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: MOLECULAR CHARACTERIZATION OF NEARSHORE BAITFISH POPULATIONS IN BERMUDA TO INFORM MANAGEMENT
Review round: 1
Reviewer: 2

Output:
Basic reporting: The article "Molecular characterization of nearshore baitfish populations in Bermuda to inform management" by Goodbody-Gringley et al. meets all the standards of the journal.
The work shows the importance of baitfish for management, not only as a food source for larger predators higher up the food chain but also to protect species in isolated areas.
Experimental design: The experimental design appears to be very good.
However, I am insisting on changing some of the wordings. On page 17 at the beginning of the discussion the authors mention the difficulty to identify two of the target species (O. oglinum and S. aurita). Although this is a possibility with other species, this is not the case with O. oglinum and S. aurita because the meristics of the dorsal and anal fins are clearly different with no overlap between the two species. Even before the threadfin develops in O. oglinum they should easily be distinguishable. Even the larval stages have different pigment patterns. I strongly recommend to rephrase this section (see comment in pdf)
Validity of the findings: No comment
Additional comments: I really enjoyed the paper and believe it is of high importance for the management and therefore protection of commercially less important species/baitfishes.


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

Input:
Title: THE PROGNOSTIC IMPACT OF AGE IN DIFFERENT MOLECULAR SUBTYPES OF BREAST CANCER: A POPULATION-BASED STUDY
Review round: 1
Reviewer: 1

Output:
Basic reporting: The authors attempt at addressing the association between age and breast cancer prognosis is appreciable. The reporting in general is comprehensible and contains appropriate references to existing published studies. Details about the methodology have been included wherever applicable and the data well presented.
I have minor comments with respect to the writing:
Line 47 - the authors fail to mention which proliferative markers they are talking about
Line 48 - the word surrogate to describe the classification of TNBC is not appropriate
Line 54 - ER- has been mentioned twice
Line 58 - was currently controversial should be replaced with subject of discussion or speculation
Line 89 - vital stats instead of vital status
Experimental design: The only, and an important, shortcoming that exists is the fact that age and treatment have not been accounted together and their association has not been evaluated for TNBC deaths for <40 years patients.
Validity of the findings: The authors have addressed the shortcomings of their study, to their full credit. They have also ensured that their interpretations are robust due to the sheer enormity of the sample size taken into consideration along with the large number of variables that have been covered. However, the study falls short on certain minor criteria:
1) No association studies have been performed for patients under 40 who have undergone chemotherapy/ radiotherapy, especially for TNBC samples. This holds important clinical relevance since the authors have concluded how age is an important factor. Their OS survival will thus shed important light in how effective the current therapeutic regimen is.
2) Not much discussion of the results with respect to the clinical implications.
3) I would the authors to speculate (in the discussion section) on the reasons they think TNBC seems to be more prevalent in patients under 40.
Additional comments: Please refer to my specific comments for my minor concerns regarding 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: THE PROGNOSTIC IMPACT OF AGE IN DIFFERENT MOLECULAR SUBTYPES OF BREAST CANCER: A POPULATION-BASED STUDY
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript by Dai and Zhong et al. shows the correlation of age with different breast cancer subtypes. The article is too short. Introduction could have been more detailed with more background on different cancer subtypes- for example, which type is more common, aggressive and affects what percent of all breast cancer patients. Table presented is comprehensive though. There are grammatical and typographical errors throughout the manuscript that are stated below in the “Comments for the author” section.
Experimental design: No comment
Validity of the findings: No comment
Additional comments: Some comments, corrections and suggestions are as follows:
1) Line 44: Change “luminal-A and luminal-B” to “luminal A, luminal B”.
2) Line 45: Change “was determined” to “were determined”.
3) Line 54: Change “ER-, ER-” to “ERα, ERβ”.
4) Line 54-55:  Replace “higher expression of EGFR” with “higher mRNA expression of HER-2 and EGFR”.
5) Line 57: The suitable word is “older women” instead of “elder women”. This needs to be checked throughout the manuscript.
6) Line 72: Change “United State” to “United States”.
7) Line 74: Change “breast subtypes” to “breast cancer subtypes”.
8) Line 82:  Correct by adding underlined word “we involved it with”
9) Line 89: Correct by adding underlined word “should be over”
10) Line 89: Spell-check “recorded”
11) Line 90: Correct by adding underlined word “Patients who did not meet”.
12) Line 104: It looks like something is missing in “the value tumor size” It should be “the values of tumor size.
13) Line 120: Change “was listed” to “is listed”.
14) Line 129: Change “follow-time” to “follow-up time”.
15) Line 137” Did you mean “BCSS” (breast cancer specific survival) instead of “BSCC”. If not, please state what is BSCC.
16) Line 173: Change “deaths rate” to “death rates”.
17) Line 183: Change “cease increasing” to “cease to increase”.
18) Line 189: Correct by adding underlined word “delay in diagnosis”.
19) Line 200: Correct by adding underlined word “found that the”.
20) Line 207: Change “was missed” to “was missing”.


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

Input:
Title: THE PROGNOSTIC IMPACT OF AGE IN DIFFERENT MOLECULAR SUBTYPES OF BREAST CANCER: A POPULATION-BASED STUDY
Review round: 1
Reviewer: 3

Output:
Basic reporting: no comment
Experimental design: It should be noticed that which classifaction is used for defining moleculary subgroups clearly in methods.
Validity of the findings: no comment
Additional comments: Dear Authors,
This study is well designed and found out valuable results about breast  cancer spesific mortality comparing different range group of age in terms of moleculary subtypes. Figures are well described and have enough quality.
Results are concordant with lieterature;  breast cancer patients with TNBC and HR (+), HER 2 (+) moleculary subgroups had higher over all survival in age over 40 years comparing those in age ≤ 40 years.  Despite of retrospective design of study patient numbers of study kohort are enough.
It should be noticed that which classifaction is used for defining moleculary subgroups clearly in methods.


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

Input:
Title: THE PROGNOSTIC IMPACT OF AGE IN DIFFERENT MOLECULAR SUBTYPES OF BREAST CANCER: A POPULATION-BASED STUDY
Review round: 2
Reviewer: 1

Output:
Basic reporting: No Comments
Experimental design: No comment
Validity of the findings: No Comment
Additional comments: My primary concerns have all been addressed. I would still urge the authors to proof read the manuscript properly to ensure there are no typographical errors. I can still spot a few of them.


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: COLOCR: AN R PACKAGE FOR CONDUCTING CO-LOCALIZATION ANALYSIS ON FLUORESCENCE MICROSCOPY IMAGES
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1. The manuscript by Ahmed et al. reports their software for calculating two major colocalization coefficients, i.e. Pearson’s Correlation Coeffient (PCC) and Mandars Overlap Coefficeint (MOC), in multicolor imaging of biological samples. The manuscript is readable and english grammar is mostly fine. However, there are a few paragraphs with unclear intensions or discussions at inappropriate places. The manuscript is inclined too much toward software description, but lacks insights into the practical issues or theories in microscopy. For these reasons, this reviewer requests a major revision before publication from PeerJ.
2. The manuscript did not mention the practical problems in multicolor microscopy and thus failed to add key references in this field. Firstly, the authors should mention in their Introduction or Discussion section about chromatic shift correction, which is an important pretreatment before measuring colocalization of microscopy data. The milestone papers to cite are:
  (i) Manders, E. M. M. Chromatic shift in multicolour confocal microscopy. J. Microsc. 185, 321–328 (1997).
  (ii) Churchman, L. S., Ökten, Z., Rock, R. S., Dawson, J. F. & Spudich, J. A. Single molecule high-resolution colocalization of Cy3 and Cy5 attached to macromolecules measures intramolecular distances through time. Proc. Natl. Acad. Sci. U. S. A. 102, 1419–1423 (2005).
  (iii) Matsuda, A., Schermelleh, L., Hirano, Y., Haraguchi, T., and Hiraoka, Y.  Accurate and fiducial-marker-free correction for three-dimensional chromatic shift in biological fluorescence microscopy. Sci. Rep. 8: 7583 (2018).
3. Secondly, the authors should cite the original theoretical paper describing application of PCC and MOC in biological microscopy.
  (i) Manders, E. M. M., Verbeek, F. J. & Aten, J. A. Measurement of co‐localization of objects in dual‐colour confocal images. J. Microsc. 169, 375–382 (1993).
4. The manuscript may require additional data sets for testing the validity of the approach. Detailed suggestions will be made in “Validity of the findings”.
5. Presentations of tables and figures need to be re-considered. Detailed suggestions will be made in the “General comments” section.
Experimental design: This work reports software development using R programming language. Their software helps to measure the amount of colocalization, that is widely required in biological microscopy. The software is open-source and its quality is validated by the ROpenSci community. This work fits with aims and scope of PeerJ.
Validity of the findings: 1. The difficult step toward quantifying colocalization is the determination of ROI. While authors developed a software facility to select ROI, the image presented in the manuscript is only a 2D image. Does it work for 3D images? In the case of 3D images of wide-field microscopy or confocal microscopy using large pinhole sizes, out-of-focus blur makes simple thresholding difficult. The authors should test if their approach works for 3D images, or if it doesn’t then mention its limitation about the dimension.
2. ‘Additional file 1’ is mentioned at the end of “Materials and Methods”, but it was not found; only the manuscript was found.
Additional comments: 1. Specific names of packages or functions (Such as ‘Shiny’, ‘cimg’, ‘imager’, ’image_load’, ‘data.frame’) should be in different fonts, capitalized if applicable, or enclosed by quotations to discriminate from conventional words.
2. What image file formats are accepted by the software? Are there any restrictions for image data (data size, bit depth, number of channels, number of time frames etc..)?
3. The word “statistics” is used throughout to indicate ‘statistics’ or ‘coefficient’. When talking “colocalization statistics”, biologists would expect a statistical analysis of the amount of colocalization measured for many cells. Therefore, I would recommend to use ‘colocalization’, ‘measurement of colocaliztion’ or ‘quantifying colocalization’ for a single measurement, and ‘statistics’ for many cells.  In this context, ‘statistics’ seems inappropriate, for example, in line 62 and probably line 130, and many other places if they are intended to mean ‘quantifying colocalization’.
4. Line 82, “Testing for statistical significance” this subsection is a discussion which is inappropriate for the “Materials and Methods” section. Furthermore, the conclusion of this subsection is unclear. Please explain the last sentence more carefully. Why “auto-correlation in the image pixels” in the null model is a problem? What is “the varying nature of the probe interaction”? Do the authors finally recommend to use t-test?
5. Line 73, this sentence needs proof-reading.
6. Line 89, Table 1 and Line 164. My personal impression is that ‘how to use’ of the code or too much detail of individual functions should be written in the document of the source code and unnecessary in the main body of a scientific manuscript. Alternatively, can these be moved to supplemental files? I think Figure 4 is sufficient to introduce their functions.
7. Line 102, “cancer cell line were stained…” should be changed to “cancer cell line stained…”
8. Figure 1-2. Is the blue color in the merged image really required? Because of this blue color, it is very difficult to see the red color. The images are too small to validate that the colocalization coefficients are really true at the pixel level. A magnified view would be helpful. Also since the combination of red and green may be difficult to see for those with color-blindness, a combination of magenta and green or red and cyan is recommended. Scale bars are missing.
9. Line 109, “However” is inappropriate here. Just removing it makes the sentence more readable.
10. Figure 3. The color code in the left plot is ambiguous. It is difficult to see how many colors are used in the plot, therefore, it is unknown how many ROIs are used here. Do these ROIs correspond to ROIs in Figure 2 and Table 2? If so, please indicate as such. Also in this figure legend, at the end of the first sentence, ‘show’ should be changed to ‘shown’.
11. Line 131-132, ‘We described … elsewhere.’ Please indicate where it is described.
12. Line 156, the message in this paragraph is unclear. Does the author describing or discussing about the “other image processing packages in R”? The last sentence needs proof-reading (where is verb?).


