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: SPECIES RECOVERY AND RECOLONIZATION OF PAST HABITATS: LESSONS FOR SCIENCE AND CONSERVATION FROM SEA OTTERS IN ESTUARIES
Review round: 1
Reviewer: 1

Output:
Basic reporting: Comments to Author:
Overall, I think this was a well-written manuscript on a worthy topic. The authors present a very interesting study on the potential for estuaries to support sea otter populations and aid in the recovery of southern sea otters. The authors created a habitat specific population model for sea otters in Elkhorn Slough and applied that model to estimate the number of sea otters that could be supported in San Francisco Bay, and estimated it could support 66,000 sea otters. They also found that prey populations in San Francisco Bay are sufficient to support a healthy sea otter population. I believe this study adds important information on sea otter recovery by studying sea otters in estuaries, for which fewer studies have been conducted when compared with studies of sea otters in outer coast habitats.
Major comments:
1.	I don’t think that the historical importance of estuaries as sea otter habitat is best presented in a literature review in the Methods and Results. I think the results of this literature review are better suited for inclusion in the Introduction. The addition of this information to the Introduction will strengthen the background provided to give context for this study. Along these lines, on Lines 104-106, I suggest adding some additional discussion of the historical importance of estuarine habitats to sea otters, including beyond California, as sea otters have historically occupied estuaries in other areas, such as in Washington State.
There are a few areas where the phrasing could be improved for comprehension and/or clarity. I have listed these below, along with other minor comments/suggestions.
Minor comments/edits:
1.	The abstract does not include any information on the results of the prey surveys that were conducted. I suggest adding a sentence or two to the abstract to summarize the results of this part of the study, given that the current abstract is 250 words of the allowed 500.
2.	Line 61: Suggest changing “Recovering species often are limited to…” to “Recovering species are often limited to…”
3.	Line 63: Suggest changing “accident” to “artifact of”
4.	Line 66: Suggest changing “estuaries provide” to “estuaries can provide”
5.	Line 79: Suggest changing “for successful restoration ecosystem functions and processes” to “for successful restoration of ecosystem functions and processes”
6.	Line 87: Suggest changing “missing keystone predator” to “top predator whose decline has dramatically altered ecological relationships as a keystone predator” or something similar to more directly connect this statement about sea otters to the topic of the paragraph.
7.	Line 96: Consider deleting “in the early 20th century” and “small” as they are repetitive with the 1914 reporting date and 50 individuals
8.	Line 98/99: Suggest changing “As of 2017, this population has grown to over 3,000 individuals and has expanded to the north and south along a range that extends more than 400 km (USGS, 2018).” To “As of 2017, this population has grown to over 3,000 individuals and has expanded its geographic range to include more than 400 km of coastline (USGS, 2018).”
9.	Line 101/102: Estes and Palmisano 1974 and Simenstad et al. 1978 are cited for “foundational studies on the ecology of southern sea otters” that have focused on kelp habitat, but these studies are on Northern sea otters in the Aleutians. I suggest either deleting these citations and replacing them with relevant citations specific to southern sea otters, or broadening the statement to say something along the lines of “because many remnant recovering populations occurred in kelp forests along open coastline, foundational studies of the ecology of sea otters have focused on this habitat”.
