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: CHITIN DISTRIBUTION IN THE OITHONA DIGESTIVE AND REPRODUCTIVE SYSTEMS REVEALED BY FLUORESCENCE MICROSCOPY
Review round: 1
Reviewer: 1

Output:
Basic reporting: -	In general, the manuscript is nice to read, however, there are some minor language issues. Therefore, the assistance of a native English speaker for proof reading would be helpful.

-	The current state of anatomy research of copepods is described insufficiently and the background should be significantly refined.
Particularly, since this study focuses on development of the new staining method, it is especially important to compare it with other staining techniques used for studying digestive and reproductive systems of copepods. However, references to many approaches, which were used in previous studies are lacking, e.g. to gonad observations of formalin preserved individuals (without staining), staining with the borax carmine, fast green, molecular fluorescent probes, gut fluorescence analysis, etc. (e.g. Tande & Hopkins, 1981; Batchelder, 1986; Wolfram et al., 2014; Niehoff & Runge, 2003). So I strongly recommend to briefly review these methods in the introduction and point out the advantages/disadvantages of the proposed double staining (DAPI+FITC) in the discussion section.

-      The ms structure is clear. I appreciated the high figures quality, which is usually difficult to get when studying such small organisms. One minor omission – the absence of explanation of the abbreviation (“go” – gonad?) in the legend to the Fig. 4.
Experimental design: There are two important points related to the methodology, which substantially decrease the value of this study:

-	The results of the study did not convince me that the double staining with FITC and DAPI is the appropriate method for the study purpose.
To my opinion, figures 1-3 indicate that DAPI staining did not reveal more details of anatomy of the reproductive system than FITC staining (except of the presence of semen). The ovary, oviducts and seminal receptacles are well seen after FITC staining, so additional DAPI staining seems to be excessive. However, my conclusion is based on image analysis, maybe under the microscope it looks different. Nevertheless, in the present form the advantages of staining with DAPI are not obvious for readers and it should be either carefully justified in the text or the findings should be considerably re-considered.
The need for using FITC is well explained in the Introduction (lines 33-40), but the reasons for subsequent DAPI staining are not discussed there (only briefly in Methods, line 57), so I suggest to insert this information to indicate clearly, whether DAPI staining is really necessary. DAPI is a non-specific fluorochrome causing fluorescence of all tissues and it is not obvious from the text why it has been chosen to study the reproductive system instead of, say, lipid-specific stains.

-	The quantity of material, which has been analyzed, is not clear enough.
The number of samples should be indicated as well as numbers of individuals stained (for both Oithona species) and their age (copepodite stages/adults).
Please, clarify also, why such sampling periods (November 2016, January 2017, March 2017) was chosen. To my knowledge, copepods usually do not reproduce actively at low temperatures and these months do not seem to be well suited for studying their reproductive system (in the peak of reproductive season much more gonad stages are available).
Validity of the findings: -	Some generalizations in Discussion are not supported by references and the presented results.
For example, one of the findings discussed (lines 129-130) is that the presence of chitin throughout the digestive system, distinguishes Oithona from other arthropods. However the authors provided references only about decapods and insects for comparison (lines 128-129), whereas phylum Arthropoda includes also many others taxonomic groups (e.g. subphylum Chelicerata, Myriapoda). So the statement about “other arthropods” (line 130) should be removed or supported by appropriate literature data.
In addition, in the Introduction it was mentioned (lines 28-30) that some free-living copepods contain chitin in the anterior and posterior midgut peritrophic membrane (PM) according to previous studies. Therefore, if I understand properly, the chitin localization is similar to observed in Oithona (throughout the digestive system)? If so, this finding (lines 129-130) should be revised.

-	The description of some results is not sufficiently detailed:

1.	It is reported that chitinous elliptical structures of unknown function were observed AT the exopods (line 81) and IN the exopods (line 122). Therefore, it is unclear whether they are internal structures or localized at the surface. In addition, please indicate were these chitinous structures  observed only in O. nana females (and what is about O. similis)? Did they present in males, copepodits and nauplii? Besides of luminous glands (line 123) these structures could be for example, a result of moulting. Anyway, more detailed data about these structures should be provided to make assumptions.

2.	Since two related copepod species were studied, it would be good to discuss similarity/dissimilarity in their internal anatomy separately.  This will be a valuable addition to the discussion.

3.	It is unclear what hypothesis was tested by analysis of correlation between the presence of intestine shrinkages and fecal pellets (lines 139-140). If the shrinkages are temporal structures visible only at the moment of gut contraction, the absence of shrinkages does not necessarily reflect that they are not involved in the pellet motion. Therefore, the correlation analysis seems to be useless here. In addition, the fact that some observed individuals did not have shrinkages is not indicated in the results (lines 94-97). Please, clarify also whether there were copepods with shrinkages and without pellets and vice versa (without shrinkages and with pellets), if you discuss this item.
Additional comments: Dear Authors,

I found your manuscript about anatomy of important but yet understudied Oithona species interesting, but requiring a serious revision. The proposed WGA-FITC staining procedure gives very nice results and seems to be the helpful tool for studying digestive and reproductive systems of copepods. However the rationality of subsequent DAPI staining causes some doubts (please, see my detailed comments). Unfortunately, the gaps in explaining the relevance of the new staining method, serious inaccuracies in literature review and discussion listed in my comments reduce the potential impact of this study, thus I would recommend an additional round of careful editing. Hopefully, my comments will help you to improve the manuscript.

Best wishes,
Katya


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: CHITIN DISTRIBUTION IN THE OITHONA DIGESTIVE AND REPRODUCTIVE SYSTEMS REVEALED BY FLUORESCENCE MICROSCOPY
Review round: 1
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: The authors write that the samples of zooplankton after collection were immediately placed in ethanol (line 50-51). This indicates that the procedure for defecation of the copepod intestine was not performed. The question is «why»?
The Copepod are filters and can consume food from a water that differs in composition and genesis. The composition of which may include marine fungi plankton and detritus containing chitin. The authors write "In both Oithona species, we detected chitin throughout the digestive system, which distinguishes it from other arthropods." (line 129-130). Probably, this coloring gave not only chitin lining the walls of the intestine, but also chitin, contained in the food of crustaceans. I would like to clarify this point. Why the authors in the discussion do not consider the possibility that the full intestine of the crustaceans has distorted the data on the distribution of chitin in the digestive tract of copepods.
Validity of the findings: No comment
Additional comments: line 154-155
"The application of the method to Oithona extended the knowledge of the biochemistry and structure of its digestive and reproductive systems".

replace with

"The application of the method to Oithona extended the knowledge of the structure of its digestive and reproductive systems".

because the biochemical structure in this work was not considered. In fact, the article is devoted to the morphology digestive and reproductive systems and methods of their investigation.


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: CHITIN DISTRIBUTION IN THE OITHONA DIGESTIVE AND REPRODUCTIVE SYSTEMS REVEALED BY FLUORESCENCE MICROSCOPY
Review round: 2
Reviewer: 1

Output:
Basic reporting: General comments:

The ms has been substantially improved, however some important points are still not fully addressed. As I understand, the authors agree that the proposed subsequent DAPI staining provides only minor additional information to the results of FITC staining and seems to be excessive when studying structure of digestive system and gonads. The double-staining is rational for addressing the narrow range of tasks (e.g. to check the presence of semen). So, it is necessary to focus on the appropriateness of the proposed double-staining method (particularly, DAPI step) in results and discussion (not only in the response to reviewer's comments). I can't find this information in the text, however it is one of the main results of this study.
I also have several minor comments concerning the introduction section (some parts should be reformulated), methods and results, please find them below.
Experimental design: Unfortunately, the line numbers indicated in the authors' response do not correspond to the actual changes...

Introduction

The paragraph about methods of studying copepods is still confusing and difficult to read, reformulate it, please:

Lines 58-61: It could be compiled since electronic microscopy has been widely used for studying different copepods (not only calanoids and Oithona). It is also important to indicate that these methods are additional to light microscopy observations. For example: “The details of external anatomy of copepods has been also analyzed through Congo red fluorescence [18] and electronic microscopy [19], [2].”

Lines 61-64: Should be shortened (formalin is an aldehyde, so “or” could not be used here; only cell, but not gonad can have nucleus and cytoplasm).  I suggest something like: “Reproductive system of copepods preserved with aldehydes has been studied by direct observation of individuals [20-23], staining of gonads with borax carmine [22, 24], fluorescent polyunsaturated aldehydes (PUAs) probes [25], or with Fast Green [26].”

Lines 67-69: Please, re-formulate this sentence so that it would begin: “The anatomy of digestive system was studied using...

Lines 74-75: better to move this sentence to the beginning of paragraph (and make it the 2nd). If the mentioned diagrams are based on light microscopy observations it is necessary to notice.

Methods

Line 99: Although the authors consider impossible to provide information about the number of samples and stained individuals, at least data about the stages/sexes of stained copepods are needed here. Please, clarify this before the description of staining procedures.

Line 106: delete the point after g.
Validity of the findings: Results

It is very important to give the general information about the effectiveness of the proposed staining method in the beginning of results, before the describing of detected structures. Please, summarize it also in the beginning of the discussion.

Line 152: as I understand, not only DAPI, but also FITC staining makes the gonads visible.
Line 154: Replace Mironova et al. by Mironova & Pasternak

Conclusions

Line 231-233: it is too specific issue for conclusions, to my opinion, better to delete this sentence.
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: TESTING METHODS TO MITIGATE CARIBBEAN YELLOW-BAND DISEASE ON ORBICELLA FAVEOLATA
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript is generally well written with appropriate, professional use of English throughout.  I recommend the author consider restructuring the introduction. The opening paragraph describes Caribbean yellow-band disease distribution and its history in five sentences. The next paragraph discusses generally, coral disease outbreaks followed by a paragraph that outlines previous treatment attempts for various types of disease, then back to Caribbean yellow-band disease for the next three paragraphs. This structure is a bit disjoint and I would recommend moving the opening paragraph between the second and third paragraphs (after line 85). I believe this would provide a more logical flow from general to more specific. There likely will need to be additional text to create a bridge from the general treatments to more explicitly connect the concept of treatments to CYBD.