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: COLOCR: AN R PACKAGE FOR CONDUCTING CO-LOCALIZATION ANALYSIS ON FLUORESCENCE MICROSCOPY IMAGES
Review round: 1
Reviewer: 2

Output:
Basic reporting: This manuscript reports a new package for R for co-localisation analysis of fluorescent microscopy images called “colocr”. The package is open source, and is available on GitHib but there is no licence on the software. In this report the authors present the use of the software against one pair of individual images which seems insufficient to evaluate the utility of the software.
Experimental design: No comment
Validity of the findings: The authors compare their work on colocr to a few co-localisation packages that are part of the ImageJ plugin collection. There are several other Image J plugins and indeed several R packages already released that implement colocalization. For example, the Bioconductor package and in particular EBimage R suite contains many of the functions the authors report here. In addition, the arrival and universal adoption of super-resolution methods has raised several questions regarding the utility of colocalization at the diffraction limit, since super-resolution techniques will often resolve what is co-localised in conventional microscopy. Thus while the work appears to be well formed the rationale for yet another co-localisation package hasn’t been well presented.
Additional comments: If the author’s present a comprehensive representation of tools for co-localisation in ImageJ and R and elsewhere, a clear argument for their use and the description of colocr, the paper would then be suitable for publication.


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

Input:
Title: COLOCR: AN R PACKAGE FOR CONDUCTING CO-LOCALIZATION ANALYSIS ON FLUORESCENCE MICROSCOPY IMAGES
Review round: 2
Reviewer: 1

Output:
Basic reporting: The manuscript was improved after the revision. There still remain a few points that are confusing for readers. This reviewer requests a minor revision for this manuscript.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: At line 30, the references seem to be cited for a wrong reason. “Churchman et al., 2005” and “Matsuda et al., 2018” solved the problem of chromatic shift and therefore should be cited at line 32 together with “Manders 1997”.

Related to the citations, there are several references missing volume and page numbers in the REFERENCES section.

We can predict that there are many researchers who wish to measure colocalization of 3D images or time series. To avoid these researchers spending time for nothing and to help them re-direct to other resources, the authors should indicate the dimensions that their current software version can handle in the manuscript. Also, even though the program uses “magick” package to read image files, many file formats of microscope companies (such as czi, oib, lif etc..) does not seem to be readable. Again to save time for researchers not spending time to try directly load their image files from microscopes (which is possible in the case of, for example, ImageJ), please indicate the kind of files that are accepted by colocr. Such descriptions should help readers to make a fair judgement on software and be consistent with the idea to make the manuscript self-contained as the authors claimed in their response.

Line 78-79 needs proof reading (spelling of “therefore” and overlapping “to”s).

Figure 2 and 3. The authors can indicate the size of image in the legend instead of inserting scale bars.


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

Input:
Title: A SURVEY OF THE SPERM WHALE (PHYSETER CATODON) COMMENSAL MICROBIOME
Review round: 1
Reviewer: 1

Output:
Basic reporting: - The manuscript needs to be proof-read and edited to ensure correct language and grammar are used throughout. Currently, many sentences do not make sense and the meaning is often ambiguous.

- The introduction does not clearly lay out the aim of the study. The authors suggest that not much work on cetacean microbiomes has been undertaken, but there is a moderate amount of literature surrounding cetacean microbiomes (e.g. see Apprill et al 2014, Humpback whales share a core skin bacterial community: towards a health index for marine mammals?; Bierlich et al, 2018, Temporal and regional variability in the skin microbiome of humpback whales along the Western Antarctic peninsula; Sanders et al, 2015, Baleen whales host a unique gut microbiome with similarities to both carnivores and herbovires, etc) , albeit not of stranded individuals (although see Godoy-Vitorino et al, 2017, The microbiome of a striped dolphin stranded in Portugal) . The authors go on to suggest that stranding is a large issue in cetacean biology (I suppose specifically in conservation, although this isn’t mentioned and perhaps should be), and that this research will shed light on the microbiome related to host physiological characteristics, deep-sea adaptation and stranding. This creates an unclear perspective of what this paper is about, particularly because neither physiological characteristics nor deep-sea adaptations are further discussed. I would suggest re-writing the introduction with a focus on what is currently known about the cetacean microbiome (citing papers which have explored this already), and that this study addresses the gap in knowledge we have about the microbiome of stranded cetaceans.

- The discussion is too short and does not actually discuss the findings of the paper in depth. The authors should focus more on what their results actually show (at a fine-scale) and how this links with the broader literature of cetacean microbiomes (and microbiome work in general).

- I could not find the raw data. The NCBI accession number shared by the authors took me to the sperm whale genome, but not to the data generated in this study (that I could see)

- Figures and tables:

Figure 1A: A PCA of species with sample type overlaid would be a clearer and more informative way to visualise this data. If a Venn diagram must be used, it would be helpful to at least have the circle sizes proportional to the number of species found.

Figure 1B: clarification needed. Is this the top 20 most abundance species across all tissue types?

Figure 2A: the proportions of firmicutes and euaryarchaeota don’t seem to match the proportions reported in line 158 of the text

Table 4 is not mentioned in the text

Figure 3C is missing

Figure 3B: it would be good to have species names here (could still be coloured by clade e.g. dolphin/whale).
Experimental design: - This is an opportunistic study which took advantage of a rare event occurring - the stranding of a sperm whale with the opportunity to sample it - and the results, although interesting and providing new knowledge to the field of microbiome research, must be seen within this context. In particular, it must be acknowledged that there is a sample size of 1 in this study, and therefore the scope of what it can tell us about the sperm whale microbiome is extremely limited.

- The research question is not well-defined by the authors. As previously mentioned, the introduction needs to be rewritten in order to put this study in context, and it must also clearly define the research question. Arguably, the aim of the study is to characterise the blood, muscle and fecal microbiome of a stranded sperm whale, compare the results with published cetacean microbiomes to understand whether the microbiome of a stranded sample still shows similarity to other cetacean samples, and explore *possible* pathogenic bacterial species and parasitic species present in the whale.

- Comments on the methods:

My largest concern in the methods is the lack of control for, or discussion of, contamination.

Contamination is a common problem in microbiome studies (e.g. see Salter SJ, Cox MJ, Turek EM, Calus ST, Cookson WO, Moffatt MF, et al. Reagent and laboratory contamination can critically impact sequence-based microbiome analyses. BMC Biol. 2014;12:87.), and can be at least partially controlled with the inclusion of blanks at the sequencing stage. There is no discussion of blanks in this paper, and I therefore assume that they were not included.  As a late-stage method to understand whether contamination from human skin may influence results, the authors could align the unfiltered data to the human genome to understand whether human contamination (and therefore human-associated microbiome contamination) has occurred (for an example of this and further ways to address possible contamination see Hooper R, Brealey JC, van der Valk T et al. (2018) Host-derived population genomics data provides insights into bacterial and diatom composition of the killer whale skin. Molecular Ecology). Given that some of the bacterial species identified in the study are common to human skin (e.g. Streptococcus species, which is also a common lab contaminant) I think this is a step which must be taken in order to understand whether we need to interpret the results with the caveat of contamination.