10.	Line 105: I suggest deleting the Laidre et al 2001 citation. I think it is potentially misleading to cite Laidre et al 2001 to support the statement that “southern sea otters historically occupied estuaries as well as the open coast” as the use of the word “historical” in this context is assumed to mean pre-fur trade, while the citation is well after fur trade and does not discuss estuaries historically, but in the mid-1990s.
11.	Lines 109-111: I suggest adding relevant citations here
12.	Lines 109-111: This sentence appears to be out of place, as it refers to “these open-coast studies” when the beginning of the paragraph describes sea otter use of estuaries. I suggest deleting “these” from this sentence.
13.	Lines 111/113: Both “open-coast” and “open coast” are used throughout. I think “open coast” is correct. Either way, it should be consistent throughout.
14.	Line 118: Suggest adding citations (Hessing-Lewis et al., 2017; Hughes et al., 2016, 2013) after “recent and ongoing research”
15.	Line 122: Add “to” between “sea otters” and “better”
16.	Line 122-124: I am not sure that this work actually accomplishes the goal of using “the recovery of southern sea otters to better understand how a recovering species interacts with historically occupied ecosystems..”. I suggest removing this sentence.
17.	Line 143: I think the asterisk should be moved to directly after “embayment” instead of after “embayment or”
18.	Line 154/155: Suggest adding a citation to support the assertion that “it is likely that other estuaries (including San Francisco Bay) will eventually be colonized”. Has any population modeling predicted this?
19.	Line 168: Consider adding “growth” between “stochastic logistic” and “model”
20.	Line 172: Suggest removing “hidden or”, as “hidden” and “latent” are synonymous. If the goal was to provide a definition of the term “latent”, I would suggest saying “…true abundance, Nt, is an unobserved (latent) variable…” instead.
21.	Line 176: Should equation referenced be 0.1 instead of 1? Similar issue throughout if so.
22.	Line 177/178: I am confused by the inclusion of both  and p, as only p is included in Equation 0.1. I think it would be more clear and correct to say “…N(0, p) represents a random normal variable with mean 0 and standard deviation p, where p represents process error or environmental stochasticity (a fitted parameter).”
23.	Line 189: Consider adding a citation for JAGS and R.
24.	Line 190: Consider adding a comma after K, as it sounds like K and N1 are variance parameters as written.
25.	Line 191: I think it is misleading to say that this is a weakly informed prior. I would remove “weakly” here.
26.	Line 205-208: The four habitat types should be listed explicitly here, or earlier near line 180.
27.	Line 206: Would like to see a citation here for degradation of San Francisco Bay habitats.
28.	Line 209: capitalize bay
29.	Line 212: The use of the phrase “somewhat lower densities” seems at odds with the decision to assume that offshore densities would be ~10% of nearshore densities. Perhaps this could be addressed by explicitly stating average distances from shore or percent of time spent offshore in embayments in AK or other areas instead of saying “somewhat lower densities”. These citations could be useful here (although are foraging specific):
Bodkin, J. L., G. G. Esslinger and D. H. Monson. 2004. Foraging depths of sea otters and implications to coastal marine communities. Marine Mammal Science 20:305–321.
Laidre, K. L., R. J. Jameson, E. Gurarie, S. J. Jeffries and H. Allen. 2009. Spatial habitat use patterns of sea otters in coastal Washington. Journal of Mammalogy 90:906–917.
30.	Line 220: Suggest adding “derived from Elkhorn Slough” after “habitat-specific equilibrium densities”.