The discussion summarizes the findings with additional commentary that provides explanations/interpretations for the treatment outcomes or suggested improvement.

Line 100-101 – The logic behind the statement that indicates CYBD is caused with weakened host immunity seems weak. Lines 87-88 describe CYBD as primarily a disease of the symbiotic dinoflagellates. It doesn’t necessarily follow that it is the host’s defenses that are compromised. There are numerous references that describe various defense systems in algae, including dinoflagellates, thus it is just as likely that it is dinoflagellate's defense system that is compromised as that of the host. Recommend reconsidering this statement since it is speculation and modify.

Line 349 – please clarify whether the epoxy application was intended to suggest a mixture of peroxide and antibiotics or should it read ‘peroxide or broad spectrum antibiotics…’?

Lines 376-378 – Recommend that the authors consider other alternatives to just a decrease in virulence when comparing Puerto Rico to the Buck Island populations. For example: Were the measurement methods comparable? Were the genotypes of Orbicella sp. comparable?

Figure 4 – The image in the shaded x July 15 appears to be a different magnification than the others in the shaded series of photos. For other images, particularly in the chiseled series add small arrows to depict areas described in the text to assist reader in orienting to the particular area of interest being described. I would suggest also for the authors to investigate whether color correcting to remove some of the blue hue in these photos could help increase the contrast and clarity for the reader, since this panel of photos is relatively small. The methods do not describe the photography, but it appears that the images could have been improved with proper lighting, for these photos as well as others in the manuscript.

Figure 6 – Similar comments as for Figure 4 regarding color correction. Also arrows or other notations that correspond to the text description of these images would be helpful.
Experimental design: Overall the experimental design is sound, is sufficiently replicated and demonstrates three different approaches to treating Caribbean yellow-band disease and the treatment outcomes. It is also worth noting that the extended observation time was critical to the final conclusion regarding the efficacy of the chiseling method. Although a fully successful treatment was not discovered, means of treating priority, high valued coral in the field is critical for coral conservation given the current state of disease in many coral populations, which exceeds the normal carrying capacity of disease in a population and this work moves us a step forward in identifying treatments that work.

Most of the methods are described in sufficient detail to evaluate their soundness and allow replication. However, this falls short in the description of the digital photography and details for the image analysis and software used. Given the photos provided in the various figures it is unclear how measurements were made to sub-centimeter accuracy. Though the text indicates photos were taken from the same angle, the scale used seems quite coarse and often at various angles in the examples provided. The distance from the subject is also important in a repeated measures design. A more thorough explanation by the authors on how they achieved the precision of the reported rates of lesion movement is important to include in the manuscript as well as any caveats. It is well known that even slight differences in angles between camera and lesion, placement of the scale or ruler and distance from the image can generate significant differences in the resulting measurements. The authors are referred to Chapter 35 of the book Diseases of Coral by Moses and Hallock. This chapter provides details of the digital photography methods and image analysis for assessing wound healing in coral. Also suggested is that the authors review the method of digital planimetry (e.g., Plos One 10(8): e0134622 and other references). For precise measurements there are specific criteria that need to be followed, it is not clear that these were used here.
Validity of the findings: Although there is concern regarding the precision of the measurements reported, the overall findings and conclusions appear sound. There is sufficient replication of the treatments in the design and statistical analyses are sound. I believe the suggestions and recommendations detailed in other sections of this review can provide improvements to the manuscript.
Additional comments: 1.	Lines 46-59 – the sentence beginning on line 57 seems redundant with a previous sentence beginning on line 52. Recommend rewording sentence on line 57 to: While it is seldom reported prior to the 2005 Caribbean coral bleaching event, CYBD is now common on many Caribbean reefs.
2.	Line 70 – Suggest using a more specific description of the ‘modeling clay’ to avoid confusion. I believe it is a specific artist clay with defined characteristics (Roma plastilina #2) that was used in the reference and subsequently recommended by the FL Keys National Marine Sanctuary for backfilling divots or bare skeleton resulting from sampling or treatments.
3.	Line 73 – suggest breaking the sentence beginning on line 72 after ‘mixed success’. And begin next sentence with “These include; …”
4.	Line 91 – suggest breaking sentence after the citation list and begin the next sentence “Further, Koch’s postulates…”.
5.	Line 92 – suggest revising sentence to reduce wordiness to “However, studies clearly show that the bacterial communities…”


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: TESTING METHODS TO MITIGATE CARIBBEAN YELLOW-BAND DISEASE ON ORBICELLA FAVEOLATA
Review round: 1
Reviewer: 2

Output:
Basic reporting: This is an interesting paper because it explores one of the most neglected fields on coral disease research which is the development of treatments to mitigate the effects of disease on their host, to cure or to expand the life spam of infected hosts. It is also valuable because the model-host-pathogen selected is without hesitation among the most worrisome problems on Caribbean reefs nowadays. The paper is well-written, literature review is pertinent, hypotheses are clear as well as results. There are some minor statements which I consider to be wrong (e.g. CYBD lessons extend perpetually). While this may actually happens, it is not always like that, some times the disease arrest, progression slows down and even stops for years and in some cases the disease reactivates. Yet, I have seen colonies that might fully recover. This pattern is actually an unexplored field perhaps determined by a combination of environmental-genetic factors affecting host-pathogen interactions, resistance, susceptibility and virulence. I think this needs to be further discussed  in the manuscript. Like in many host-parasite systems, there are some host that can cope with the infection better than others, and that also applies to treatments because not all hosts respond in the same way to them. Question is why?

There are other interesting findings of CYBD that are missing in the introduction that can be used for this paper. For example, Guerra et al. (2014) found that the gross (relative) chemical composition of CYBD tissues are different compared to healthy tissues.

In Material and Methods, there is an assumption for selecting the treatment of shading: "shading reduces light and the potential built up of ROS which should increase immune competence" In my opinion this assumption is not quite well supported for CYBD. There are a couple of papers showing that immunologic responses in CYBD tissues are compromised (e.g. Mylardz et al. 2009, Montilla et al. 2016). So I do not think, that shading will improve immunological responses in this case, cause CYBD tissues have been shown to be compromised already.

As for experimental design. There are some points that in my opinion deserve some discussion and in other cases some relevant considerations that need to be clarified in order to better judge the validity of the results presented here. Briefly:

(1) I am happy with the application of the treatments but I am not clear with the controls that were used. Authors talked about controls, but I am not sure if these are absolute controls (i.e., diseases colonies without the treatment, no shading for instance) or procedural controls (e.g. a set of colonies with a frame similar to the one used for shading but allowing light to penetrate). For the aspiration treatment, the control would be no aspiration (untouched corals) and the procedural control the same mechanical treatment but without removing the tissue completely (i.e., vacuum off). As for the fire wall, the procedural control would be scratching the tissues without creating a deep trench in the tissue.

(2) According to the results, authors did not find an effect. Given the low umber of replicates used (3 colonies), I think it is necessary to estimate the achieved statistical power. In other words, it is imperative to convince the reader that authors did not found a false negative result (i.e., retain the null hypothesis when it is wrong).

Discussion and conclusions: the literature review is Ok, the discussion is focused on author's results. However, I think it is necessary to better explain the nature of controls used in the experiment (i.e., absolute or procedural) to better judge the validity of these findings.
Experimental design: The paper is original and interesting. The questions are clearly stated and well supported from theoretical backgrounds, ethical standards are fulfilled, the technical quality of the paper, however, needs to be revised for more detail on methods and experimental design used needs to be presented (see below).

Please explain the use of controls in the experiment. I am not sure if these are absolute controls (i.e., diseases colonies without the treatment, no shading for instance) or procedural controls (e.g. a set of colonies with a frame similar to the one used for shading but allowing light to penetrate). For the aspiration treatment, the control would be no aspiration (untouched corals) and the procedural control the same mechanical treatment but without removing the the tissue (i.e., vacuum off). As for the fire wall, the procedural control would be scratching the tissues without creating a deep trench in the tissue.
Validity of the findings: The results are novel. Replication seem to be low, i.e., only three colonies per treatment. While this is not a problem per se, I believe that power analysis must be presented. For instance, what is the maximum achieved power given a size effect and the actual total variance? What would be the sample size that was required to achieved the power desired? It is important to show the readers that there no effects for two out of three treatments and this is not a problem of a huge type 2 error due to low replication.

Conclusions will be validated if authors convince me why the lack of procedural controls like the ones used, for instance, in classic exclusion experiments whereby treatments are: cage (complete exclusion), open treatments (no cage or exclusion absolute control) and cages without cap (procedural control for the device or artifact used in the experiment).
Additional comments: I think this a great idea and a great paper. The literature review is very good, the way hypotheses and models supporting the hypotheses are presented is very nice. While I think sample size for each treatment is low, i celebrate the temporal component of the experiment. It is very nice to see that colonies were monitored for long time. I think this sort of papers are needed and represent an important contribution for coral disease research.

My only concern is the use of controls, I think, there is a lot of manipulation in this experiment, therefore artifact controls (not only absolute controls) were needed.

I would like to see and looking forward to listed author's arguments before I decide whether or not this paper can be accepted for publication.