The authors must address the concern of contamination at each stage - what steps were taken to identify/remove contamination at the sampling stage (for which methodological detail must be expanded - how were samples collected? how was human contamination avoided?), at the sequencing stage (were blanks included?), and finally at the bioinformatics stage (were any bioinformatics techniques used to try to identify whether contamination had occurred?) to mitigate this potential confound.

Other concerns about the methods section are listed below:

•	Tryptophan pathway: To leap from missing KOs to the whale being susceptible to colitis and CNS inflammation is extremely speculative, primarily because these KOs may be missing only post-stranding/post-death due to changes in community composition. I do not think that this analysis should be kept in the manuscript.
•	Unsure what Figs 3a and 3b, and associated analysis, add to the manuscript. How do they fit with the study? What, of interest, are they showing us? The trees seem quite uninformative and the analysis doesn’t have any context in this study.
•	More information is needed on the building of the cluster tree. What data did the authors use? Did they download the data from each of these species (and if so, where from?). Are they all gut microbiomes? How does this fit with the rest of the study? This is not discussed at all later – it is a very interesting result but is barely touched upon and not integrated into the manuscript. The authors should also explain why they use 16S data for this analysis, as opposed to shotgun data used in the rest of the paper (I assume so that a direct comparison between datasets can be made)
•	Is gut synonymous with fecal in the methods? Confusing terminology; should be clarified.
•	More details on sample collection needed. How were the samples collected?
•	Line 122: do you mean adapters were removed? And why only from gut (presume fecal) and not blood/muscle?
•	Typo in line 132/133: two mentions of blood and no mention of muscle
•	Line 150 – 151 belongs in the discussion
•	Line 165 – 166 belongs in the discussion
•	Line 169: far too certain of causality
Validity of the findings: Although this study contributes novel and interesting knowledge to our understanding of the sperm whale microbiome, I have two key issues with the manuscript.

Issue 1) the interpretation of results and conclusions drawn

The authors assert that this microbiome gives clues as to why the whale stranded. The muscle and gut samples were both taken post-death of a stranded whale. After death, the microbiome is likely to change due to chemical changes in the body. For example, species of bacteria which respire anaerobically are likely to dominate (such as clostridium spp.). Thus, the microbiome seen in the recently deceased sperm whale could reflect post- mortem changes in the microbiome, as opposed to, as the author’s suggest, the microbiome of a sperm whale which is unwell and likely to strand. The blood sample was taken post-stranding, but while the whale was still alive. This is likely to show the microbiome of an individual that is stressed and, possibly, dying, but it is purely speculative to suggest that the blood microbiome of a post-stranded individual shows what the microbiome of a pre-stranded individual may look like. It is unlikely that the microbiome (blood, muscle or gut) of pre-stranding whales is likely to be similar to those of post-stranding whales given that internal conditions of the whale post-stranding are likely to differ dramatically to that of pre-stranding, and therefore different bacterial species are likely to dominate.

In brief, the authors assert that the pathogenic bacterial species (and associated virulence genes) found in this whale’s microbiome are likely to have caused its stranding. This is flawed reasoning because i) the microbiome of the stranded whale is likely to have changed post-stranding, and thus this research only sheds light on what the microbiome of a stranded sperm whale looks like, and ii) see issue 2. The manuscript therefore needs to be rephrased to take this into account. This will require a re-framing of both the introduction and the discussion.

Issue 2) pathogenic bacteria

The authors suggest that they have identified pathogenic bacterial species and parasites, which likely played some role in the death of the individual. However, many bacterial species which have been found to have pathogenic properties are actually also often found as commensal species. For example, Fusobacterium nucleatum is a commensal of the human oral microbiome, but can play a role in disease. The same is true of many Streptococcus species (which also are often found as part of the human microbiome). Whereas parasites are pathogenic, and the high quantity of them in this individual probably suggests ill health (although whether they were the cause of stranding/death is unknown as there is no healthy control whale to compare with), the bacterial species that are identified could very well be commensal species. I therefore think that the identification of possible pathogenic bacterial species should be included only in the discussion and should be highlighted as quite speculative, with a need for further study (with a healthy control for comparison).

The finding of a high abundance of parasites is interesting, but without a healthy control, it cannot be known whether this is an unusually high abundance of parasites. The authors have again made some very strong assertions that these parasites played a role in the death of the whale, but this is unfounded. At best, the authors can say that the high abundance of parasites may be related to poor pre-stranding health of the individual, however comparisons with other individuals would be needed to understand whether such high parasite loads are atypical and related in any way to the frequency of strandings.
Additional comments: No additional comments


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

Input:
Title: A SURVEY OF THE SPERM WHALE (PHYSETER CATODON) COMMENSAL MICROBIOME
Review round: 1
Reviewer: 2

Output:
Basic reporting: The English sentence structure and grammar, word usage, and use of plurals is often not correct. For example: Line 21 ‘Mammals commensal microbiota’ should read ‘A mammal’s commensal microbiota’. The word habit is used instead of habitat, e.g. Line 45.

More discussion on the stranding of whales and the microbiome of mammals would provide a better background.
Experimental design: As this stranded whale was very near to death and the pathogens in the body were a cause it would be interested to discuss further the pathogens and the symptoms observed. This study is very opportunistic and as a result is more of a case report on a single individual.

I could not see Animal Ethics documented clearly.
Validity of the findings: The design In the materials and methods in isn’t clear what samples from which part of the animal’s body were collected. In the methods it isn’t clear how the raw sequences were treated. It is good practices to clean and trim your sequences and also to process and filter raw data such as the OTU table. I think the results you have could be discussed in the context of the current body of literature of stranding events and whale health.
Additional comments: No comment


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 SURVEY OF THE SPERM WHALE (PHYSETER CATODON) COMMENSAL MICROBIOME
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: A survey of the sperm whale (Physeter catodon)
commensal microbiome
Many cases where plural words need to be singular or the alternative, e.g. Line 22 – plays to play; Line 72 – microbiomes to microbiome
Several lines seem to be missing words, e.g. Line 27 – that may due needs revising; Line 51 – microbes mainly needs revising
Line 49 – habit to habitat
Line 55 – has to have
Line 62 – apply these knowledge, needs revising
Line 67-68 – revise as doesn’t make sense
Many situations past and present tense are mixed, e.g. Line 71 – has not been clear, change to are not clear
Line 71 – not clear meaning not known? Please revise the wording for clarity
Line 75-77 – this sentence is better suited to the results, not the introduction
Line 96 – plural and past tense – extraction was to extractions were
Line 99 – is this Qubit 3.0 flto dissol the correct term?
Line 101 – some words seem incorrect as this sentence isn’t clear – dialed? And same?
Line 122 – use of same inn’t clear what you mean here


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: TOWARDS OPTIMIZED VIRAL METAGENOMES FOR DOUBLE-STRANDED AND SINGLE-STRANDED DNA VIRUSES FROM CHALLENGING SOILS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Language was in English and mostly clear throughout.  There were a few places where word choice or phrasing could be modified to increase clarity or reduce ambiguity:

l. 29; “sequencing data for soil viruses are hard to come by…” I do not think this is an accurate characterisation, as several (at least 5) soil metagenomes exist in public databases.  Thus sequence information for soil viruses is available and accessible.  I think the case could be made that comparatively less has been done with respect to soil viruses as opposed to, say, marine viruses – but this is a different message.

l. 41: “While little empirical data are available for inhibitors in soil viral extractions” I am not sure what you mean here.  That few suggestions are available in the literature for dealing with or removing inhibitors? Please clarify.

l. 266-267: “These findings suggest that DNA yields are best when not purifying the resuspended viral particles and when lysed using heat.”  This phrasing was difficult for me to follow.   How about, “…DNA yields were highest when CsCl density gradients were omitted and viral particles were lysed using heat”?

Some important literature references have been left out:
l. 24: I found this potentially misleading, as more is known about viruses in soils than the few facts presented here.  Viruses are dynamic and responsive to change (Srinivasiah et al. 2015. Dynamics of autochthonous soil viral communities parallels dynamics of host communities under nutrient stimulation. FEMS Microbiol. Ecol. 91: fiv063)

Several soil viral metagenomes have been analysed:
Fierer, N., Breitbart, M., Nulton, J., Salamon, P., Lozupone, C., Jones, R., Robeson, M., Edwards, R.A., Felts, B., Rayhawk, S., et al. (2007). Metagenomic and small-subunit rRNA analyses reveal the genetic diversity of bacteria, archaea, fungi, and viruses in soil. Appl. Environ. Microbiol. 73, 7059–7066.
Green, J.C., Rahman, F., Saxton, M.A., and Williamson, Kurt E. (2017). Quantifying aquatic viral community change associated with stormwater runoff in a wet retention pond using metagenomic time series data. Accepted MS ID: 201703009.
Kim, K.-H., Chang, H.-W., Nam, Y.-D., Roh, S.W., Kim, M.-S., Sung, Y., Jeon, C.O., Oh, H.-M., and Bae, J.-W. (2008). Amplification of uncultured single-stranded DNA viruses from rice paddy soil. Appl. Environ. Microbiol. 74, 5975–5985.
Reavy, B., Swanson, M.M., Cock, P.J.A., Dawson, L., Freitag, T.E., Singh, B.K., Torrance, L., Mushegian, A.R., and Taliansky, M. (2015). Distinct Circular Single-Stranded DNA Viruses Exist in Different Soil Types. Appl. Environ. Microbiol. 81, 3934–3945.
Zablocki, O., Adriaenssens, E.M., and Cowan, D. (2016). Diversity and Ecology of Viruses in Hyperarid Desert Soils. Appl. Environ. Microbiol. 82, 770–777.