31.	Line 220-222: This seems like it should be in the results instead of the methods. I think “of 6607 otters (Table 1)” should be removed, and the methods should be discussed generally. I also suggest deleting reference to Fig. 2A-B, as these are results as well.
32.	Line 223: It is unclear to me as written if r and p are the parameters estimated from the Elkhorn Slough data, or if they are estimated for San Francisco Bay, although I believe they are the parameters estimated from the Slough data. I suggest explicitly stating this.
33.	Line 236-238: The current phrasing of this section makes comprehension difficult. I suggest making it clear on line 237 where prey species lists were “assembled” from by moving the information about the prey species lists on lines 240-242 earlier. I suggest then combining the information about crab sampling and sea otter prey surveys into one sentence.
34.	Line 251: Define CPUE here.
35.	Line 259: Does “conspicuous” mean they are larger than other crabs? Or present in the Slough at higher abundances? Suggest adding more detail here.
36.	Lines 260/261: CPUE definition can be deleted here if it is defined when first used on line 251.
37.	Line 261: Add “s” to “linear mixed model”, or change to “using a linear mixed model”
38.	Line 272: Change citation for “Seachore” to “Seashore”
39.	Line 287: Perhaps “continued to be published” should be changed to “continue to be published”
40.	Line 327: Suggest specifying that the range referenced is for Southern sea otters – as written, this statement could be misinterpreted to mean all sea otters, not just Southern sea otters.
41.	Line 330-332: This sentence seems more appropriate for the Discussion rather than the Results
42.	Line 338-339: This sentence also seems more appropriate for the Discussion rather than the Results
43.	Line 338: Here both SF Bay and Drakes Estero are listed as “focal sites”, but above on line 331 SF Bay is listed as the focal recovery site. On Line 331, I suggest saying “one of the focal recovery sites”
44.	Line 339: Suggest adding “to support a sea otter population” after “supply of crab prey”
45.	Lines 342-347: The current phrasing of this section makes comprehension difficult. How can crabs in Drakes Estero be of similar size to crabs in Elkhorn Slough pre-sea otter, and crab sizes in SF Bay also intermediate between Drakes Estero, Morro Bay, and Elkhorn Slough (pre and post sea otter)?
46.	Line 343: Suggest replacing “greater” with “larger”, and adding “crabs from” between “larger than” and “sites”
47.	Line 368: Is “topographically” the appropriate term here? I think it is if you are referring to the shallow water in estuaries, but I think a broader term would be better suited here that describes the habitat complexity that estuaries provide.
48.	Line 382: It is unclear what is meant by “natural experiment” in this context, although I have an idea based on the use of recolonization as a natural experiment in other sea otter research. Do you mean sea otter recolonization of the Slough as a natural experiment to test if sea otters can thrive in an estuary? I suggest clarifying this.
49.	Lines 415-418: The current phrasing of this section makes comprehension difficult. Suggest changing “When comparing the coverage of the two dominant habitat-forming species (estuaries with eelgrass) and the outer coast (with kelp, e.g., Macrocystis pyrifera) we conservatively estimated there to be 1750 ha of eelgrass habitat within the projected range, and most of the habitat is from the northern side of the otter range (see Bernstein et al. 2011).” To: “When comparing the coverage of the two dominant habitat-forming species within the sea otter range – eelgrass in estuaries and kelp (e.g., Macrocystis pyrifera) on the outer coast – we conservatively estimated there to be 1750 ha of eelgrass habitat within the projected range, and most of the habitat is from the northern side of the otter range (see Bernstein et al. 2011).”