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

Input:
Title: TESTING METHODS TO MITIGATE CARIBBEAN YELLOW-BAND DISEASE ON ORBICELLA FAVEOLATA
Review round: 1
Reviewer: 3

Output:
Basic reporting: This is an excellent manuscript in terms of writing and editing, making it a pleasure to read! Background research on the problem studied is solid and focused; photos of the field experiment and detailed illustrations are included, and raw data are shared. However, coral scientists continue to use terminology that is inappropriate or incorrect to describe what is happening as coral tissue disappears from the skeleton in diseases. Terms and descriptions from pathology are needed. For example, the definition of "disease progression" is “The worsening of a disease over time” https://www.bing.com/search?q=disease+progression+definition&form=EDGHPT&qs=AS&cvid=bef1590c7876433c9aa6a5747bff3eb1&cc=US&setlang=en-US. “Progression” and “advancement” have been used in papers on coral diseases when explaining the continuing loss of tissue from the skeleton, as seen in several diseases given various field-identifying names, such as white plague, white-band disease, and Caribbean yellow-band disease. However, the disease is not worsening in severity or intensity over time, just its effects, i.e., the amount of coral tissue killed at the colony margin (not “front”) has increased. "Front" is not being used correctly in the phrase “disease front.” “Front” refers to an orientation, as in what someone or something is facing, or faces forward or in a particular direction, or an area along the edge of something, not the edge itself. That should be changed to “tissue-loss margin.” In multiple places "disease [or diseased] lesion" is used. A lesion is any area of abnormal tissue and it is abnormal when it has a functional impairment of any kind, therefore it is diseased, by definition. Also, lesions do not “advance,” as in “move,” but enlarge as more cells die, lyse, and disappear from the skeleton. Finally, the term "virulence" is often used with "disease," when virulence, which is a pathogen or microbe's ability to damage or infect a host, should refer to the pathogen (any virus, microorganism or factor, biotic or abiotic, that causes disease).  Detailed edits on necessary wording changes are provided in the general comments section.
Experimental design: This manuscript reports on well-designed experiments to test hypotheses of methods that might be used to control tissue loss in CYBD-affected corals. The literature review supports the gap in knowledge and the experiments could probably be replicated based on the information provided. They were performed with high technical and ethical standards. The field permit is identified in line 130.

Technique 1: Number of lesions on number of colonies treated is not provided in text (is in results figure, but could be stated in text, too).

Technique 2: Supplemental Figure 1, why not put this with the main paper? In middle text box, “syphon” should be spelled “siphon” (the latter is the more common or usual spelling, at least in the US, also change it elsewhere, as in Line 167)

Line 225: not exactly sure what is meant by “had reconnected with the disease lesion.” In Figure 3b, it appears that the tissue remaining outside of the chiseled groove has regained normal color and what was apparently healthy tissue at the time of the chiseling has now filled in that groove to reconnect with the formerly diseased tissue. So, may be best to write “had filled in the chiseled groove and reconnected to the formerly diseased tissue”?
Validity of the findings: The findings are consistent with the methods used and the resulting data obtained. The discussion is well done, except for including a paper that is not relevant. The supporting literature is otherwise appropriate and the conclusions are valid, with good suggestions for future research and application of tissue loss control techniques for diseased corals. The conclusions are well stated, cover the original research question, and limited in scope to supporting the results.
Additional comments: Line 26: change “lesions advance slowly” to “lesions enlarge slowly”; not all cases result in complete loss of tissue from a colony, so change “eventually leading to” to “often resulting in”

Lines 27-28: change “The slow advancement of CYBD, however,” to “The slow loss of tissue in CYBD, however,”

Line 30: change “mitigate disease progression” to “mitigate the disease” or “minimize disease impact” (last preferred, if leaving “to mitigate CYBD” on line 9)

Line 32: change “progression” after CYBD to “tissue loss”

Line 33: change “rate of disease progression” to “rate and amount of tissue lost”

Line 37: change “slows disease progression” to “slows tissue loss”

Lines 42-43: change “slow and variable progression” to “slow but variable rate and pattern of tissue loss in this disease.”

Line 76: change “white-sign” to “tissue-loss”

Line 93: change “on diseased lesions” to “on and in diseased coral tissues” AND change “on healthy colonies” to “on and in apparently healthy coral tissues”

Line 106: change “the lesion front” to “the tissue loss margin”

Line 125: change “thick” to “wide”

Lines 126-128: confusing description of the lesions here, suggest changing to “A gradient of tissue discoloration is present, with apparently normally thick and pigmented tissue progressively thinning and becoming paler yellow at the dying margin, which is usually smooth with an indistinct edge. The algae biofouling community that colonizes the denuded skeleton is visible adjacent to this edge, i.e., areas of stark white recently denuded skeleton are rarely seen.”

Lines 128-129: change “progresses slowly, either” to “proceeds slowly, as the tissue-loss lesion expands, either”

Line 137: change “progresses slowly” to “tissue loss proceeds slowly”

Line 139: change “disease progression” to “the rate of tissue loss”

Line 143: change “progression” to “tissue loss”

Line 146: change “disease progression” to “tissue loss”

Line 152: change “of coverage of healthy tissue beyond the disease front” to “coverage of apparently healthy tissue adjacent to the thinning yellow tissue margin”

Lines 153 and 374, 375: Need en dashes instead of hyphens between the numbers of cm to mean “to” in the range of values

Line 162: change “sealing the diseased-tissue front” to “sealing the tissue loss margin”

Line 190-191: change “diseased lesion” to “lesion”, insert “apparently” between “adjacent” and “tissue” and delete “that did not show visible signs of disease.

Line 191: “a bacterial pathogen” but in some papers the disease might be caused by multiple bacteria species?

Line 193: change “If CYBD is” to “If the pathogenic microorganisms causing CYBD are”

Line 199: change “and the disease front” to “and the yellow tissue margin”

Line 204: change “of progression of both control lesions and treated lesions” to “of tissue loss from control lesions and treated lesions”

Line 210: change “estimate of linear disease” to “estimate of the rate of linear tissue loss”

Line 211: change “progression” to “during this time (disease progression)”

Line 212: change “shaded bands progressed more slowly than” to “tissue loss on the shaded parts of bands was slower than”

Line 213: change “progression” to “tissue-loss”

Line 223: change “barrier between the diseased lesion and the remaining coral tissue” to “barrier between the lesion [OR the diseased tissue] and the remaining apparently healthy coral tissue”

Line 224: add “apparently” before “healthy”

Line 225: not exactly sure what is meant by “had reconnected with the disease lesion.” In Figure 3b, it appears that the tissue remaining outside of the chiseled groove has regained normal color and what was apparently healthy tissue at the time of the chiseling has now filled in that groove to reconnect with the formerly diseased tissue. So, may be best to write “had filled in the chiseled groove and reconnected to the formerly diseased tissue”?

Line 230: “condition” would be better term than “health,” since that should be determined by multiple parameters, not just gross observations

Line 231: here ‘healthy’ has been defined as being based on gross observations, so this is fine; change “no macroscopic” to “no grossly visible” (also on line 281)

Line 233: change “disease progression” to “tissue loss”

Lines 233-234: “the diseased lesion” is unclear. Would the tissue have reconnected to diseased tissue, was this seen (not apparent in Figure 3b)? Would changing this to “the formerly diseased tissue” or “the now apparently healthy tissue” be better here?

Line 236: delete “disease” before “lesions”

Lines 237–241: Change “progression rates” to “tissue-loss rates” or “tissue loss-rate data”

Line 255: change “to progress” to “to enlarge”

Line 256: change “The disease front advanced….and the rate of progression was not significantly reduced” to “The tissue loss rate was approximately 0.4 cm per month and was not significantly reduced….”

Line 258: change “diseased lesions also appeared to widen and bleach” to “width of the discolored tissue at the lesion margin increased and bleached…”

Lines 263-266: A very interesting observation! Did the tissue include polyps and coenenchyme, and were these visually normal in structure and behavior, if not also color? Perhaps the only zooxanthellae that were available to repopulate the tissue was the clade reported to be resistant to the pathogen(s) (whatever it is), which is why the discoloration is translucent yellowish in this disease. Need to describe in more detail what was observed, to qualify the terminology. Change to “having resheeted as diseased or having resheeted as apparently healthy but subsequently became diseased.” End of line 264: change “advance and cause tissue loss on” to “enlarge with additional tissue loss on”; line 266: change “advancing” to “continuing at”

Lines 271-272: change “disease lesion” to “lesion”; change “main” to “remainder of the colony”

Lines 274-277: change “with high rates of progression” to “at a higher rate”; change “The progression of the disease lesion” to “The enlargement of the lesion”; and “had progressed” to “had developed”

Line 284: delete “diseased” before “lesion”

Line 285: insert “apparently” before “healthy”

Line 287: change “health” to “condition”; “over” to “more than”

Line 290: change “rate of disease progression” to “rate of tissue loss”

Line 294: change “progression” to “tissue-loss”

Line 295: delete “progression” after “disease” (tissue loss is a key feature of the disease)

Line 297: change “progression rates” to “tissue-loss rates”

Line 308: change “disease progression rates” to “tissue-loss rates”

Line 308: change “outlook” to “prognosis”

Line 310: change “progression of various” to “tissue loss resulting from”

Line 312: change “progression” to “rate of tissue loss”

Line 313: change “rate of disease progression” to “rate of tissue loss” or “rate of lesion enlargement”

Line 317: change “infections of endogenous pathogens” to “infections of endogenous pathogenic microorganisms” [pathogens can also be non-infectious]

Line 320: change “of the disease” to “of the pathogens”

Line 321” change “progression” to “tissue loss”

Line 330: insert “apparently” before “healthy”

Line 330: change “disease” in both places to “pathogenic microorganisms” (and “was” to “were” after the second one)

Line 341: change “health” to “condition”

Line 343: insert “apparently” before “healthy”

Line 345: delete “diseased” before “lesion” OR change “lesion” to “tissue”

Line 346: change “lesion” to “tissue loss” AND “progress” to “proceed”

Line 349: change “disease front” to “tissue loss margin”

Line 350: Note that the coral disease community is not advocating use of antibiotics in the field, perhaps this should not be mentioned or state that this will only be tried in controlled laboratory conditions?

