assembled and how many were able to be scaffolded using your mate-pair information? I think that would be an interesting piece to add here to demonstrate how well your final scaffolds look.


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: TRANSPOSON MUTAGENESIS REVEALS PSEUDOMONAS CANNABINA PV. ALISALENSIS OPTIMIZES ITS VIRULENCE FACTORS FOR PATHOGENICITY ON DIFFERENT HOSTS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript "Genome-wide transposon mutagenesis reveals Pseudomonas cannabina pv. alisalensis  (Pcal )optimizes its virulence factors for pathogenicity on different hosts" by Sakata et al, reports a genetic screen to identify virulence factors in the bacterial pathogen. This work is important to understand the virulence of Pcal towards cabbage and oats. The study identified 60 Tn5 insertions that compromised virulence towards cabbage. Thirty one of those insertions also also impacted virulence on oats. The authors used the Tn5 insertion sites to identified the genes in Pcal and found that those genes belong to several functional categories and included genes known to be important in bacterial pathogenicity.
While the manuscript is easy to understand, the writing needs to be improved for clarity. Specific comments on the writing are:
Lines 29-30. A better statement for the different virulence factors needed in cabbage vs oats is: "Interestingly, almost half of the mutants that showed reduced virulence on cabbage, also showed reduced virulence on oat, …...
Line 58. Pseudomonas is not spelled correctly.
Line 61. Replace in this study, for in such study or, in that study.
Lines 73-83.  It is not clear if the authors are referring to different names for the Pcal, or if they consider that Pcal is different from P. syringae pv maculicola.
Line 111. Concentration is missing information on the units.
Lines 187- 188. The authors found 60 insertions sites but only referred to 57 insertions being in coding regions,  where were the other 3 insertions?
Lines 188-189. Please clarify the statement "Our results showed no virulence factors which accounted for a great ratio". What ratio are they referring to? and how is that related to the expected number of virulence factors?
Lines 209-210. The authors mentioned again 60 mutants, but from the previous section only 57 were further analyzed.  It is not clear why 3 mutants appeared to be neglected and then reappear again.
Line 218-219. Does the statement "three in four" refers to the four mutants associated with amino acid biosynthesis? If that is the case, why is that assumed that those are important in colonization?
Lines 222-223. If mutations in AraC does not have a virulence defect, why are they listed as important in colonization?
Line 246. Pcal is the name of the pathogen, not the name of the disease.
Lines 251-252. Not clear how discovering those genes is evidence for the reliability of the inoculation method.
Line 279. The reference (Rico and Preston, 2008) is not relevant supporting that hypothesis.
Lines 281-282. Not sure what is the value of that statement.
Lines 306-309. It is a very strong statement that is not fully supported by the authors evidence. Not clear the link between amino acid biosynthesis and pathogenicity. Those could be detrimental for bacterial survival regardless of infection processes.
Lines 326-340. Can be simplified and focused on HexR. Superfluous information makes it hard to follow. The same can be applied for lines 345-352 and LysR.
Lines 353-361. Simplify to just mention that the study identify virulence genes that can be investigated further to dissect what is their function in the interaction between Pcal and its hosts cabbage and oats. The reference cited is not relevant.
Lines 364-365. The authors do not know that is going to be the case.

Regarding the tables and figures:
Table 1. The same name KB211 is given to two different versions of P. cannabina pv. alisalensis.
The manuscript will be improved if supplementary Figure 1 is included as a main text figure. It facilitates the understanding of the manuscript.
A picture comparing disease symptoms between wild-type bacteria and mutants is needed in the main text, especially because the authors assigned different scores of disease severity, but the pictures included in Supplementary Figures 2 and 3 showed that all the mutants are alike.
The title of Table 2 is misleading when referring to the 57 genes as "predicted to be essential" . Essential genes are required for life, which is not necessarily true for pathogenicity or virulence genes. Are the authors referring to genes required for pathogenicity?
It will be more helpful if the authors actually include the gene names and their respective functional categories in Table 2, just like they did with Table 3. That way is easier to follow the text when they are referring to specific genes (ie lines 199-206).
Methods state that mutants with reduced virulence were those with a score of less than 2 in cabbage (line 144), however, the title of table 3 indicates that reduced virulence on cabbage was scored as less or equal to 1.
Experimental design: No comment
Validity of the findings: The authors stated in the abstract that their work is insightful to understand the infection process by Pcal  regarding the epiphytic phase  and endophytic phase but their results do not support that. The authors should limit to state the discovery of potential virulence factors with some reference to what is known from the literature in other systems.
Lines 249- 251. The manuscript did not conducted those experiments to demonstrate that the mutated genes in Pcal are actually involved in the processes described. In lieu of the evidence, please explain which specific genes are involved in the processes described with supporting evidence from the literature. Table 2 and Figure 2 do not show that.
Lines 235. States that mutants NL37 and NN13 are not significantly different from wild-type. However, bacterial growth curve assay in Figure 1A shows that all the mutants have 2 or more logs difference in comparison with wild-type strain including NL37 and NN13. Those differences would be considered significant. It looks like there is a problem with the statistical analysis.
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: TRANSPOSON MUTAGENESIS REVEALS PSEUDOMONAS CANNABINA PV. ALISALENSIS OPTIMIZES ITS VIRULENCE FACTORS FOR PATHOGENICITY ON DIFFERENT HOSTS
Review round: 1
Reviewer: 2

Output:
Basic reporting: A very solid manuscript for publication with PeerJ. Definitely has potential to lead to new discoveries regarding specific host adaptation factors in a tractable broad host system.