In addition, it is unclear what is meant by “projected range” – do you mean the 100km to the north or south of the current range? Finally, this sentence does not actually compare the coverage of kelp and eel grass, despite saying “When comparing the coverage of the two dominant habitat-forming species…”. I suggest adding the ha of kelp habitat here, although I do see these discussed later in the paragraph.
50.	Line 431: Suggest changing “At present” to “As of 20XY” (applicable year).
51.	Line 433: Should it be “has a relatively pristine watershed” instead of “had”?
52.	Line 455-458: Suggest changing from “Results showing positive effects of sea otters in estuaries has regional conservation implications...” To: “Results showing positive effects of sea otters in estuaries have regional conservation implications...”
53.	Line 458-460: This is not a complete sentence. Suggest changing “Severe recent losses of eelgrass to the south of Elkhorn Slough in Morro Bay (> 95% since 2007; (MBNEP, 2014)), with the exception of areas near the mouth where sea otters permanently reside.” To: “Recently, severe losses of eelgrass to the south of Elkhorn Slough in Morro Bay (> 95% since 2007; (MBNEP, 2014)) have been observed, with the exception of areas near the mouth where sea otters permanently reside.”
54.	Line 475: Suggest adding applicable citations here.
55.	Line 482 and 485: Capitalize estuary
56.	Line 491: Is there applicable work you can site here on sea otter disturbance? Like work by Sea Otter Savvy?
57.	Line 515-516: Suggest adding applicable citations here
58.	Line 534: Suggest changing “where they have been documented to hunt” to “and have been documented to forage in a range of estuarine habitats…”
59.	Line 540: I have never heard of sea otters eating oysters. As such, I think it is important to mention (and cite applicable literature) if sea otters have ever been observed eating oysters anywhere throughout their range, and if they have not been observed eating oysters, say so.
60.	Lines 563-581: This section seems repetitive with Lines 477-491. Consider combining and consolidating.
61.	Line 588: Re-introduction is hyphenated here but not elsewhere. Suggest not hyphenating and being consistent throughout.
62.	Line 599: Unclear what is meant by “comparison between estuaries…”. Suggest changing to “Comparison of estuaries occupied (Elkhorn Slough and Morro Bay) and unoccupied (San Francisco Bay and Drakes Estero) by sea otters offer the potential for valuable insight on socio-ecological effects of coastwide recovery”
63.	Line 614: The current phrasing of this section makes comprehension difficult. Do you mean “Engaging in restoration of both species and habitats?”, or “both habitats” as in eelgrass and kelp? In addition, “implementation requires an active, adaptive approach” is vague. Implementation of what?
64.	Lines 625: should read “assisted with crab surveys in Drakes Estero”
65.	Line 848: The figure legend refers to “protected coastal habitats” but the manuscript uses estuary throughout. I suggest being consistent with the use of estuary, and changing this in the figure legend.
66.	Line 849: Suggest changing the caption to better describe blue points to say: “(A) Range of southern sea otters in California estuaries as of 2016. Red points indicate protected coastal habitats where permanent populations currently exist. Blue and black points indicate estuaries within 100 km of the current range (USGS, 2018), and blue and red sites are focal sites used in this study…” In addition, the red line is not explained in the figure caption, and I suggest doing so for clarity.
67.	Line 858: The caption states “equilibrium abundances” but the figure legend says “density at K”. Which one is correct?
68.	Population model code does not appear to have been attached.
Experimental design: Major comments
1.	The Introduction needs more discussion of the knowledge gap being filled by this research, in addition to that provided on Lines 117-121. Along those lines, one way to provide justification and context for this work is to discuss the current status of southern sea otters in the Introduction. I suggest moving Lines 464-472 into the Introduction (or stating something similar in the Introduction), as it helps to clearly articulate the knowledge gap and impetus for this work. Similarly, in order to provide additional context, I suggest discussing the results from Silliman et al. 2018 (i.e. that many animals, rather than occupying these seemingly unique habitats for the first time, are in fact reoccupying historical habitat). I could see this fitting in well around Line 100.
2.	The methods are currently not written with sufficient detail and information to replicate. The sizes of crabs in the 1970s are not brought up in the Methods, and are first mentioned on Line 345. I suggest making these time periods more explicit in the Methods. As the methods are currently written, it appears that current prey species composition and crab biomass and size were compared between sites within and outside the current sea otter range. This would be an issue, as sea otter presence over time at sites within the current range would likely change the biomass and size of crabs available, hence mentioning the pre-sea otter colonization sizes and biomass is important.
3.	Similarly, there is not enough detail included in the crab capture methods on Lines 244-256. What effort went into surveying crabs? How many days were pots put out? In what seasons? At different points in the tidal cycle?
4.	It is not clear as written on Lines 238-243 and Line 332 if the prey in the “prey species lists” have been observed as sea otter prey items, or if these benthic prey were identified as “possible” sea otter prey items during benthic surveys. Have all the prey types listed been observed being consumed by a sea otter? I think sea otter diet data and benthic invertebrate surveys should go into making this list. In Table 2, oysters are listed, which are not a known sea otter prey item to my knowledge, and the literature cited are for benthic surveys alone. I think Supplementary Table 2 is much more informative and clear in this regard.
5.	I am not convinced that assessing the biomass and size of two crabs and the presence/absence of other prey in each estuary is a justifiable metric of sea otter prey availability and therefore potential for expansion into an area. I think this choice needs further discussion and justification on Lines 238-240.