Line 353: change “lesion” to “diseased”

Line 355: insert “apparently” before “healthy”

Line 358: change to “yellow-band disease slowly affects tissue, but the rate of tissue loss responds…”

Line 359-362: The “yellow-band disease” described in the Korrubel and Riegl paper was not at all similar to what is now known as CYBD, not only in location and species affected, but also in the description of the lesion. In personal communication with B. Riegl, this reviewer learned that the “yellow band” was more like the fine filaments seen in black-band disease (BBD), but brightly yellow, and that yellow particles remained on the recently denuded skeleton after the tissue disappeared. A large pile of mined elemental sulfur was onshore near this site. Because the microbial consortium in BBD includes all the members of the sulfur cycle, including Beggiatoa spp. that excrete particles of elemental sulfur on the surface of their filamentous cells, what might have happened is that sulfur from the pile had run off or blown into the seawater there and black-band microbes had been metabolizing the excess sulfur. Unfortunately, no other studies were done on these affected corals, including microbiological or histological evaluations, so we cannot be sure. However, this reviewer suggests deleting this reference, because it is not at all related to CYBD, and that rate of tissue loss was similar to what occurs in BBD, another clue to its identification.

Line 362: delete “also”

Line 363: change “progression” to “tissue loss”

Line 368: change“progression” to “tissue loss”

Lines 369 and 371: change “progression” to “tissue-loss”

Line 372: change “CYBD progresses” to “CYBD tissue loss is”

Line 373: “virulence of CYBD” is not correct. In pathology, a disease is not virulent, but a pathogen (abiotic or biotic) has a quality known as virulence, which is a pathogen or microbe's ability to damage or infect a host (“virulence of a disease” may be found in general dictionaries, but this is not used in pathology). This could be changed to “the virulence of the CYBD pathogen(s)”

Line 375: change “progression” to “tissue-loss”

Line 377: change “virulence of CYBD” to “the virulence of the CYBD pathogen(s)”

Line 379: change “coral-disease infections” to “coral diseases” [pathogen may not be biotic]

Line 382: change “infection occurs” to “tissue loss starts”

Excellent ending!

References – some consistency issues in capitalization and formatting

Line 425: “Vibrio” should be “vibrio”

Line 439: fix to “Current Microbiology”

Line 442: for the record, disease names are not capitalized, but yes, this was unfortunately done in this paper title.

Lines 444, 486, 487: book chapters, should follow formatting of reference on Line 410

Line 460: suggest deleting from text and here

Line 477: italicize journal title and volume number

Line 480: fix to “Coral Reefs”

Line 495: italicize “Acropora”

Figure 1 legend begins with “Three disease mitigation techniques.” Then next paragraph says “Three disease-mitigation methods…” Need to put hyphen between “disease” and “mitigation” at beginning to be consistent. The middle 3 lines contain the words “disease lesion” but that only needs to be “lesion”


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: TESTING METHODS TO MITIGATE CARIBBEAN YELLOW-BAND DISEASE ON ORBICELLA FAVEOLATA
Review round: 2
Reviewer: 1

Output:
Basic reporting: I have conducted a second review of this manuscript after revisions.  I find all of the revisions acceptable and the manuscript suitable for publication with the following exceptions:
Line 75 “application of chlorine embedded within epoxy to mechanically and biologically block” It is unclear the intent of ‘mechanically and biologically’ but chlorine is a chemical and thus is not a biological agent for blocking. Rephrase to clarify meaning of biologically or change to chemically or other relevant term.

Line 79 – the reference should be Gignoux-Wolfsohn et al. 2012.  Unless specified by PeerJ it is conventional to use et al. if there are more than two authors.

Line 128 – ‘apparently normally thick…’ should be rephrased.  Suggest ‘….with apparently normal thickness and pigmented….. OR ‘apparently normal, thick and pigmented tissue’ depending on the intent.

Line 129 – ‘which is usually smooth with an indistinct edge’  Suggest clarifying, what is smooth and how is it then an edge indistinct?

Line 129 – correct spelling of ‘biofouling’

Line 131-132 – though I think I understand “centrally located lesions vs peripherally located lesions”, I’m not sure a reader unfamiliar with CYBD would know what this means.  Suggest either providing a figure of the two or improving the description.

Lines 472-474 – This is not the most appropriate reference (in addition to being a single author and the text reflects Fisher et al.) Use instead Marine Ecology Progress Series (2007) 339:61-71.

I believe these are minor and could clarify the manuscript.
Two of the above are corrections that need to be made.
Experimental design: meets standards
Validity of the findings: meets criteria
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: TESTING METHODS TO MITIGATE CARIBBEAN YELLOW-BAND DISEASE ON ORBICELLA FAVEOLATA
Review round: 2
Reviewer: 2

Output:
Basic reporting: Overall I am happy to see that authors undertook major issues of experimental design and data analysis. I am quite happy with their review.

There is however minor issues that needs to be considered before the paper goes into print. First, the paper now acknowledge the lack of procedural controls (i.e., controls for the artifacts used during the experiment), and I think that is honest and great. Authors mentioned that they did not notice any effect of these artifacts (e.g. nails, shading devices, etc) and I believe them. Yet, I think that a paragraph or at least a sentencein the discussion arguing about the potential effects of ignoring these sort of controls on the results is needed.  I strongly advice authors to include procedural controls in their future experiments.

Secondly, there is a conceptual problems (minor one) with the power analysis they performed. Power analysis only proceeds when you don't find significance in your main test. Authors included power analysis in cases where statistical significance was found. They need to delete this from the manuscript.

One can calculate the rate of a false positive for a test that becomes significant, if it is desired, but this is not a power analysis.

I will be happy to endorse the publication once these minor changes are incorporated.
Experimental design: The problem of experimental design cannot be solved unless the experiment is repeated, but at least the authors acknowledge the problem. I think that is the way to go. However, I think that a sentence in the discussion explaining how this may hamper the conclusions is needed. As a reader, I believe in the "qualitative-observational" argument that says "we do not observe any effect" but I also want to read that if artifacts used in the experiments had an effect, how these could have had hampered the results and conclusions.
Validity of the findings: Again, anyone could say that the lack of procedural controls invalidate the results. In this case I don't; but authors need to go a bit over this issue in the discussion.
Additional comments: I congratulate authors for this paper, I do think is a great contribution. I strongly recommend to delete power analysis in cases where statistical significance is found. I celebrate how authors undertook my comments, I am quite happy with that. Yet, I think a sentence in the discussion is need it to argue how the lack of procedural controls would hamper (or not?) the results.

If these minor changes are included, I will be happy to endorse this paper 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: TESTING METHODS TO MITIGATE CARIBBEAN YELLOW-BAND DISEASE ON ORBICELLA FAVEOLATA
Review round: 2
Reviewer: 3

Output:
Basic reporting: I approve of the revisions made to the manuscript, it is much improved.
Experimental design: Additional details and explanations provided in the revision have answered the questions posed by reviewers.
Validity of the findings: The data validity remains sound and additional details on the analyses continue to support the findings.
Additional comments: The revisions made are acceptable and the paper is now in excellent shape! Just a couple of minor things:

Use of "spread" vs. "enlargement" of lesions: Seeing that the co-authors used the former, I looked at papers online to see if there is a preference or particular definition of these terms for pathology. It appears that "spread" is appropriate, sometimes these terms are used interchangeably, or to mean different things (as in a focally, size-limited type of lesion appearing in an increasing area is spreading, whereas the increase in size of a singe lesion is enlarging). So, this reviewer would prefer if distinction could be made for the lesions studied here, but understands that may not be possible.

Line 382: change "progression" to "spread"?
Line 385: add "d" at the end of "disease"
Line 399: break into a new paragraph at "While the...", because it is rather long.

Line 404: the authors note the prevalence of CYBD has decreased in some areas with time. This reviewer notes that fewer cases were seen on Looe Key Reefs in 2011 and it was absent on Bonaire reefs she visited in 2017, whereas it had been rampant there in the early 2000s, but surviving Ofav colonies had continued to grow, building skeleton and live tissue around suspected former lesions.

Line 61: the sentence change made here is good; this reviewer has a photo of an Ofav colony with suspect CYBD taken in 1979 on Grand Cayman reefs and had heard that others were reviewing their photo collections to look for it. (Thought the lesions might have been due to the clionid sponge appearing in the exposed skeleton, but of course, this was before we would think much about coral diseases!)


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 STRATEGY TO FIND NOVEL CANDIDATE ANTI-ALZHEIMER’S DISEASE DRUGS BY CONSTRUCTING INTERACTION NETWORKS BETWEEN DRUG TARGETS AND NATURAL COMPOUNDS IN MEDICAL PLANTS
Review round: 1
Reviewer: 1

Output:
Basic reporting: This manuscript describes the molecular docking of more than 30000 molecules (present in plants used in traditional chinese medicine) to 33 proteins proposed to be involved in the biogenesis of Alzheimer disease.  This impressive amount of docking work is, however, not presented in the most favorable way nor analysed to the fullest:

A) the quality of the language in this manuscript is extremely poor. The manuscript must be checked very thoroughly by a professionally-proficient English speaker.