Leaving Schrieber et al 2012 out of the introduction doesn't make sense though. This is the closest and most relevant study to the current work with the major differences being the host plants used. Please include in the intro alongside the discussion of other Tn studies in P. syringae

Table 2. I'm confused by the use of "essential" Do you mean essential for virulence? If so that wording may be far too strong for some of these genes (e.g. NL37).
I would suggest eliminating table 2 completely and porting the information for the 57 genes into table 3 with additional columns noting the actual virulence scores for both cabbage and oat.
Experimental design: How were strains verified as carrying single transposon insertions, or more specifically how were multiple insertions excluded? Were multiple genomic rescue clones sequenced to ID insertion location per strain?

The KB growth testing is not very robust and should have some statistical analysis performed (FigS4). About 1/3 of the strains do not look as though they grow similarly to WT which was reported (e.g. NN13, NL3...) I do not think this is necessarily a huge problem if growth in rich media is lower, but the differences in growth characteristics are worth noting so readers can robustly interpret the results.
Validity of the findings: The claim that cabbage requires more Pcal  virulence factors than oat seems premature based on the data collected. If the Tn screen had been conducted in both cabbage and oat in parallel and fewer "vir down" strains had been found in oat overall, then maybe that claim would make more sense. There may be just as many oat-specific virulence factors that then would not show an effect in cabbage if oat had been screened first.  I think it would be more valid to say that approximately half of genes making virulence contributions to disease on cabbage also make contributions to virulence in oat rather than saying oat requires fewer virulence factors.
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: TRANSPOSON MUTAGENESIS REVEALS PSEUDOMONAS CANNABINA PV. ALISALENSIS OPTIMIZES ITS VIRULENCE FACTORS FOR PATHOGENICITY ON DIFFERENT HOSTS
Review round: 1
Reviewer: 3

Output:
Basic reporting: The quality of the writing was generally clear and straightforward. “In this study” in line 61 should be changed to “Somlyai et al inoculated…” or some analogous construction. On first reading I mistakenly thought the authors had switched to describing the conclusions of their own work.
Line 96: If the authors are going to mention the existence of “few studies reporting on in interaction… under field conditions” those few should be cited. However, the sentence could also be deleted given that the conditions being used in this study do not qualify as “field conditions” and consequently do not contribute new information to that particular gap in our knowledge
Line 99: I was also puzzled by the statement that large scale surveys have not been performed with bacteria that cause serious disease in the field. This statement is definitely not correct if the authors are indeed referring to all plant pathogenic bacteria!
Lines 249 and 364: “several genes involved in the whole infection process” infers that individual gene products are playing a role at multiple stages. It would be more appropriate to state that several genes were identified with apparent impact on virulence. “Predicted functions based on sequence analysis suggest that different members of the set may be involved at different stages of the infection process”
Experimental design: According to the manuscript, 1040 Tn5 mutants were evaluated for reduced virulence on cabbage. 60 were found to have reduced virulence on cabbage. These same 60 were then evaluated on oat and 31/60 showed reduced virulence on the monocot. The authors conclude (line 30) that more virulence factors are needed to infect cabbage than oat”. But by only testing the 60 on oat, it can’t be ruled out that there are others that are required in oat but not cabbage. The abstract (line 27) states that there are “common and specific virulence factors of both monocot and dicot plants” but the experiments were not set up to identify those specific to virulence on oats.
Additionally, given that with 1040 mutations, the study is likely evaluating ~ 20% of the total number of ORFs in the Pcal genome (assumed from genome sizes of sequenced P. syringae strains), a comprehensive statement about numbers of genes involved in virulence is not possible here. Consistent with this, the breadth and definitiveness of the title is not supported by the data presented.
The authors demonstrated that decreased virulence correlated with decreased multiplication in planta, but I did not see evidence that they had evaluated growth in culture for the virulence-impacted mutants.  Not including the outright auxotrophs, could the decreased virulence result from an overall diminished fitness, independent of a specific, pathogenic interaction with the host plant?
Validity of the findings: 1.	The study identifies genes which, assuming unimpaired growth in culture, are involved in virulence of Pcal on cabbage.
2.	There appear to be differences in genes involved in virulence on cabbage and oat, though the extent of these differences cannot be determined given the relatively limited number of mutations screened and uncertainty to which particular mutations may be generally compromising the fitness of the bacterium
3.	Given the significant role of and place of Type III effectors in research on P. syringae and the evidence presented that the Type III secretion system is important for virulence, type III effectors should be addressed more directly than just lines 291-292. Did any of the 60 genes identified show similarity to known effectors?  Did any sequencing of insertion sites reveal evidence of upstream hrpL binding sites? If not, this underscores the concern that 1040 mutations was too few to make a meaningful advance in our understanding of Pcal virulence factors.
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: ROLE OF MIR-10B-5P IN THE PROGNOSIS OF BREAST CANCER
Review round: 1
Reviewer: 1