The collective metagenomic data for soils suggest that genetic diversity of soil viruses far exceeds that of other environments for which virome data are available.  It also suggests that viromes are localized – that viruses break out into habitat-specific groups.
I think it is important to mention these references and observations in order to provide a more complete view of our current understanding of viruses in soils.

I think the language regarding ssDNA virus metagenomics needs to be revised:
l. 318- 319: This is a bit disingenuous, as it seems to suggest that previous researchers were somehow negligent – when in fact, methods that specifically target library construction of ssDNA viruses have only recently been developed!   Unless you want to count GenomiPhi, which has been around considerably longer, but whose selective amplification of ssDNA templates is more of an unwanted byproduct than a deliberate feature.

l. 322: Several soil viromes have been published featuring ssDNA viruses: Kim et al (2008), Reavy et al (2015), Green et al (2017), see suggested list of additional citations, above.
Experimental design: l. 34: pH and soil organic matter are likely to be important factors as well.  While not an explicit part of your experimental design, I think it could/should be acknowledged that they are likely to play a role.

l. 42: one seemingly obvious solution has been omitted: why not just perform more virus extractions and then pool and concentrate the extracts until sufficient DNA has been obtained for sequencing?  Such an approach would avoid all issues described from MDA and SISPA.  Later on, in the methods (lines 88 – 90), it is described that 7-g to 10-g soil samples were used for DNA extractions.  Is there any limitation on extracting larger masses of soil, or multiple 10-g replicates and then pooling the extracts?  Why couldn't this be part of the experimental design?

l. 67: what is/are the specific hypotheses being tested?   Part of the explicit reviewer instructions for the journal ask me to check that the “Research question well defined, relevant & meaningful. It is stated how research fills an identified knowledge gap.”  I think the specific objective(s) of this work could be made more clear.  Right now it seems to be to determine what happens if virus particle extraction conditions are changed – is that correct?  If not, how would you make it more clear to your reader?

l. 91 and l. 124: I am not sure what the difference is between “optimizing DNA extraction” and optimizing particle lysis and purification,” the two different headers for these subsections.  It appears to me that both subsections are trying to do the same thing – elute virus particles from soil samples and extract DNA from these particles for metagenomic library construction.  But the different labels would appear to suggest that different things are happening?  Could you please clarify?

l. 125 – 133: could you provide any rationale for these changes?  For example, why omit CsCl density gradient centrifugation as a means of separating virus particles from potential co-eluted contaminants?  Were these random adjustments to “see what happens” or was there a rationale behind these choices?  Please make your reasoning for these adjustments more transparent to the reader.  (I see that this is addressed in the Results/Discussion section.  You could consider providing an explanation in the Methods either instead, or as well, to reduce reader confusion.)

l. 139: these libraries were sequenced at 151 bp, paired end, while the libraries sequenced from the previous section were done at 100 bp, paired end.  I was highly concerned that this additional variable would affect your outcomes, making it more difficult to say for certain which factors account for differences in outcomes.  I had done quite a bit of reading into the manuscript before determining that the steps done in the first part of your methods were not being directly compared to the steps done in the second part.  This goes back to the headers (lines 91 and 124) and lack of clarification as to what is being done in these two sections and how they differ.  I am still not entirely clear on what is different in the ultimate objectives of these two subsections so any way you can distinguish the two sections and how their goals might differ would be extremely helpful to my understanding.

l. 244: could this uneven coverage arising from PCR be avoided if we simply put more effort into obtaining a larger amount of starting material (and avoiding amplification altogether)?  (see previous question about this, as well)

l. 260: loss of virus particles in CsCl density gradient centrifugation.  Were different density layers tested or optimized?  As mentioned in Thurber’s protocol paper (Thurber, R.V., Haynes, M., Breitbart, M., Wegley, L., and Rohwer, F. 2009. Laboratory procedures to generate viral metagenomes. Nat. Protoc. 4, 470–483, cited in this manuscript) some care must be taken in selecting density gradients, depending on the particle distribution (and genome types) of a particular sample.  Thus, this may not be an insurmountable problem.  In addition to cells that may pass through 0.22 um filtration, CsCl density gradients provide additional benefits by removing particulate contaminants (clay particulates, for example, have no lower limit on their dimensions, but differ greatly from virions in their density).  So omission of the CsCl step seems to have an incompletely characterised cost-benefit sheet.  I think it would bolster your results to try to determine more about what is gained vs. what is lost when CsCl is used (or not) in the purification of virus particles from soils.
Validity of the findings: While I do not doubt the authors observed what they reported, I think the paper would benefit overall from some healthy self-skepticism and careful consideration of the limitations of the experimental design:

l. 195: I think this is an important caveat of this paper.  This work is impressive, to construct metagenomic libraries to determine how different methods skew the perception of the metagenome composition.  But we can really only say that these impacts occur in the specific soils sampled and analysed.  It is not clear to what extent, if any, these results may be generalised for other soils.  And not to denigrate the work already done here, but each method is represented by only one library/virome and this does not enable us to differentiate within-group from across-group variation.  In other words, in spite of the great amount of work done here, each specific treatment has N=1.  While I would not go so far to say that it negates and findings, it is a limitation of the design that should at least be acknowledged.

l. 207: only bog and fen soils were tested.  So it might be more accurate to say, “in the soils tested (bog and fen).”  I am curious, why not the palsa soil?  Was there a rationale behind this choice or was it more due to external constraints?

l. 210-221: it is probably worth noting that A260 reading for viral nucleic acids could be tricky, as the absorbance-to-DNA concentration factor varies depending on dsDNA, ssDNA, and RNA, and virus capsids are likely to have a mix of these different nucleic acid types.  Further, there are many compounds that can interfere with spectrophotometric quantitation of nucleic acids, and many sequencing facilities favour fluorometric quantitation (e.g., PicoGreen) over spectrophotometry for this reason.  I am curious why fluorometry was not used in this case.  I think it would be reasonable to at least account for the strengths and weaknesses of these approaches, and to consider the possible caveats of the results as computed using spectrophotometry here.

l. 231: good point. In the end, if you can successfully generate sequence, does it matter if the A260/A280 ratio is not optimal?  Still, part of the end-goal of this paper seems to be to determine how these factors (e.g., differences in DNA clean-up) affect perceptions about viral metagenome richness and composition.  So I guess we will find out if it matters.

l.252: could you please provide more explanation here about how you are assigning the variance to the DNA extraction method and how you can be sure that it is not explained by amplification bias?  It would seem that these two factors are entangled and I am not sure how one assigns a greater role to a particular factor over another.  Any help here would be appreciated.

l. 279: I am not completely convinced by 16S PCR results alone as the definitive check for cellular contamination in your DNA preps.  This is a helpful metric, and should be done, but transducing phage have been shown to carry copies of the 16S gene from bacterial hosts.  Careful screening of your viral extracts under fluorescence microscopy or TEM could (perhaps should?) be used to bolster your 16S PCR results, and can quickly reveal potential cellular contamination in virus extracts.  While inclusion of a CsCl step will undoubtedly result in some losses of your target viruses, it certainly reduces sources of non-viral DNA – both cellular and extracellular.  I am dubious of omitting the CsCl step, which has been part of standard protocols for viral metagenome preparation for over a decade, with good reason.  The simplest explanation for the increased yield for these (-)CsCl samples is the inclusion of non-viral DNA.

l. 278-290: as shown here, a perceived low contamination based on low 16S PCR copy number could easily be due to impure samples that hamper efficient PCR – and not, in fact, due to low contamination levels at all.  Paradoxically, the contamination, itself, causes the samples to appear less contaminated!  At least using a PCR-based method.  Again, bolstering this with a secondary detection method such as microscopy would help reduce doubts here.

l. 293-294: a natural question here is: if the additional DNA gained by omitting the CsCl step is NOT viral, then how were additional contigs obtained?  Or, in other words: Since you got more contigs by omitting CsCl step, you must have gotten more viral DNA this way.  This is a fair interpretation, but other possibilities should be considered, and ruled out if possible.  Much of this interpretation comes down to the specifics of your algorithm.  It has been observed that, due to coevolution, many virus sequences mimic those of their hosts, down to G+C content and even homologous genes.  So if you have contaminating host DNA in a (-)CsCl DNA extract, its removal from bioinformatic analysis is only as good as the specificity of sequence-sorting algorithm.  One way to test this is to spike your viral DNA extracts with varying concentrations of prokaryotic DNA and determine when you can no longer parse host from virus: how much contaminating DNA is needed to fool your algorithm?  Have such controls been performed, and perhaps reported elsewhere?  (Indeed, I am wondering: if the VirSorter program is able to parse virus sequence from other microbial sources, why go through all the trouble to generate virus-enriched samples for analysis?  Couldn’t you just extract total DNA and pull the virus sequences from the resulting metagenomes, using deep sequencing to get at rare taxa?)  I don't wish come across as if I am pulling this all apart!  I do wish to recognize the amount of effort that’s gone in here and the need to understand how our perceptions of microbial diversity may be biased by our methods.  But I also believe that, as scientists, we must take a critical eye to our own work and take pains to consider other explanations, not simply the most convenient ones.  And I believe the manuscript would benefit from a more critical analysis in this regard.
Additional comments: l. 32- 34: This seems an accurate characterization.  But it begs the question: will the results you obtain here be applicable to any other soils, save the specific ones being analysed here?
l. 304- 306: this is an interesting finding.  Different methods generate subsets rather than different sets with different degrees of overlap.