B) Precise information of the docking poses/putative interactions between ligand and proteins is completely absent, and this strongly limits my enjoyment of this paper. A description of the molecular features of the best ligands must be provided, at least highlighting whether they are relatively strightforward analogs of  previously-known ligands or whether they are novel scaffolds (and in this case, describing the interactions common among members of the same cluster)

C) Figure 4 (and all figures in the Supporting Information) is very hard to read and not very informative due to the large number of labels, small font and lack of legends. I understand that networks are intrinsically hard to display, but I cannot help thinking that figure 4 would be much more informative if it were divided into different figures and (especially) included the chemical structures of the compounds.
Experimental design: line 105 "The docking binding site center for each target is the structural binding center of ligand embedded." I think that authors mean:

 "The docking binding site center for each target is the structural binding center of the ligand present in the crystal structure." If that is so, authors should confirm that they used only structures of complexes of putative AD-targets with their natural ligands (or confirmed inhibitors). Otherwise, there is a possibility that authors have mistakenly used, for example, the structure of a protein with a non-specifically-bound molecule as initial guess for the correct binding site.

line 134: authors should clarify that using fingerprint FP2 a Tc=1 can be obtained from different molecules even if they differ among themselves by isolated instances of C, N or O atoms.

line 146:was the clustering based on the computed Tanimoto coefficients? Please clarify.

line 148: how did the authors select the number of clusters? Was it based on, e.g. , maximum distance allowed between members of a cluster, minimum distance between members of different clusters, etc?

line 148 "If the sum of one member distancing to other members reaches the minimum value, this member is selected as the cluster center."  I think the authors mean: "The molecule with the lowest total distance to all other members of the cluster was taken as the cluster center"
Validity of the findings: Table 1: Please include the identity of the ligand present in the crystal structure as a new column before the "Ligand Energy". I think the table would benefit  from  relebeling the "Ligand Energy" column to "Binding energy of original ligand" and by moving the  "Compound number" column to the extreme right, to ensure that readers do not mistakenly read the "Ligand energy" column as referring to the docked compounds.

line 163: the massive number of compunds  (20000 in 30438)  binding strongly to caspase-3, QC, IDO and GLP-1R is surprising and (if correct) seems to imply that those enzymes would be readily blocked in vivo by 2/3 of the secondary metabolites found in plants. This does not seem very plausible, since enzyme variants  which are easily inhibited would tend to be  deleterious to survival and therefore selected out of the population. Did the authors confirm that those compounds do block the active site, i.e. if their docking poitions superpose with those to the original ligand instead of binding to the protein surface without interfering with the active site?

line 168: typo I think the authors mean "0.5%" instead of "5%"

line 174 and following: authors often use the word "target" when they mean "target protein". This becomes confusing. The description of the number of compounds in two-target and three-target networks is not very informative. I would have preferred  to have found , for example, the structure of the compound which binds 25 target proteins, an analysis of the chemical diversity of the 76 compounds which bind SIRT1, identification of pharmacophores, etc.

Table 2:  The plant best associated with RAR is included, in spite of the poor binding of all compunds to RAR (compared to the original ligand).

Table 3 could be made more informative by highlighting, in each structure of the molecule at the cluster center, the precise core shared by all members of that cluster.The binding energy of the  molecule at the cluster center should also be compared to the binding of the best member of the cluster, to help identify key portions of the molecule
Additional comments: Replace "AD"  by  "Alzheimer disease" at first mention

Supporting Information:

The structures of the compounds in SMILES format are not half as useful as 3D-structures would have been. A link to the precise query used to download those 3D-structures from the http://tcm.cmu.edu.tw/ would probably be more useful. I do not know if the TCM database allows re-distribution of their data: if they do, I would advise you to include the 3D-structures of the 1654 molecules instead of their SMILES strings. If such redistribution is not allowed, I think that providing the SMILES strings of the molecules in each cluster in separate files would be much more useful than the current massive SMILES file.

The Supporting information includes some data on clinical reports of the efficay of traditional Chinese Medicine prescriptions for Alzheimer Disease. The most important data (titles of the studies, their references/sources and results) are unfortunately missing, which severely hampers the usefulness of that information.


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 STRATEGY TO FIND NOVEL CANDIDATE ANTI-ALZHEIMER’S DISEASE DRUGS BY CONSTRUCTING INTERACTION NETWORKS BETWEEN DRUG TARGETS AND NATURAL COMPOUNDS IN MEDICAL PLANTS
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comments
Experimental design: There aren't sufficient informations to replicate
Validity of the findings: no comments
Additional comments: in my opinion, the authors should better describe AD, justifying the choice of targets. They also lack information on how they were obtained.


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 STRATEGY TO FIND NOVEL CANDIDATE ANTI-ALZHEIMER’S DISEASE DRUGS BY CONSTRUCTING INTERACTION NETWORKS BETWEEN DRUG TARGETS AND NATURAL COMPOUNDS IN MEDICAL PLANTS
Review round: 1
Reviewer: 3

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The paper is clearly written, well organized and should be accepted for publication after minor revision.
The detailed validation procedure of the docking results should be presented. The docking results should be visualized, at least in the Supplementary material. Also, it would be useful if the lipophilicity of the selected compounds is presented (logP).


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 STRATEGY TO FIND NOVEL CANDIDATE ANTI-ALZHEIMER’S DISEASE DRUGS BY CONSTRUCTING INTERACTION NETWORKS BETWEEN DRUG TARGETS AND NATURAL COMPOUNDS IN MEDICAL PLANTS
Review round: 1
Reviewer: 4

Output:
Basic reporting: see attached annotated PDF
Experimental design: see attached annotated PDF
Validity of the findings: see attached annotated PDF
Additional comments: see attached annotated PDF


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 STRATEGY TO FIND NOVEL CANDIDATE ANTI-ALZHEIMER’S DISEASE DRUGS BY CONSTRUCTING INTERACTION NETWORKS BETWEEN DRUG TARGETS AND NATURAL COMPOUNDS IN MEDICAL PLANTS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The new version of this manuscript is much improved. I think, however, that the presentation of results and conclusions still needs some more work to ensure that your research is appropriately communicated.
Experimental design: line 285: I am not completely convinced that dividing the full 1476 molecules in clusters is very informative. Wouldn't it be more intuitive to search for clusters in the sets of ligands that bind to each individual protein (i.e. to see if there were very different scaffolds able to bind SIRT1, lyn,....,etc.) ?
Validity of the findings: lines 204-207: Authors state" The docking results of these compounds indicated that almost all of the docking energies of the top 0.5% of compounds bound to target proteins were superior to those of their embedded ligands (Fig. 1). Thus, the docking results are reliable, and the 1,476 compounds can be considered candidate compounds for AD." I am afraid that the second sentence does not necessarily follow from the first one, and that at most you could say " [...]. Assuming the docking results are reliable, these 1,476 compounds can be considered candidate compounds for AD".  To show that your docking results are reliable, you would have to show that experimentally-confirmed weak-binding analogues of the original binding ligands do not have better docking energies than the original ligands. Such benchmarking  has probably  been performed by the original developers of the docking algorithm used, either in the original publication of the method or in closely related work. I think you should refer to the false-positive rates (or receiver operating characteristics) described in those works for an estimate of the likely percentage of true positives among the 1476 compounds.

lines 222 ff.: Authors compare docking energies of their candidates to those of four AD drugs. They do not, however, state whether those drugs bind to any of the tested proteins, and the relevance of these comparisons is therefore not clear.
Additional comments: lines 210-217: I do not think that listing the residues in the binding pocket of a single protein is very useful: an image with a side-by-side-comparison of best TCM ligand and original ligand would probably be easier to understand (actually, I think that providing the PDB coordinates of each protein with embedded original ligand and best TCM ligands as Supporting information would be ideal). Anyway, you should report the range of similarities you find (Tc betwwen 0.06 and 0.48) instead of the single Tc for one protein.

lines 218-221 . State the reason behind the choice of only SIRT1 for pharmacophore detection

lines 262: authors refer to "existing drugs". Do they mean "existing anti-AD drugs" or "existing molecules present in at least one of the world's pharmacopoeias"? Please clarify.


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

Input:
Title: A STRATEGY TO FIND NOVEL CANDIDATE ANTI-ALZHEIMER’S DISEASE DRUGS BY CONSTRUCTING INTERACTION NETWORKS BETWEEN DRUG TARGETS AND NATURAL COMPOUNDS IN MEDICAL PLANTS
Review round: 2
Reviewer: 2

Output:
Basic reporting: In my opinion, the manuscriptt can be published.
Experimental design: In my opinion, the manuscriptt can be published.
Validity of the findings: In my opinion, the manuscriptt can be published.
Additional comments: In my opinion, the manuscriptt can be published.


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 STRATEGY TO FIND NOVEL CANDIDATE ANTI-ALZHEIMER’S DISEASE DRUGS BY CONSTRUCTING INTERACTION NETWORKS BETWEEN DRUG TARGETS AND NATURAL COMPOUNDS IN MEDICAL PLANTS
Review round: 2
Reviewer: 3

Output:
Basic reporting: The authors corrected the manuscript according to the suggestions.
The paper should be accepted for publication.
Experimental design: The authors corrected the manuscript according to the suggestions.
The paper should be accepted for publication.
Validity of the findings: The authors corrected the manuscript according to the suggestions.
The paper should be accepted for publication.
Additional comments: The authors corrected the manuscript according to the suggestions.
The paper should be accepted for publication.