Output:
Basic reporting: In general english is clear and background literate is well-cited. Authors should provide more details in figure legends.
Experimental design: Overall computational analysis is well structured however it lacks the experimental validation.
Validity of the findings: Experimental validation needs more strength.
Additional comments: In this manuscript authors report down-regulation of miR-10b-5p in breast cancers and its role in tumorigenesis. However, I failed to see a single experiment which describe the role of miR-10b-5p in 'tumorigenesis'. Tumorigenesis means initiation/formation of tumor but none of the experiment or analysis address this in manuscript. I will suggest either authors change the title accordingly or provide evidence for 'Role of miR-10b-5p in the tumorigenesis of breast cancer'.

Specific points:
1) What is expression pattern of miR-10b-5p in Basal, Luminal, and Her2+ breast cancer cell lines?
2) Does knock-down of miR-10b-5p reduce the expression of BIRC5, E2F2, KIF2C, FOXM1 and MCM5?
3) Does miR-10b-5p down-regulation impacts tumor initiating property of breast cancer cell lines?
Minor point:
1) Fig.4 x and y axis font size should be bigger, difficult to read in current size.


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: ROLE OF MIR-10B-5P IN THE PROGNOSIS OF BREAST CANCER
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript by Wang et al predicted that miR-10b-5p a micro RNA is downregulated in breast cancer cells and it targets five important genes which are involved in cell growth cycle. The level of these predicted target genes as expected upregulated in breast cancer cells. All these analyses were done by using various bioinformatic tools. The paper is well written, and conclusions are mostly supported by data. The paper is interesting. However, the novelty of this paper is not clear at all considering it is well known that miR-10b is involve in tumorigenesis (Li Ma, 2010 review breast cancer research).

 Introduction; its not up-to-date. Please add references from recent articles as there are many interesting studies on breast cancer and miR-10b relationship.

Ref. Dai et al. does not have DOI.
None of the address has vol no, issue no, and page numbers which are critical. Please add them as per journal’s format.
Considering combining table 1,2, and 3 under three subheadings such as biological, cellular and molecular functions. Having extra tables with just two values does not improve the manuscript.
Although the manuscript is written in clear English, there are some sentences that need to be modified such as Line 31, 32, and 197 “ was found to be significantly 32 correlated to worse outcome”.
L 53 needs reference/citation
Fig3 ligand (A) label should go at the end of the sentence to maintain the consistency.
Fig5 ligand order of alphabet numbering are at the beginning which is different than other ligands.
Fig8 ligand needs more information. Full form of PPI should be written as Fig should stand by itself. The detail of 3 colored line should be elaborated.
More information required for some figure ligands. Such as figure 1 how many samples were considered? What was the basis of selection as some of the cancer cell line does not have even normal cells data? Authors may consider changing “Abnormal expression” in Figure 1 ligand.
Experimental design: Need  to add more how the question identified the gap area as miR-10b was reported as possible cancer marker by multiple groups. Author can emphasis on the target search.
Validity of the findings: Figure 1. Thanks for providing this very simple but meaningful figure. However, there are some cancer cell data such as LAML, FPPP and LGG does not have their control (normal cells). In absence of control I don’t think these data are adding any value to the result.
Since reports on miR-10b expression in cancer cells are controversial in literature, please provide reason why expression of micro RNA is high in PCPG cancer cells than normal cells?
Figure 2. Why sample numbers for cancer cells are almost 10 time less than normal cells(control) were considered for box plot analysis?
Controversy on miR-10b level can be partially explained due to Heterogeneity of miR-10b expression in circulating tumor cells (Gasch et al, scientific reports 2015).
All the data are predictions, I would strongly suggest to at least check the expression of these 5 targes(BIRC5,E2F2, KIF2C, FOXM1, and MCM5) by either northern blot or RT-PCR under overexpression of micro RNA 10b in breast cancer cell line. This very simple experiment will give a direct evidence of regulation.

Since there are multiple reports on high level of miR-10b RNA which may be due to heterogeneous cell population but still deserve further analysis. positively correlated genes should also be analysed and added as a supplemental material. This knowledge will help researcher in the field.
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: ROLE OF MIR-10B-5P IN THE PROGNOSIS OF BREAST CANCER
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: Authors and carefully address all the concerns raised in previous version.


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

Input:
Title: ROLE OF MIR-10B-5P IN THE PROGNOSIS OF BREAST CANCER
Review round: 2
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: New RT PCR results should be discussed under the discussion section showing the bioinformatical analysis prediction is Indeed true.
Additional comments: The manuscript has improved but still, need some corrections such as the result of RT-PCR has not been discussed. Author needs to write both how these RT-PCR result helped in overall conclusion and compare these result with the literature of other target genes reported.