The key finding here is that omission of a CsCl step may enhance recovery and assembly of viral sequences in soil metagenomes.  Since CsCl purification has been a central piece of viral metagenome library construction for over a decade, this strikes me as a bold claim requiring considerable support.  Part of the challenge here is that no one knows what the viromes “should” look like, what their true structures are.  While it is convenient to adopt the position that the method that gives us “the most” must be “the best,” there are reasons why this might not always be the case.  For example, inclusion of non-viral sequences interpreted as an increase in viral richness.  Is it possible to conceive of an experimental design wherein the true viral community composition is known, and therefore, you can determine how close your methods got you to the actual answer – a kind of control?

Related to this, it is not at all clear whether the omission of CsCl will give similar results in other soil types for example, highly mineral soils or high clay soils.  The soils surveyed here are all similar in terms of their formation in tundra, with lots of organic content.  Indeed, the authors state that samples were collected within 120 m of each other.  So, as interesting as the results are, it is very difficult to know whether these methods could be applied to any other soils with the same result.

Fig. 5. I am not sure why the square root of copy number shown and not actual copy number.  What is the reasoning behind this transformation?


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: TOWARDS OPTIMIZED VIRAL METAGENOMES FOR DOUBLE-STRANDED AND SINGLE-STRANDED DNA VIRUSES FROM CHALLENGING SOILS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The is a well written, clear manuscript which deals with the generation of viromes from soil samples. Several common DNA extraction and purification methods are systematically evaluated against DNA yield, purity, successful metagenomic library preparation and assembled virus contigs.  The finding will be very useful to anyone working in the field of virus metagenomics, as an optimum extraction method is identified for soil samples, and one common virome purification methods (CsCl purification) is shown to significantly reduce DNA yield and the number of virus contigs assembled compared to other methods. This study also addresses ssDNA and dsDNA virus ratios by using a relatively new library preparation kit to infer that dsDNA viruses make up the vast majority of particles in the soils tested.
Experimental design: see General comments
Validity of the findings: see General comments
Additional comments: L26  superscript on 10^7 and 10^9 didn't come through on my copy.  Virus-like-particles or confirmed viruses?

L102 What were the units of DNase used?

L105 superscript on cm3

L197 - Can you label this header "Experiment 1: Different DNA extraction......" for clarity.

Figure 2A/B: Nanodrop absorbance ratios: I'm not clear how you managed to get reliable 260:280 ratios given such low DNA extraction concentrations shown on Figure 2A? They are often unreliable at such low concentrations. The total DNA yield would also be of interest in the text somewhere, I didn't see the volume written.

Figure 2C - You make a valid point (L213-214)  that A260:280 is a better predictor of library success than A260:230 (which is the main reason for doing this study). Given you don't use the 260:230 ratios any further in the study I would consider removing Figure 2C. It is a busy figure and distracting with the wide error margins. Also, ANOVA would be preferable to doing multiple t tests on all the different combinations. Possibly the low DNA concentrations are throwing off these ratios and producing the high error values or some unimportant impurity is left in the sample. Either way, I don't think you need 2C as the success criterion is library generation which you deal with later on.

L217-218 "For Bog.....PowerSoil extracts consistently exhibited the highest A260:230 ratios (i.e. inferred to be cleanest)."  - When  I read Figure 2B, in Bog there is no significant difference between powersoil and the others? CTAB + Ampure fits the ratio limits of 1.6-2.1 best.

L255 Can you label the header "Experiment 2: Heat-based lysis...." so its easier to follow.

L273-274 "CsCl purification had the most impact: yield was higher without it than with it for all but one sample (Palsa, –CsCl[BB])" - Palsa - CsCl BB shows ND (non-detectable DNA concentrations) in Figure 4. "Without CsCl" treatments look consistently the best in all samples from Figure 4.

L291 on the viral signal

L302 - These are the 12 viromes from figure 4? 9 of these had no detectable DNA in them via Qubit. It might be worth mentioning then in L244 onwards that the library prep kit amplifies DNA from the picogram range.

Figure 7 - Can the C suffix be labeled "soil core" or similar in the legend to aid in understanding the groupings.

Figure 3 is not referenced or discussed in 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: TOWARDS OPTIMIZED VIRAL METAGENOMES FOR DOUBLE-STRANDED AND SINGLE-STRANDED DNA VIRUSES FROM CHALLENGING SOILS
Review round: 2
Reviewer: 1

Output:
Basic reporting: I am satisfied with the changes the authors have made in terms of improving clarity/meaning, and inclusion of additional references.
Experimental design: Likewise, I am satisfied with the edits made to address the previously stated concerns.
Validity of the findings: Thank you for the perspective on the nature of the peat soils - particularly the damage incurred by sampling.  This is something that is often overlooked - or at least unmentioned - in field studies, and might be worth mentioning explicitly in this manuscript.

I am satisfied with the edits made, save one specific item.
With regard to confirming increases in DNA yield with (-) CsCl approaches (l. 270-290):  You seem to insist that PCR screening is sufficient to check for non-viral contamination. I am not sure why you could not screen your viral extracts using fluorescence microscopy to check for the presence of cells (or cell-sized bodies).  This would provide a level of confidence in the origin of the subsequently extracted nucleic acids that cannot be achieved with PCR alone.  I really think this should be done (and example images provided in the supplemental materials) to help characterize what is in the (-) CsCl extracts.  It is simple, cheap, and would quickly reveal the extent (if any) of bacterial contamination in these samples, and extinguish any lingering concerns from readers such as myself.
Additional comments: I appreciate your thoughtful consideration of and responses to my questions and concerns.  It can be difficult work to endure criticisms, or to be asked to do even more, when so much effort has already been invested.  Thank you for bearing with the process.  I really think that in the interest of transparency and good reporting, you ought to perform fluorescence microscopy on the (-) CsCl samples and make the resulting data available in the manuscript. I look forward to seeing this manuscript in print after attending to this last concern.


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

Input:
Title: MONITORING THE CRITICALLY ENDANGERED CLANWILLIAM CEDAR WITH FREELY AVAILABLE GOOGLE EARTH IMAGERY
Review round: 1
Reviewer: 1

Output:
Basic reporting: Clear and unambiguous, professional English used throughout:
This is a very well written and coherent paper with almost no English language errors. The few I found are listed below:
“Fynbos Biome” on line 37 should be lower case.
“6” on line 47 should be written as “six”.
Line 88: “satellite based” should be “satellite-based”
Line 89: "Clanwilliam Cedar’s” should be “Clanwilliam cedar’s”
Line 105: “from other species” should be “of other species”.

Literature references, sufficient field background/context provided:
I thought the introduction was succinct, but well contextualised.

Professional article structure, figures, tables. Raw data shared:
The paper conforms to the journal’s requirements with respect to structure.

The one figure also largely conforms to the journal requirements, except needs uppercase labels for the subfigures.

The raw data are made available via Figshare.

Self-contained with relevant results to hypotheses:
The paper represents a coherent, self-contained body of work.
Experimental design: Original primary research within Aims and Scope of the journal.
The research question of this paper, testing the viability of using Google Earth imagery to map and monitor individuals of the Clanwilliam cedar, is clearly defined and very relevant to, among others, conservationists, ecologists and remote sensing scientists.

Rigorous investigation performed to a high technical & ethical standard.
The investigation is highly rigorous.

Methods described with sufficient detail & information to replicate.
In lines 59-60 you state that tree identifications were “verified with ground photographs from a publicly contributed archive accessible through Google EarthTM.” Would it be possible to provide more details on this archive? For example, are you referring to the “Google Maps Photos” and/or “360 Cities” layers in Google Earth?

Could you provide more details of the “three circumscribed field sites” (line 28) used to validate your “satellite enumeration approach”? Perhaps a table with coordinates, or even better a KML file with the boundaries of these sites.
Validity of the findings: Impact and novelty not assessed. Negative/inconclusive results accepted. Meaningful replication encouraged where rationale & benefit to literature is clearly stated.
No issues in this regard.

Data is robust, statistically sound, & controlled.
The data seem to be robust. However, I would like to see a few more details reported to support some of the conclusions:

Line 77: Did you get horizontal mapping accuracy for each individual/clump identified in the satellite imagery? If so, the mean and variability in horizontal mapping accuracy should be reported.

Line 79-80 (and actually 82-84): Can you provide some statistics together with these numbers? For example, what was the total accuracy? Ommission error? Can you make it clear that none of the dead trees were detected in the Google Earth imagery (lines 82-84).

Linked to the above, can you provide an indication of the number of clumps of individuals that were identified as individual trees using the satellite imagery (otherwise your results do not support your conclusion in lines 92-93 (“and cannot discern between clumps of trees and single individuals”).

Although the sample sizes are very small, it would be helpful to report the results per field site (perhaps as a supplementary table, or by subdiving the bars in Fig. 1A to represent the three field sites). This is especially important to be able to support your statement in lines 99-103:  “Our small field survey and work by White et al. (2016) both suggest that population structure, canopy health, recruitment (presence of seedlings) and mortality (presence of dead stems) are highly varied across the Cederberg, cautioning against extrapolation without sufficient sampling, stratified across environmental gradients and spanning the species’ full range”.