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

Input:
Title: A STRATEGY TO FIND NOVEL CANDIDATE ANTI-ALZHEIMER’S DISEASE DRUGS BY CONSTRUCTING INTERACTION NETWORKS BETWEEN DRUG TARGETS AND NATURAL COMPOUNDS IN MEDICAL PLANTS
Review round: 2
Reviewer: 4

Output:
Basic reporting: Authors have done some improvement, mostly on the editorial and formatting side.
Experimental design: I am still concerned with the experimental design of this work. authors have used a large number of TCM compounds to dock against large number of targets - it is like you comparing apples with tomatoes. That is why it is hard to draw out a concise conclusion or outcome of this work. I see that authors did not provide a proper conclusion with the key findings/outcome. I find it hard to come out with a message from this work that can help medicinal chemists or biologists do further research or expand on the outcome/results created here. Authors stated in the discussion/results that "Most of these
compounds are abundantly found in plants used for treating AD in China" - so where is the novelty here. the outcome of the study was found to be exactly what is actually known for these compounds and what they have been used for as anti AD!! the study did not provide any additional information, no additional structural or molecular description of the identified compounds, no precise description of the binding mode/mechanisms of the identified compounds.I am sorry, yes authors have done quite a bit of calculations with lots of numbers, however, these numbers don't provide any useful information to the scientific community.
Validity of the findings: Please see above comments
Additional comments: please see above


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 STRATEGY TO FIND NOVEL CANDIDATE ANTI-ALZHEIMER’S DISEASE DRUGS BY CONSTRUCTING INTERACTION NETWORKS BETWEEN DRUG TARGETS AND NATURAL COMPOUNDS IN MEDICAL PLANTS
Review round: 3
Reviewer: 1

Output:
Basic reporting: see below
Experimental design: see below
Validity of the findings: see below
Additional comments: All my objections have been appropriately answered. I think the manuscript can be published as-is.


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: LIGHTEN UP THE DARK: METAZOAN PARASITES AS INDICATORS FOR THE ECOLOGY OF ANTARCTIC CROCODILE ICEFISH (CHANNICHTHYIDAE) FROM THE NORTH-WEST ANTARCTIC PENINSULA
Review round: 1
Reviewer: 1

Output:
Basic reporting: The English usage is generally good, but the punctuation, particularly the use of commas, requires some editorial attention in some sections. The authors show a good knowledge of the literature apart from that on the recent changes in classification of cestodes. The structure of the manuscript conforms to the usual standard sections.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: This is overall a well written and presented manuscript and I have only a few comments and suggestions for improvement. They are as follows.

Among the parasite taxa reported are “Pseudophyllidae indet.” The cestode order Pseudophyllidea is no longer recognized; it was suppressed by Kuchta et al. (2008), who proposed two new orders – Bothriocephalidea and Diphyllobothriidea. Considering the sites of infection of these cestodes in the fish, I assume that they were postlarvae, in which case they would have been diphyllobothriideans, maturing in marine mammals. The references in the text (lines 192, 276, 285) and in Table 3 to pseudophyllidean or Pseudophyllidae should therefore be changed to diphyllobothriidean or Diphyllobothriidea respectively. It would also be helpful in Table 3 to refer to both the diyphyllobothriidean and tetraphyllidean cestodes as postlarvae, and to distinguish the nematodes as either larvae or adults.  Also in Table 3 Acanthocephala is wrongly spelt. Here is the full reference to the Kuchta et al. paper.
Kuchta, R., Scholz, T., Brabec, J and Bray, R.A. (2008). Suppression of the tapeworm order Pseudophyllidea (Platyhelminthes: Eucestoda) and the proposal of two new orders, Bothriocephalidea and Diphyllobothriidea. International Journal for Parasitology 38, 49-55.

In the Conclusion (line 401), 15 new host records are mentioned for the first time. This information should be given earlier at the beginning of the Results section. The new records should also be indicated in Table 3, for example with asterixes and a footnote.

Lines 239 to 243. This paragraph repeats what has already been stated in the Introduction and is unnecessary.

Line 255. Birds can also be final hosts for the genus Contracaeceum.

Line 350. It seems very unlikely that fish could be final hosts for Corynosoma bullosum. References to records from mammals and birds would be useful.

Congratulations to the authors on a valuable contribution to our knowledge of the diet and parasite faunas of a group of fish about which relatively little is known.


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: LIGHTEN UP THE DARK: METAZOAN PARASITES AS INDICATORS FOR THE ECOLOGY OF ANTARCTIC CROCODILE ICEFISH (CHANNICHTHYIDAE) FROM THE NORTH-WEST ANTARCTIC PENINSULA
Review round: 1
Reviewer: 2

Output:
Basic reporting: A) The abstract is written in a manner that promotes interest in reading the manuscript. However, the manuscript would benefit from further synthesis and improved clarity in both the writing and some data presentation.  The writing is generally good but it is not unambiguous throughout the manuscript.  Some sections would benefit from improvements to writing and greater attention to important details.  Examples are provide below:
1.	Lines 388-389:  “However, sequences were assigned to the species Parascaris equorum by comparisons with GenBank.”  This statement is not accurate and subsequent writing acknowledges this fact.  GenBank does not assign species identification through BLAST.  The GenBank BLAST simply provides top BLAST hits based upon sequences available in that database.  Assigning these nematodes an identification of Parascaris equorum would be erroneous based on that BLAST analysis.  This parasite is clearly not a horse nematode but it is related to that parasite phylogenetically.  Subsequent writing acknowledges this fact but some statements are incongruent with this fact if viewed in isolation (i.e., Table S2).  The column heading in Table S2 should be changed from Organism (= species identified by GenBank) to Organism of Highest Identity for accuracy and clarity.
2.	Lines 194-195: “Tetraphyllidean cestodes occurred at high intensities, with a mean intensity of mI=77.5±63.5 averaging all fish species.”  This is misleading as written.  According to Table 3, only one of the five host fishes actually harboured Tetraphyllidean cestodes.  Sentence structure contributes in part to this particular issue.
3.	Line 198: “There were no differences in diet composition of the five examined fish species”.  There might have been no statistical differences, but the paragraph that follows this statement highlights several differences that were observed in diet composition.
4.	Lines 91-93: This sentence would likely be more effective if re-written to convey more effectively both the current state of knowledge and the significance to the current investigations.  Neither of the two papers cited discuss sampling effort and so perhaps you could reposition the citations within that sentence and then add another citation that makes the case for sampling effort.  Is an analysis from 80 fish hosts distributed among 5 genera sufficient – justified from other studies?
5.	Line 386-388: “Samples came exclusively from nematodes found in the host species Chaenodraco wilsoni and Champsocephalus gunnari.”  Isn't this referring to the “Nematode indet” that was also found in the host P. georgianus?  Perhaps the single specimen from P. georgianus was not sequenced but it should be indicated that another fish host also had an unidentified nematode – is this likely to be the same parasite and with a broader host range?
6.	   Lines 146-149: This section provides no indication of what 19 reference species were included, where their sequence data was acquired, or how/why these Anisakid nematodes were selected.  Additional details would improve understanding here.
7.	   Line 157: What is the GenBank Accession number for the sequence that was included as an outgroup for the tree?  This isn’t in the text or in the tree.  How/why was this species chosen?
8.	  Lines 427:  Insufficient citation.  The software producer suggests citing the user manual as follows: Clarke, KR, Gorley, RN, 2006. PRIMER v6: User  Manual/Tutorial. PRIMER-E, Plymouth, 192pp.
9.	Lines 97-98: It is not obvious how this current study was performed to increase knowledge of parasite life cycles per se but rather it clearly expands our knowledge of potential intermediate/paratenic hosts that could participate in the life cycle.  Line 280 makes brief mention of life cycle knowledge acquired in the current study.
10.	Lines 39-40:  “The stomach content, with only Crustacean (Euphausiacea, Amphipoda) in all examined species, was less diverse.” The sentence structure could be improved – perhaps: “The stomach contents were less diverse with only Crustacea (Euphausiacea, Amphipoda) recovered from all examined fishes.”
11.	Line 211-213: “Blastn-analyses partially yielded equivocal results.  The 117 obtained sequences showed highest congruence with eight different sequences that were in parts not annotated to a species level in GenBank (Table S2).”  Both of these sentences could be improved easily to convey the information more effectively.
12.	Line 275: “Parasite species diversity was low and considered mainly…” Writing could be improved here.
13.	Line 325: “due to the intake of already infected other fish”  Writing could be improved here.
14.	Lines 370-371: “Also representatives of the Channichthyidae were known to be infested with larval forms of…” The sentence structure could be improved.
15.	Lines 376-378: “Only one sequence was obtained and could be compared with a GenBank reference. It was assigned to the species P. decipiens E which is a sibling species of the P. decipiens complex, known from Antarctic waters.”  Merging sentences could improve the writing here, perhaps: “Only one sequence was obtained and it was assigned to the species P. decipiens E, a sibling species of the P. decipiens complex known from Antarctic waters.”
16.	Line 378: “It genetically differs…” perhaps change to “It differs genetically…”
17.	General:  Most Figure, Table, and supplementary Table titles would benefit from more clarity in the writing. Line 27:  “the Antarctica” change to “Antarctica”
18.	Line 38:  “six taxonomic groups Digenea” change to “six taxonomic groups including Digenea”
19.	Line 67:  “cold adapted” change to “cold-adapted”
20.	Line 68 and throughout: “Cheng, di Prisco & Verde, 2009” to “Cheng et al., 2009”
21.	Lines 79-80: Eight families stated but only seven listed.  Artedidraconidae is missing.
22.	Line 80 and throughout: “Near, Pesavento & Chenge, 2003” to “Near et al., 2003”
23.	Line 83 and throughout: “Near, Pesavento & Chenge, 2003” to “Near et al., 2003”
24.	Line 92 and throughout: “Klimpel, Seehagen & Palm,, 2003” to “Klimpel et al., 2003”
25.	Line 94: “content” to “contents”
26.	Line 138: “were controlled” perhaps a better descriptor “visualized”?
27.	Line 146: “the BLASTn” change to “a BLASTn search”
28.	Lines 160-161: Parasite abundances?  Parasite intensities?
29.	Line 191: “sampling” change to “sample”.
30.	Line 194: “which occurred in all five species with overall prevalences” perhaps change to “which occurred in all five host genera/species with overall prevalences of”
31.	Line 211: “Blastn” change to “BLASTn”
32.	Line 211-218: “were” change to “was”
33.	Line 222: Should spell out on first use of the acronym - Operational Taxonomic Units.
34.	Line 222: Should indicate that the sequences being compared (study and reference) are Anisakid nematodes.
35.	Line 224: Delete period after Pseudoterranova
36.	Line 226: Italicize P. azarasi
37.	Line 231: “Blastn” change to “BLASTn”
38.	Line 242: “content” to “contents”
39.	Line 250: Delete one of the double brackets
40.	Line 251: “table” change to “Table”
41.	Line 256: “Acanthocephala” change to “acanthocephalan”
42.	Line 259: “the low infection parameters”  It is unclear what this means specifically.
43.	Line 264. Italicize C in C. wilsoni
44.	Line 280: “role of the fish as final hosts in the life cycle of these parasites.”  Perhaps change to “role of the fish as final hosts in the life cycle of these digenean parasites.”
45.	Line 288.  “Palm, Klimpel, & Walter, 2007” to “Palm et al., 2007”
46.	Line 292: Delete “two”
47.	Line 321:  Should “three” be “four”?
48.	Lines 334-335:  Generic names can be abbreviated after first use.
49.	Lines 340-342:  Generic names can be abbreviated after first use.
50.	Line 360: “nematods” change to “nematodes”
51.	Line 366: “detailed” perhaps change to “accurate”
52.	Line 393: “by the low values of sequence identity” perhaps change to “by the low sequence identity”
53.	Line 404: “host” change to “hosts”
54.	Line 480: Need to italicize genus and species name.
55.	Line 541: Need to italicize genus and species name.
56.	Multiple citations require the first letter in the journal name components to be in upper case: Line 414, 424, 429, 478, 481, 485, 505
57.	Multiple citations require additional information to be an acceptable as a citation. Volume number, page numbers, etc: Lines 429, 430, 446, 454, 456. 463, 478, 548, 553
58.	Line 589:  Need a colon after “grey”
59.	Line 592:  Capitalize Table
60.	Line 600: “results for the” perhaps change to “identity”
61.	Line 605:  Capitalize Table
62.	Figure 3 title could be more specific regarding anisakid nematodes.  Capitalize t in Table S2.
63.	Table 1. Figure legend title could be improved:  “Information of” change to Information on”;  “caught individuals” change to “individuals caught”;  Need a period after the species initial in the five column headers to the far right of the Table.
64.	Table 2. Figure legend title could be improved:  “data of the investigated species” change to “data on the host species investigated”;  Tg is actually TW elsewhere – change to TW for consistency;  “Arimetric” change to “Arithmetic”;  C = condition factor but it is usually abbreviated as Cf or K factor
65.	Table 3. Figure legend title could be improved:  “Parasitological data of the examined channichthyid Pseudochaenichthys georgianus” change to “Parasitological data from the channichthyid Pseudochaenichthys georgianus”;  S = surface but the table uses SU;  For worms from stomach/intestine, are these from the lumen or are they embedded in the mucosa/serosa of these tissues?
66.	Table 4. Figure legend title could be improved.  See suggestion for Table 3.  S = surface but should use SU;  For worms from stomach/intestine, are these from the lumen or are they embedded in the mucosa/serosa?;  Column row “Cestoda”.  Column to the right states L/F/T but should be L/FT