While Hughes et al. 2013 and Garshelis and Garshelis 1984 are cited, I do not think that these citations support the assertion that crabs “are considered to be an essential prey item for successful sea otter recolonization”, even though Hughes et al. 2013 did find that the majority of the diet in Elkhorn Slough was made up of crabs in the last 10 years. I think the jump between crabs being the majority of the diet in some currently occupied areas and therefore being “an essential prey item for successful sea otter recolonization” is too big. While I agree that crabs are an important prey item for sea otters in many areas, I think this assertion needs further support from the literature in addition to Garsehlis and Garshelis 1984 and Hughes et al. 2013. What about areas that sea otters have recolonized where bivalves are the primary prey item? How can crabs be an “essential prey item for successful sea otter recolonization” in areas where they are rarely eaten?

Minor comments/edits
1.	If you decide to leave the literature review in the Methods/Results, I suggest a visual of the divisions used for categorizing the literature on Line 143-148, like a flowchart.
Validity of the findings: Minor comments/edits
1.	Line 420: It is unclear how the 31% is calculated. If eelgrass habitat (1750 ha) and kelp habitat (~5700 ha) is what constitutes sea otter recovery habitat for this calculation, then eelgrass habitat would make up approximately 23% of available sea otter habitat.
2.	The results of the literature review for northern and southern sea otters in different ecosystem types and habitats mostly focus on southern sea otters, does not discuss different habitat types within the ecosystems, and only briefly discuss northern sea otters. If you choose to keep the literature review as it’s own section of the Methods and Results, the results of the literature review should be discussed in greater detail.
Additional comments: Major comments
1.	The authors fail to mention that surrogate reared pups have been and are currently released into Elkhorn Slough. It is misleading to leave this out and not address how this has or has not influenced the population in the Slough, especially given that surrogate reared animals account for the majority of the population in the Slough. How does the population model account for these surrogate reared pups? Are they included or excluded? Similarly, the reintroduction of sea otters to other areas of historical occupation (like SF Bay) through the surrogate program has very much been a part of the conversation in recent years, especially with the Slough appearing to be at or near K, but the potential reintroduction of surrogate reared pups to SF Bay is not mentioned in the Introduction. This could provide additional justification and context for this work by exploring the potential of other estuaries to support sea otters.
2.	If the Introduction is edited to include additional discussion of potential sea otter reintroduction to SF Bay, I suggest also bringing up historical reintroductions to SE AK, BC and WA around Lines 91-94. I think this will be important to mention, given that sea otter reintroductions have been instrumental in sea otter recovery post fur trade (pages 44-59 of Sea Otter Conservation book, 2015).
3.	Line 539-540: I think sea otter/fisheries interactions warrant further discussion, and additional citations beyond Garhselis and Garshelis (1984). I suggest a brief but more comprehensive summary of the perceived conflict (and in some cases documented impacts) of sea otters on fishery species, and citing the work below.
Fanshawe, S., G. R. VanBlaricom, and A. A. Shelly. 2003. Restored top carnivores as detriments to the performance of marine protected areas intended for fishery sustainability: a case study with red abalones and sea otters. Conservation Biology 17:273–283.
Larson, S. D., Z. N. Hoyt, G. L. Eckert, and V. A. Gill. 2013. Impacts of sea otter (Enhydra lutris) predation on commercially important sea cucumbers (Parastichopus californicus) in southeast Alaska. Canadian Journal of Fisheries and Aquatic Sciences 70:1498–1507.
Hoyt, Z. 2015. Resource Competition, Space Use and Forage Ecology of Sea Otters in Southern Southeast Alaska. 152 pp.
Minor comments:
1.	Line 125-127: Food for thought: The amount of research on sea otters in different coastal ecosystems (open coast vs. estuaries) is also determined by where estuary habitat is available for them to occupy/reoccupy and how much of it is available, in addition to where sea otters have been reintroduced/where the extant population remained, and/or research priorities. It would be interesting to also include a review of the literature that synthesizes where sea otters have estuary habitat available and have or have not reoccupied those areas. It seems to me that there would be much less habitat in estuaries than open coast habitat, but it would be interesting to see the numbers on that.
2.	Line 362-364: Food for thought: This “narrow configuration” also seems to be the case in Washington, where sea otters are limited to a predominately one-dimensional habitat with range expansion only occurring at the range edges. However, as stated above, northern sea otter populations (including in Washington) are growing faster that southern sea otters in California. How do you account for this discrepancy, despite similar shoreline complexity/habitat configuration?
3.	Lines 398-399: I suggest further discussion of sea otter haul out activity elsewhere to provide a broader context for this point. Is this haul out frequency unique to the Slough due to the availability of salt marsh habitat? What is the haul out frequency of sea otters elsewhere in their range (not just on the outer coast of CA)?
4.	Line 454: After the discussion of the sea otter trophic cascade in eelgrass habitats, I am left wondering how this connects to sea otter recolonization, especially of San Francisco Bay. I suggest explicitly mentioning the projected benefits of sea otter recolonization into SF Bay, with potential for mediation of eutrophication and expansion of eelgrass beds, in the last sentence of this paragraph (even though they are discussed further in later paragraphs of the discussion).
5.	Lines 251-256: Suggest specifying that green crab is not included in Oftedal et al 2007, thus necessitating the use of a function from another crab.
6.	Line 486: Suggest adding year that sea otter was hit