Conclusions are well stated, linked to original research question & limited to supporting results.
In general the conclusions were very clearly stated and linked to the original research question. I think the opening sentence of the discussion, “Our satellite based approach did very well to provide a near-perfect fine-scale description of the Clanwilliam Cedar’s distribution...” will be better supported once the above changes are made to the results.
I felt there were only two other statements that need further clarification of the results in order for these to be robustly supported, as described in the previous section:
Lines 92-93
Lines 99-103
I also think that lines 199-122, although undoubtedly true, could use a reference to support this statement.

Speculation is welcome, but should be identified as such.
I felt the speculation in the discussion was reasonable and well supported. For example, in lines 97-99, the authors state “With good field estimates of the species’ size class distribution and canopy health it would be feasible to provide a relatively accurate estimate of population numbers based on the locality data”.

In the lines following the above statement, lines 99-103, I have already highlighted that I feel the authors need to provide a breakdown of the results by each of the three field sites in order to support  their suggestion that “population structure, canopy health, recruitment (presence of seedlings) and mortality (presence of dead stems) are highly varied across the Cederberg...”
Additional comments: Widdringtonia cedarbergensis Marsh is now recognised as Widdringtonia wallichii Endl. ex Carrière (e.g. http://www.theplantlist.org/tpl1.1/record/kew-2466623)

I wonder if it would be helpful to have an additional few subfigures in Figure 1 where each of the field sites are shown together with the trees identified in the field, and those identified using Google Earth highlighted in another colour / point shape.


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: MONITORING THE CRITICALLY ENDANGERED CLANWILLIAM CEDAR WITH FREELY AVAILABLE GOOGLE EARTH IMAGERY
Review round: 1
Reviewer: 2

Output:
Basic reporting: Slingsby & Slingsby present an original approach to mapping critically endangered Widdringtonia cedarbergensis trees. The paper provides an interesting case study for the manual monitoring of large trees using open access satellite imagery and the fine-scale location data provided is of great value for conservation, research and teaching purposes.

The paper is generally well written and structured in professional English and reads well throughout. Figure 1a is relevant and well described, though Figure 1b needs both axes labels and a scale bar and could also possibly benefit from an inset to show regional context and if enlarged as a separate figure, the 3 field sites used could be added on.

The introduction could benefit from additional context on whether similar methods have been used before or the novelty of this approach to monitoring large organisms, such as trees.

The raw kml file was successfully downloaded from figshare.
Experimental design: The methods need further detail and information. Specifically, the method validation and ground surveys need clarification. I feel a reference or explanation of horizontal mapping accuracy as a validation method is necessary. It is unclear what constitutes a healthy or unhealthy tree relative to percentage canopy cover.
Validity of the findings: The results needs more detail on the method validation, specifically presenting data on the horizontal mapping accuracy comparisons. I am not experienced in mapping applications, but I assume each sampled tree has an accuracy value in m, meaning a mean ± sd could be reported.
Additional comments: The strength of this paper is the excellent fine-scale, healthy adult, location dataset produced from a relatively efficient and low-cost method. As it currently stands, the method validation has not been clearly demonstrated and requires clarification from the authors.

Specific comments below:

L2. Consider changing ‘from space’ to ‘using high resolution satellite imagery’

L57. Resolution in m/arcsec?

L57. Add a note on the spatial extent of the cedar ‘search’. Only within the Cederberg WA or within suspected historical extent?

L71. “Explore the effect of canopy health.” On what? Satellite detection?

L77. This seems like one of the most important metrics in the paper, yet was not explained in the methods. See previous comments.

L78. This is an arbitrary scale – change to km/km2 covered.

L80. The method identified 21/61 adults when considering all ground surveyed sites, but 9/10 from two of the three sites. Could you explain the discrepency here? Were the trees at the site not included in the canopy health survey particularly unhealthy, with lots of adults with sparse canopies?

L81. I’m unsure why canopy health was included in the analysis, as it is only measured at 2/3 sites and it is not clear how it relates to the original objectives of the paper.


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

Input:
Title: MONITORING THE CRITICALLY ENDANGERED CLANWILLIAM CEDAR WITH FREELY AVAILABLE GOOGLE EARTH IMAGERY
Review round: 2
Reviewer: 1

Output:
Basic reporting: I am happy with the changes made by the authors
Experimental design: I am happy with the changes made by the authors
Validity of the findings: I am happy with the changes made by the authors
Additional comments: I am happy with the changes made by the authors.

On the issue of the scientific name of the Clanwilliam cedar, I am not a taxonomic expert, so would be loathe to make a recommendation in this regard. I think it would be helpful that this issue is clarified with IPNI so that further taxonomic confusion does not ensue. I see that Marsh (1966; Bothalia) does not recognise Widdringtonia wallichii as the accepted name, giving it the assignation of "nom. nud."

Nomen nudum refers to a species "without a description or diagnosis or reference to a description or diagnosis" (https://www.iapt-taxon.org/icbn/frameset/0120AppendixVII.htm). However, the reference for W. wallichii does seem to include a description: https://www.biodiversitylibrary.org/item/106503#page/78/mode/1up (page 62)
This would suggest to me that perhaps Marsh (1966) was incorrect in assigning W. wallichii as "nom. nud.", and because this name is the older of the two potential names, it should be given preference.

However, as I said, I would suggest that this be taken up with a taxonomic expert and, if IPNI and The Plant List need to be modified, these changes be recommended to them.


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: MONITORING THE CRITICALLY ENDANGERED CLANWILLIAM CEDAR WITH FREELY AVAILABLE GOOGLE EARTH IMAGERY
Review round: 2
Reviewer: 2

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: I feel the authors have adequately addressed the issues raised by both reviewers. I can now recommend this manuscript for publication and I look forward to seeing its impact in the field.


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

Input:
Title: A POSSIBLE NEW SPAWNING AREA FOR ATLANTIC BLUEFIN TUNA (THUNNUS THYNNUS): THE FIRST HISTOLOGIC EVIDENCE OF REPRODUCTIVE ACTIVITY IN THE SOUTHERN GULF OF MEXICO
Review round: 1
Reviewer: 1

Output:
Basic reporting: There are a number of issues with the reporting of the results of the study:
(1) title: the title needs to refer to Atlantic bluefin tuna
(2) abstract: the first sentence needs to be refer to Atlantic bluefin tuna. Also, the abstract does not explain why it is important to know whether spawning occurs in Mexican waters (e.g. the importance of this information for fisheries management); the abstract needs a concluding sentence;
(2) line 42: explain the meaning of ICCAT;
(3) lines 51-52: there needs to be some more biological background to the sentence (i.e. it suggests that the tuna migrate into Mexican waters but no information on migration pathways is provided in the Introduction, therefore the relevance of knowing this information is not obvious);
(4) the Introduction does not conclude with statements about why the information collected by this study is needed or important;
(5) some of the information in some figures is unclear: Fig 1 (what is the darker blue shading?), Fig 2 (the units of length on the X-axis are missing); Fig 5 (what types of error bars are shown?);
(6) lines 83-105: the descriptions of the ovarian stages would be better represented as a table rather than text in a paragraph.
Experimental design: I have 3 concerns about the experimental design that cast doubt on the validity of some of the study’s findings and conclusions:
(1) The study used fish that had been caught as by-catch in the fishery for another species of tuna in only a single season. As a result, only a small number of fish were studied, with histological evidence coming from 46 fish in one year. The sampling will not address the possibility of year-to-year variation in timing of spawning, and the sample size is substantially smaller than other recent studies of tuna spawning e.g. Ashida and Horie (2015) used 438 skipjack tuna; Farley et al (2015) used 640 southern bluefin tuna, and Okochi et al (2016) used 1040 Pacific bluefin tuna. The small sample sizes, and the limited spatial extent of the sampling do not provide sufficient information about the relative importance of spawning by this species in Mexican waters.
(2) The conclusion that the southern area of the Gulf of Mexico is ‘a new possible spawning area’ cannot be justified because sampling was not done extensively enough to show that this area is sufficiently distant from the spawning area in the northern Gulf of Mexico i.e. there could be a single large spawning area that covers the entire Gulf of Mexico. The data in the study only show that spawning appears to occur in Mexican waters.
(3) Sampling occurred only between January and April. Therefore the study’s conclusion #1 (line 160) that there is a ‘marked seasonal occurrence’ is not justified. The results show that the species was present for the months sampled but there is no data to show that the species was not present in other months.
Overall, the study provides some preliminary evidence that suggests Atlantic Bluefin tuna spawn in Mexican waters.
Validity of the findings: Covered in previous comments
Additional comments: This manuscript reports the results of a study into the reproductive status of Atlantic bluefin tuna in Mexican waters in the Gulf of Mexico. Fish were collected, as by-catch of the yellowfin tuna fishery, over 4 months in one year and their maturity and gonad state assessed histologically. While i can foresee the need and importance of this study, it was not suitably described in the text. The authors made a number of conclusions about from the limited data set on the timing of migration, the existence of a previously unreported spawning area, and the timing of spawning. The study was undertaken in a scientific manner; however the limited number of samples, and the spatial and temporal extents of the sampling, make this a preliminary study and do not sufficiently justify the authors’ conclusions.