B) The authors focus on examination of helminth and ectoparasites of 80 fishes representing five genera of channichthyids.  They recovered and identified (morphological/molecular) helminth parasites representing four digeneans, four nematodes, two cestodes, and one acanthocephalan as well as ectoparasites including three leeches and one copepod.  These fishes have not been examined extensively for parasites previously and this study presents 14 genera of parasites representing 15/16 species with 15 new host records for these parasites.  Partial sequencing of the cox2 gene was used to identify a subset of nematodes and served to identify one new genotype/species of larval nematode.  The introduction provides context and the literature is both well referenced and relevant.

C)  The manuscript structure conforms to PeerJ standards.

D)  All figures are relevant.
1.	Figure 2 could be improved for clarity by inclusion of a legend rather than a description of colors/patterns in the text of the figure legend.
2.	Figure 3 needs to be revisited.  This phylogenetic tree needs substantial attention before it could be published.  It requires synthesis to aid the reader in understanding the key contributions without investment of substantial effort.  The tree is presented using raw data that could be synthesized to simplify data presentation for ease of understanding.  In its current form the reader is required to spend substantial time decoding the parasite lineages and specimen identifications.  The specimens represented in the tree from the current study could be presented more simply to facilitate cross-referencing with the supplemental data.  As an example, each “Ant” and underscore is unnecessary and simply takes up space with no net benefit for the reader.  As per Table S5, ID can be simply host species and number (Cw1, Cw2, etc.) and followed by reference to worm species/number.  Repositioning the outgroup to the top of the page would eliminate need for such a long vertical branch.  The Figure should be a higher order synthesis of the data.  The term reversed should be eliminated from the tree.  It is understood that sequences must often be reversed to facilitate DNA sequence comparisons.
3.	Tables 1, 3, 4, 5 are relevant to the manuscript.  However, Table 2 could be incorporated as a supplementary data file.  There is no integration of host morphometrics from Table 2 with any of the data generated regarding diet or parasite diversity.
4.	Tables 3 and 4 should include a column that indicates the life stage of each parasite (adult or larval) to facilitate ease of comparison.  I believe that the Nematode indet represent larval parasites?  The Discussion section would benefit from earlier introduction of the concept of larval versus adult helminths regarding diet and observations on parasite diversity in these fishes.

E) Raw data is included in the form of five files.  Specific comments on these Tables is provided below:
Table S1.
1.	 The currently listed “this study” should probably be changed to “this study – new host record”.  It would seem appropriate to add “this study” to the other rows where appropriate – others reported it previously but your study also reports it.
2.	Some cestodes are referred to as “indet” but others not.  This should be a uniform descriptor throughout this Table.
3.	Reference Kock et al., 1984.  Journal name should not be upper case.
4.	Two Laskowski references need the “p” in Systematic parasitology to be capitalized.
5.	The Munoz reference needs Ascarophis to be capitalized.
6.	The Rhode et al. 1995 reference needs the Journal name to be corrected – add capital letters.
7.	Santora et al., 29013 needs Dis Aquat Org spelled out.
8.	Utevsky, 2005 needs Vestn Zool spelled out.
9.	Zdzitowiecki and Cielecka, 1996 needs journal page numbers added.
10.	Zdzitowiecki et al. 1999 needs the journal name spelled out in full.
11.	Zdzitowiecki et al. 1998 needs journal page numbers added.
12.	Inconsistent use of et al. in the reference column for Table S1.

Table S2.
1.	The Table title needs improvement.  “results for the matching with the NCBI data set” is not accurate.  The title also needs some description of the terms E value and Accession as per the other descriptions.
2.	The ID column information does not easily correlate with data in Table S5.  This information should be simplified to match new IDs for use in the phylogenetic tree.

Table S4.
The Table title needs improvement.

Table S5.
1.	The Table title is in need of improvement.
2.	N. noto should not be italicized for consistency with other abbreviations.
Experimental design: A) The manuscript focuses on primary research that fits within the scope of PeerJ.
B) The research question is defined and focuses on a survey of the parasites and diet of Antarctic fishes to help understand aspects of the ecology of the host fishes (i.e., trophic interactions).  This research serves to fill a knowledge gap regarding fishes that have evolved in an extreme environment using parasites to help understand trophic interactions.  Accordingly, the data are relevant and meaningful.
C) Methods are generally described with sufficient detail to replicate the work.  Examples of deficiencies in methodology are listed below.
1.	   Line 127-128: From Tables 3 and 4, 625 non-Ascarophis nematodes (597 Anisakids and 28 Nematoda indet) were collected in this study.  Lines 120-122 indicate that some nematodes were kept for morphological identification and the remaining were preserved for molecular identification.  117 nematode larvae were ultimately assessed for nematode species identification using DNA sequencing of cox2 but details are lacking regarding how 117 were selected from the population of 625 nematode larvae.  Furthermore, 572 of the 625 nematodes have species level identification in Tables 3 and 4.  Does the 117 represent a certain number of representatives from each individual fish species?  What morphological features enabled the remaining larvae to be identified to species in the absence of DNA sequencing?
2.	   Lines 132-134: These PCR reaction mix volumes add to more than the 25 ul reaction volume that is reported; 12.5+1.5+1.5+14+2.5 = 42 ul.  You should supply the final concentration of dNTPs and you must identify the specific Taq formulation that you used from this specific supplier (proofreading, high fidelity, hot-start, etc.).
Validity of the findings: The authors provide an interesting data set that clearly required considerable time, energy, and expense to collect and synthesize.  The overall finding and conclusions seem valid.  There are some challenges with data interpretation that represent corresponding challenges inherent in the collection of biological specimens.  In this study, 82.5% of the sampled fishes were collected from 25% of the sample sites (sites 7, 8).  Furthermore, 72.5% of the sampled fishes were from two of the five species examined.  In Line 188:  The authors report “a high similarity in parasite fauna of C. wilsoni and C. gunnari (81.6 %)”.  Both fish are specialists that feed on invertebrates but they were also collected predominately in one specific region (sites 7/8) that is some 200-250 km from the other six sample sites where the remaining 17.5% of fishes were collected.  The study serves to provide some important baseline observations on parasites but higher order analyses are complicated by low host sample sizes in some groups and by regional considerations regarding fish host distribution/samopling.  According to Munoz and Cortes (2009), only those fish species with sufficient sample size should be included in a cluster analysis “because low sample sizes can give a parasite spectrum unrepresentative for a host species.” They applied accumulation curves to evaluate the host sample size necessary to have at least 90% of the parasite species represented from a particular fish species.  It is understood that the success of catches is beyond the control of the investigators.  It is also understood that the complete parasite records for these fishes remains outstanding but the current state of knowledge is very nicely summarized in Table S1.  It is appreciated that the authors express that caution should be exercised in interpreting their cluster analysis results (Lines 190-192).  A sample size of three fish of one species and four fish of another species is likely insufficient to present an accurate picture of parasite diversity within a host species.  In lines 330-331 the authors make the statement that “The small sample size for both species is not representative in order to interpret the feeding behavior”.  Given that the helminths are acquired by feeding, the parasite diversity would similarly be not representative.  Munster et al. (2016) state that “only parasites unambiguously identified to the species level were used for Bray-Curtis calculations”.  Is this true for the current study where species level identifications were lacking for several parasites?
Additional comments: A number of surprising/interesting observations are reported.  Lines 248-251: “Low parasite diversity in four of the five fishes examined relative to other channichthyid fishes.”  Lines 260-261: “…low diversity of digeneans is also surprising given their diversity within Antarctic waters.”  In spite of rather static water temperatures, is there any evidence that time/season of collection can influence the diversity of parasites by a change in food source for Antarctic hosts?  All fishes in this study were collected Feb. 21-Apr. 1.  In line 309-310 the authors reference changes in prey species in time and space.  There was an absence of fish prey in the stomachs of even the known pisciverous host species (line 317-318).  Stomach contents and short-lived parasites can be underrepresented in studies where the data is by nature a snapshot in time.  Is the home range of these host species restricted?  Can these observations/considerations be further synthesized by incorporating additional parasite data existing from these same hosts (select regional data from Table S1)?  Parasitological data from previous studies is presented in S1 but doesn't seem to have been integrated into the manuscript.  For example, it seems readily apparent that parasite diversity correlates with the number of studies (C. gunnari; ~29 parasite species, ~14 studies).  Sample sizes are not presented but perhaps a higher order analysis possible by integrating select data with regional relevance from Table S1?