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 POSSIBLE NEW SPAWNING AREA FOR ATLANTIC BLUEFIN TUNA (THUNNUS THYNNUS): THE FIRST HISTOLOGIC EVIDENCE OF REPRODUCTIVE ACTIVITY IN THE SOUTHERN GULF OF MEXICO
Review round: 1
Reviewer: 2

Output:
Basic reporting: in the Introduction section, it is missing some more details about why this area has been previously considered as potential spawning area for bluefin tuna (Abad-Uribarren et al. 2014), for example the occurrence of this species and suitable spawning window related to shift/evolution of oceanographic condition (More details in the attached document).
Experimental design: Overall, very few samples and few months were sampled (More details in the attached document).
Validity of the findings: The obtained results can show a posible new spawning area in southern Gulf of Mexico, as it is noted in the title of the MS. However, the few individuals analyzed it weak point of this study and thus requires to be cautious in the conclusions.
Additional comments: The MS #33905 – “A possible new spawning area for bluefin tuna (Thunnus thynnus): First histologic evidence of reproductive activity in southern Gulf of Mexico” provides information on bluefin tuna (Thunnus thynnus) ovary morphology in different reproductive stages in the southern Gulf of Mexico.
The investigation is primarily focused on microscopic analysis to assess the reproductive development of ovaries and testes. According to the knowledge of this reviewer, the classification methodology used for microscopic analysis is correct but certain concepts in the classification method should be clarified. But the main concern is the low number of samples analyzed and especially those in spawning that finally support the evidence that bluefin tuna conduct spawning activity in the area proposed.

Overall, the article is written in clear English, unambiguous, and it is a technically correct text. The article has conformed to professional standards of courtesy and expression, although certain clarification is required in the Material and Methods section. Overall, the article is concise and to the point and although the context of the study is well set and it includes sufficient literature references, there are missing some more details in the introduction and discussion section about why this area has been previously considered as potential spawning area for bluefin tuna (Abad-Uribarren et al. 2014), for example the occurrence of this species and suitable spawning window related to shift/evolution of oceanographic condition. In the opinion of the reviewer, this will better set the context/rationale of the study and the comparison with northern spawning areas, more than just the observations of different spawning areas in the Mediterranean Sea. The relevance and usefulness of the figure 2 and 5 it is not clear. In contrast figure 1, 3 and 4 and tables 1 and 2 are relevant for the content of the article.

More details about above mentioned are described below:

INTRODUCTION:
As mentioned previously, in the Introduction section, it is missing some more details about why this area has been previously considered as potential spawning area for bluefin tuna (Abad-Uribarren et al. 2014), for example the occurrence of this species and suitable spawning window related to shift/evolution of oceanographic condition. In the opinion of the reviewer, this will better set the context/rationale of the study and the comparison with northern spawning areas, more than just the observations of different spawning areas in the Mediterranean Sea.

MATERIAL AND METHODS
The classification method applied to define mature from immature individuals is not clear and further clarification is required. The authors stated that “females were classified as mature if ovaries contained vitellogenic, MG or HY oocytes and/or atresia”. However, it is not described which stage of vitellogenesis (early or advance) set the limit. As it is written, it seems that E-Vit is delimiting the threshold between immature/mature, and this is not what Schaefer (1998) and Farley et al. (2013) described in their studies. In both papers presence of A-Vit oocytes was the evidence of mature or active females (in Farley et al. 2013 and Schaefer 1998, respectively). This also contribute to the confusion when in line 84 it is defined as developing, ovaries contained E-Vit oocytes as MAGO.

According to the line 82-83 these are mature individuals. A table with histological classification criteria similar to Table 2 presented in Farley et al., 2013 is recommended. This table will also help authors defining the % of atresia used to determine the sexual maturity as it was done in Farley et al. (2013) and Schaefer (1998).

Lines 83-84 and 96-97 seem to be more result and should be moved to corresponding section.

RESULTS
In lines 114-117 I would suggest including number of individuals, as having only the % does not show the few number of individuals analyzed and this information is important for the interpretation of the results and how strength of the conclusions.

In line 114 change “a few…” to one individual. There is only one individual in developing according to the table 1. In line 117 also add how many (n=1) individuals were found at spawning.

Authors did not note if POF were present in analyzed ovaries. I would suggest adding information in this respect as if there were found could better support the hypothesis of a new spawning area in southern Gulf of Mexico.

Table 1 and 2. Could the authors provide the reason to divide the months in early, mid and late periods? How can this improve the analysis of the samples?

There is missing (probably in the Discussion section) the interpretation of the different maturity stages found in female and male BFT along the months analyzed and its relation with the reproductive cycle of this species. 45% of the individuals are in regenerating stage in the months previous to the individuals found spawning capable and spawning stages.

DISCUSSION
In the first paragraph of this section (lines 127-134) the authors support the arrival of the bluefin tuna to the study region by the timing of the catch. But also, due to increased feeding behavior before the spawning season, however, this study does not provide any evidence (e.g., stomach content data) to support this statement or literature references of previous studies where feeding behavior of bluefin tuna was described and thus support the hypothesis that bluefin tuna arrive to the area between January and March to increase feeding before the spawning season.

In paragraph two (lines 135-139) could be further develop the point described in Abad-Uribarren et al. 2014 where the suitable thermal and productive condition is related to potential bluefin tuna reproductive activity. And then compare with the conditions found in the northern Gulf of Mexico during the same period.

In paragraph tree (lines 140-155) and specially in lines (146-150) the authors consider as new spawning area the southern Gulf of Mexico based on the data analyzed. I would suggest to be more conservative in the statement considering there is only one female in spawning and that there was not found other spawning markers in other ovaries from January to April.

CONCLUSION:
Considering the few individuals analyzed and the only one female in spawning found in the study I would start the section with the last proposed sentence “Additional studies are needed …” and then “However… in the present study four facts provide evidence for considering…”. And again I would be very cautious in the fourth point as only one female was found in spawning and there were not found any other spawning markers such as POF that could better support the spawning activity of bluefin tuna in the southern Gulf of Mexico.


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 POSSIBLE NEW SPAWNING AREA FOR ATLANTIC BLUEFIN TUNA (THUNNUS THYNNUS): THE FIRST HISTOLOGIC EVIDENCE OF REPRODUCTIVE ACTIVITY IN THE SOUTHERN GULF OF MEXICO
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have adequately addressed, in the revised manuscript, my comments relating to their reporting of the study.
Experimental design: The authors have adequately addressed my comments about the experimental design in their rebuttal and in the revised manuscript.
Validity of the findings: In response to my review of the original manuscript, the authors have adequately discussed the validity of their study, and its findings.
Additional comments: I am satisfied that the authofrs have adequately addressed my review comments, and their responses in the rebuttal are satisfactory.


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 POSSIBLE NEW SPAWNING AREA FOR ATLANTIC BLUEFIN TUNA (THUNNUS THYNNUS): THE FIRST HISTOLOGIC EVIDENCE OF REPRODUCTIVE ACTIVITY IN THE SOUTHERN GULF OF MEXICO
Review round: 2
Reviewer: 2

Output:
Basic reporting: Please check general comments
Experimental design: Please check general comments
Validity of the findings: Please check general comments
Additional comments: The MS #33905 – “A possible new spawning area for bluefin tuna (Thunnus thynnus): First histologic evidence of reproductive activity in southern Gulf of Mexico” provides information on bluefin tuna (Thunnus thynnus) ovary morphology in different reproductive phases in the southern Gulf of Mexico.

The investigation is primarily focused on microscopic analysis to assess the reproductive development of ovaries and testes. According to the knowledge of this reviewer, the classification methodology used for microscopic analysis is correct and the concerns raised during the first revision have been well addressed by authors (adding new tables).  However, the main concern is still the low number of samples analyzed and especially those in spawning that finally support the evidence that bluefin tuna conduct spawning activity in the area studied. The authors, following reviewers suggestions, have been more cautious in their statements and further researcher has been requested in order to support the evidence found.

Overall, the article is written in clear English, unambiguous, and it is a technically correct text. The article has conformed to professional standards of courtesy and expression, and the authors have provided required clarifications in the Material and Methods section.

Overall, the article is concise and to the point and the context of the study is well set. The authors provided further explanations (literature references, e.g., Abad-Uribarren et al. 2014) in the introduction section about why this area has been previously considered as potential spawning area for bluefin tuna.

In my opinión, although the modification of Tables 1-3 can provide more information, there is still missing  the interpretation of the occurrence of different ovary reproductive phases in certain months and its link with the reproductive cycle of the species. For example why 45% of the individuals are in regenerating phase just before the month when individuals were found spawning capable and actively spawning sub-phase.

Minor comments:
In line 102: change gonad by ovary
In line 105: change gonad by testes
I would suggest authors to keep the term reproductive "phase" when making reference to ovarian reproductive phases instead of using the term "stage", that would correspond more to oocyte development stages.


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: 2HYBRIDTOOLS, A HANDY SOFTWARE TO FACILITATE CLONE IDENTIFICATION AND MUTATION MAPPING FROM YEAST TWO-HYBRID SCREENING
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The manuscript introduces a convenient software to facilitate Y2H clone identification and mutation mapping, namely 2HyridTools. Though related software with partial similar functions exist, the authors made a good justification for the necessity of a dedicated Y2H software. The implementation and the test results were clearly stated. This tool will be useful to many users in the future. It is recommended to be published in PeerJ.

There are just four minor comments.

1.The program depends on BioJava. It would be better if it is independent, or detailed instruction should also provide for how to install BioJava.

2.Lines 158 – 161, does vector trimming allow mutations on the tag sequences, which may arrive from sequencing errors?

3.Lines 192 – 193, please clarify “If a C-terminus is not found, only the C-termnus will be translated, but not overlapped…”

4.How does the software handle the scenario that within a single experiment, different sequences correspond to different prey proteins interacting with the same bait proteins? Will it group these sequences according to prey proteins?


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: 2HYBRIDTOOLS, A HANDY SOFTWARE TO FACILITATE CLONE IDENTIFICATION AND MUTATION MAPPING FROM YEAST TWO-HYBRID SCREENING
Review round: 1
Reviewer: 2

Output:
Basic reporting: In the manuscript “2HybridTools, a handy software to facilitate clone 1 identification and mutation mapping from yeast two-hybrid screening”, the authors present a new tool for the post-processing analysis for experiments using yeast two-hybrid (Y2H) and reverse two-hybrid (RY2H).
The manuscript conforms to the structure recommended by the journal viz. Introduction, Materials and Methods, Results, Discussion, Conclusions. There is continuity in the flow for the readers. I appreciate that the authors have provided references wherever applicable. I appreciate the author for providing the source code and making the software publicly available. The English and grammar of the article are good with very few typos. Below are points that might be useful to further improve the paper:

1.	The section “Implementation” should be renamed as “Methods”
2.	Provide a documentation for installation and usage of the software on the Github page
3.	Provide full-form at the first occurrence of the abbreviation
4.	Provide a diagram highlighting the functionalities of the software and the method used to achieve it. I would suggest making it as figure 1.
5.	It would be very useful for the readers and make the software easily adopted by researchers, if you can walk-through an example Y2H and RY2H experiment.
Experimental design: The experimental design has no flaws.
Validity of the findings: The tool is an important contribution for researchers working with Y2H and RY2H. Further improvements as suggested in Basic Reporting section would be useful to the community.
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: IMPROVING CLINICAL REFRACTIVE RESULTS OF CATARACT SURGERY BY MACHINE LEARNING
Review round: 1
Reviewer: 1

Output:
Basic reporting: Some sentences are hard to follow. English editing may help.
Experimental design: Excellent, although some improvements are desirable.
Validity of the findings: Improvements are needed.
Additional comments: Based on machine learning, this study evaluated two models for IOL power calculation. It is an interesting study. However, the following comments should be addressed:

1.	Lines 297-298:  It states “The results predicted by each model were compared against the achieved “clinical results” (CR). Figures 5-8 showed that the mean errors for the CR were about -0.4 to -0.5 D, whereas the mean errors for the two machine models were close to zero. The IOL lens constant should be optimized first in order to achieve a mean error of zero for the CR, and then the MAE and % of eyes within +/- 0.25, 0.5, 0.75, and 1.0 D can be calculated and compared to the results from the machine learning models.
2.	It should be discussed that long eyes over 26.3 mm and extreme long eyes were not included in this study. As shown in table 1, eyes with axial length up to of 26.26 mm were included in this study.
3.	Both eyes of some patients were enrolled. It would be ideal to use one eye from each subject.
4.	Multiple statistical comparison issue should be addressed.
5.	If it is not statistically different, wording of comparable or equivalent should be used. Words of “insignificantly better or worse” should be avoided.
6.	Line 206: “The interrelationship between Rxpost a IOLimplanted…”, should this be “…between Rxpost AND IOLimplanted…”?


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: IMPROVING CLINICAL REFRACTIVE RESULTS OF CATARACT SURGERY BY MACHINE LEARNING
Review round: 1
Reviewer: 2

Output:
Basic reporting: - There are some grammatical and typos errors, the authors should revise it carefully.
- Fig. 2,3,4,5 should be combined together (or to become two figures)
- The network architecture in Figure 6 is not clear and it did not show fully structure of their network.
- To mention the RBF network, authors should cite more recent publications, especially in the biomedical field, such as https://doi.org/10.1186/s12859-016-1369-y and https://doi.org/10.1186/s12859-016-1163-x
- Similar to SVM, it has been successfully used in the recent publications in biomedical informatics, such as https://doi.org/10.1016/j.ab.2019.02.017
- The authors must provide their source code for reproducing.
Experimental design: - The is a need to provide independent testing in their model because overfitting is one of the most concerns for this type of problem. Overfitting needs to be addressed and discussed.
- Authors use two networks (SVM-RM and MLNN-EM), but the performance results are not much different between these two. Why did they need to use both? I suggest that the authors could combine these two networks to become a hybrid network and it should perform better than each network alone.
- The authors should report the range of parameters when they performed hyperparameters optimization.
- Hyperparameters for MLNN need to be reported clearer.
- I wonder the authors should display training MSE and validation MSE in the same graph in Figure 7. It will help readers and reviewer evaluate their model.
- When will the neurons stop to increase in the MSE?
Validity of the findings: - The authors only compared with Clinical Results (CR) is not enough, via CR is only a baseline method. Authors must compare their model with the other models using similar approaches.
- As CNN has been used successfully in different fields, especially in biomedical data such as https://doi.org/10.1016/j.ab.2018.06.011, https://doi.org/10.1002/jcc.24842, or https://doi.org/10.7717/peerj-cs.177, the authors should compare their models with a CNN model. Otherwise, the authors have to discuss this as a further direction.
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: IMPROVING CLINICAL REFRACTIVE RESULTS OF CATARACT SURGERY BY MACHINE LEARNING
Review round: 2
Reviewer: 1

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


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

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

Output:
Basic reporting: In this study, a targeted mannanase gene derived from Bacillus subtilis cAe24 isolate was successfully constructed and expressed into BL21 strain. The recombinant enzyme, RMase24, was subjected to not only examine its activities, but the RMase24 was also utilized for producing mannan oligosaccharide (MOS) from a copra meal. Based on the obtained data, it seemed that MOS5 was significantly increased tight junction integration of epithelial cells when compared with negative control. Overall, this is a very interesting manuscript with a comprehensive investigation, therefore it will certainly attract numerous citations in the future. Also, the scientific background, statistic and the research as a whole are generally sound. However, the manuscript should be thoroughly improved in writing, and some requirements should be revised to meet the standard of publication.

Introduction: Since the RMase24 was expressed from a targeted mannanase gene derived from Bacillus subtilis cAe24 in this study, not only general backgrounds of this isolate but also bacterial/microbial mannanases should be described. If there is a publication of this isolate previously, please obviously state in the manuscript.

Materials and Methods: Strains, media, general experimental conditions, chemicals, etc. should be added in a separate section. More performed information should be described for sections of SEM-EDS (line 126-128) and effects of metal ions and some chemical reagent on the activity of RMase24 (line 159-162). Writing style should be in passive (for example, revision should be applied for line 113-115, 117-119, etc.). Specific information should be stated (for example, whether “BL21” in line 142 is E. coli BL21 and which source of this strain? When is “the digestion reactions were completed” in line 166? After 24 hrs?). More performed information on TEER measurement should be supplemented.

Results: To comprehensively understand the unique features of RMase24, it is interesting and necessary to know about Bacillus subtilis cAe24 isolate and in silico information of the targeted mannanase gene derived from this isolated. Therefore, I strongly recommend adding this section in the revised manuscript.

Discussion: The authors not only mostly repeated the description of result section, but discussion and comparison with others are largely lacking. Please discuss more bacterial/recombinant mannanase, MOS production, and purification, substrate resources, etc. Any connection of enzyme activities and its bioactive function may be clarified from this study? It is also interesting to discuss and compare with other publications about the mechanism of action of MOS5 as well as its potential application in the future.
Experimental design: For a logical and comprehensive story, preliminary examinations regarding Bacillus subtilis cAe24 and targeted mannanase gene derived from this isolate, leading to the conduction of this study, should be represented as I mentioned above.
Validity of the findings: There was no data for statements from line 211-215.

Figure 1: Figure legend should provide brief information on a specific method to obtain the data. Figure 1C is confusing and may overlap with Figure 1A? Please clarify obviously the statement for this results in line 220-223. Please add statistical analysis, especially for Figure 1E.

Figure 2: If Figure 2C and D are magnifications of Figure 2A and B, respectively? If so, which parts in Figure 2A and B were magnified?

Figure 3: This figure seemed hard to see “size ranging from M2 to approximately M12” as the statement in line 253 of the text. Also, which data did the authors claim for “The percentage yield of total MOS production from S-GalMan and G-GalMan were 8.25% and 5.09% the substrate (dry basis), respectively” (line 256-257).
Additional comments: There are many typos in wording and grammar throughout the manuscript, therefore it should be revised by a native speaker.

Specific reference should be cited immediately after the statement. For example, “Several reports revealed that MOS supplement could improve growth performance and body weight in various animals” in line 52-53, “MOS is often prepared by hydrolysis reaction of a mannose-contained glucan polymer, mainly glucomannan and galactomannan” in line 68-69, etc.

Citation in the main text should be revised with the surname of reference authors. For example, “Mourao et al. 2006” instead of “Mourao JL 2006” in line 61 and 62, “Maeda & Sugiyama1980” instead of “Masaakira MAEDA 1980” in line 73, etc.

The references should be in an inconsistent format. For example, line 353, 361, 365, 406 (capitalized in journal name); line 355 (capitalized in paper title and journal name); line 377, 400 (abbreviated in journal name); line 385 (species name should be in italics); etc.