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: TEN YEARS OF PROGRESS AND PROMISE OF INDUCED PLURIPOTENT STEM CELLS: HISTORICAL ORIGINS, CHARACTERISTICS, MECHANISMS, LIMITATIONS, AND POTENTIAL APPLICATIONS
Review round: 1
Reviewer: 1

Output:
Basic reporting: This manuscript reviews the advances in the field of induced pluripotent stem cells (iPSC) during the 11 years since iPSC were first described.  Overall the review is detailed with appropriate citations, over 200, and the organization into different sections is well thought out.  Considering the growth of research in this field, it is difficult to maintain this type of review up to date although the authors have done well.  Keeping in mind that several other similar reviews on the subject have been published, this work represents a considerable body of knowledge useful both to newcomers to the field and for experienced authors.

Unfortunately, the manuscript contains a significant number of grammatical errors throughout the text, particularly concerning pluralities and tense.  For the authors help, I have included a list of potential corrections and suggestions for several phrases where the meaning was unclear.
Experimental design: The manuscript is clearly in the scope of PeerJ, with well-outlined objectives.  Even though the field is expanding continually, the analysis performed by the authors is overall of appropriate depth, detail and rigorous.

Major point:
While through most of the manuscript, the authors provide a well detailed description of the many of the advances in the field, page 6 line 167, the manuscript would be strengthened by including a deeper description of the methods enabling development of fully reprogrammed iPSC.
Validity of the findings: In the concluding sections, the authors provide a very appropriate assessment of the future potential of iPSC and the barriers we face before effective clinical deployment.
Additional comments: Clearly, the manuscript was written last year, 2016, which may account for many of the problems with tense.

Minor points:
Pg:8 line 241 The full names of DOT1L MBD3 etc. need to be provided.
Pg:12, line 344, “highly immunogenic.” needs a reference.
There are 9 possible loops in the Oct4/Sox2/Nanog transcription machinery, so while I believe the term auto regulatory loop can be used to describe this circuitry overall, in the detailed description of this mechanism, it would be more appropriate to refer to loops.
Pg: 14-16 the authors make use of both the terms iPSCs and PSCs and it is not clear if the latter is being used to describe pluripotent stem cells as a whole.


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: TEN YEARS OF PROGRESS AND PROMISE OF INDUCED PLURIPOTENT STEM CELLS: HISTORICAL ORIGINS, CHARACTERISTICS, MECHANISMS, LIMITATIONS, AND POTENTIAL APPLICATIONS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Please see General comments below
Experimental design: Please see General comments below
Validity of the findings: Please see General comments below
Additional comments: This is a very broad review of various aspects of iPSCs. It adequately covers many of the major issues relating to this technology, but because of its broad scope, lacks synthesis and critical evaluation of some of the cited work. The figures and tables are mostly adapted from previously published reviews and do not incorporate more recent work. I would recommend major revisions be carried out before the mansucripts publication.

Please see below for specific comments on the manuscript.

Line 136 : "The search started in year 2000 leading to a list of 24 candidate reprogramming factors" - This statement refers to the paper published in Cell in 2006 not 2000.

Lin 289 : "self-deleting peptide" - more common usage for this would be "self-cleaving peptide"

Instead of separating each delivery methodology to its own paragraph they should be grouped under two major headings (integrating vs nonintegrating)

Secondary IPSC systems (knock-in mouse models of Trx Factors) are not considered adequately - especially when disccussing the factors that limit the efficiency of reprogramming (Section 5.8).

Section 5.6 seems to dwell on the same topic as 5.1 and could be merged to make the text clearer

The authors use "we" in several places in the text (eg. Line 688). Since the work they cite is not theirs per se, it would be more appropriate to use "one" or the passive voice.

Section on the details of Cas9 (line727-743) seems out of  place in the context of this iPSC review.

Why is "microrna switch" on line 772 italicized?

Line 787- Should read as "believe"

Many of the figures are "adapted" from reference 117 - The authors have not really added onto these, so I wonder if they are really necessary. One can also read teh origival review

Simple adapting a table (Table 2 and 4) from an older review is not acceptable in my opinion. More recent information has to be added on these tables.


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: GLYCOSYLCERAMIDE MODIFIES THE FLAVOR AND METABOLIC CHARACTERISTICS OF SAKE YEAST
Review round: 1
Reviewer: 1

Output:
Basic reporting: See attachment
Experimental design: See attachment
Validity of the findings: See attachment
Additional comments: See attachment


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: GLYCOSYLCERAMIDE MODIFIES THE FLAVOR AND METABOLIC CHARACTERISTICS OF SAKE YEAST
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: This manuscript describes the effects of glycosylceramide on the fermentation of sake yeast. On the whole, findings are new, and it describes sufficient experimental details.

Questions (on the whole)
1. Could you explain why the addition level of glycosylceramides was 40 μg/ml? It should be added to the manuscript. The effects of higher level of glycosylceramides (more than 40 μg/ml) should be added. (The effects of glycosylceramide are dose-dependent?) It helps to consider how level of glycosylceramide is adequate on the flavor and taste of sake.

2. There is no data about the fate of added glycosylceramide in all experiments.

3. In Figure 1, author should explain why the values of “black bars” were different between A and B. For example, acetic acid was 408 % and ap. 120 % in A and B, respectively.


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: GLYCOSYLCERAMIDE MODIFIES THE FLAVOR AND METABOLIC CHARACTERISTICS OF SAKE YEAST
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript by Ferdouse et al reported their study on the effect of Glycosylceramide on metabolic profiles of sake yeast. The English is readable. However, there are major issues with the overall presentation, delivery and methods described. Please see my detailed report
Experimental design: The experimental design is not well described (see my detailed report below). I strongly suggested using a chart
Validity of the findings: It is hard to comment on this as methods used are not very suitable
Additional comments: Except the introduction section, and remaining sections are poorly written. Here are some of my specific comments

1)	L82- 83 "Glycosylceramide was purchased from Funakoshi or purified from the mycelia of A. oryzae." Then L84 - L126 almost 5 paragraphs on preparation of Glycosyleceramide from A. oryzae. This is too much focus on this part;

2)	A lot of other description is very mechanical, without transitions or explain how it is relevant to the current study

3)	Study design is very unclear and not well organized (see #5), I would suggest to use a chart from sample preparation steps to analysis

4)	Statistical analysis L190-193 "The differences among the averages of three or more groups were evaluated using analysis of variance (ANOVA), followed by Bonferroni or Dunnett's post hoc multiple-comparison test."
=> What are the 3 or 4 groups? What type of ANOVA? Bonferroni is for multiple testing, post-doc analysis is not! In particular, Dunnett's test accounts for multiple group pairwise comparisons across a single dependent variable, not across the features/metabolites. These two methods are not equivalent

5)	L199-200, "To obtain information on the specificity of the effect of chemical structures of glycosylceramide, glycosylceramides from Aspergillus oryzae, soy and Grifola frondosa were used"

=> This is a surprising.  It is not even mentioned in the experimental design, but suddenly appear in the results!

6)	In the result description L214-L219, it is rather repetitive, without any new information beyond the figure itself

7)	Fig1 & Fig2. It seems all two groups (should be t-tests and FDR, rather than Dunnett). I am not sure why Fig 1 and Fig 2 are treated differently in terms of image and statistical analysis. In addition, for many compounds, it is better to use a heatmap rather than many bar plots

8)	Fig 3. There is only one compound!

9)	Fig 4. "These normalized values were used as independent variables in principal component analysis (PCA) (p < 0.05)."
=> I am confused about PCA and p values here, I could not find other places describe this. Importantly PCA does not give p values!
=> Pathway analysis on 22 detected metabolites for all intracellular and extracellular
The enriched pathways are identical. This is not surprising as these are probably most abundant compounds due to the environment. Their differences are more interesting within the context. i.e. to perform pathway analysis using compounds that are significantly different, instead of using all detected compounds