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: SPECIES RECOVERY AND RECOLONIZATION OF PAST HABITATS: LESSONS FOR SCIENCE AND CONSERVATION FROM SEA OTTERS IN ESTUARIES
Review round: 1
Reviewer: 2

Output:
Basic reporting: I think it reasonably good. The narrative is very long and comes across more as a position of advocacy then purely objective analysis. That said, I agree with many of the arguments and believe others close to the issues will as well.
Experimental design: NA
Validity of the findings: The analyses involve a fair number of unevaluated assumptions but otherwise I find the results to be fair and reasonable
Additional comments: Review of Hughes et al. for PeerJ

I should begin with the disclaimer that I am not broadly familiar with PeerJ and thus am not well suited to judge this ms. because of my general ignorance of the journal’s standards of rigor, scope, length, etc.

Hughes et al. provide an assessment of California’s estuaries as future habitat for sea otters; review the ecological consequences of repatriating sea otters to estuarine systems; and engage in a rather length dialogue (via the Discussion), mainly as proponents, for the importance of estuaries in the future conservation and management of southern sea otters. Some of the narrative is redundant with things that have already been written but the analyses, especially those having to do with sea otter population growth and abundance potential, are rigorous, novel, and very important to any future dialogue about sea otter management in California. This in my view is the strength. Although the ms. is a long read, I found it to be factual, reasonably objective, and worthy of publication in the scientific literature. The Discussion goes on and on to discuss the sundry issues concerning the repatriation of sea otters into estuarine habitat and mostly to advocate for this course of action. But in fairness, they also mention and discuss the costs and downsides of doing this, albeit seemingly to a lesser degree.

I don’t have major concerns or suggestions, owing largely from the disclaimer above. There is nothing I can see that is technically wrong with the ms. and it does provide information that will be of interest and use to scientists, managers, and those involved with policy.

Minor suggestions or points in need to further discussion and resolution

115. high production and ample food,

122. sea otters to better understand . . .

The multiple usage of “to better understand” in this paragraph is awkward. I would revise accordingly.

Equation 0.1. This is indeed a widely known model for density-dependent population change. I have two concerns about its use here. One is the assumption of linear density dependence (i.e., as a function of N/K). Most of the empirical analyses and data I’m familiar with for large mammals seem to indicate that per capita density dependence is not linear but instead increases more sharply near K. This is not a fatal flaw but rather something to consider in the revision. My other concern is over the assumption of error with mean = 0, which implies that counts are unbiased. This is almost never true for wildlife surveys as there is always some probability of an animal being missed. There are methods for estimating the probability of sighting but they have not been applied to these data, at least so far as I am aware.

I am not competent to evaluate the statistical methods described between lines 185 and 202. I think it important for someone to check through this who is.

238. Despite Garshelis 1984 and the various observational data of sea otter diet, I’m not entirely comfortable with the claim that crabs are essential for sea otter colonization. I see no logical reason why that should be. Instead, it may simply be that most or all estuaries support decapods and thus sea otters consume decapods when and wherever they have redolonized estuarine habitats. I simply can’t understand why other groups of prey would not be suitable prey in this process, even if crabs were entirely absent. Unless the authors provide better supporting evidence, I think they need to rethink and possible reargue this element of the ms.

317. “Future work to improve precision of these parameter estimates will reduce the uncertainty in model projections.”  This statement strikes me as being almost patently obvious on the one hand and probably untrue on the other. That is, I’m not convinced that the major uncertainties and surprises in population trends are the result of parameter imprecision rather errors or deficiencies in model structure.


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: DIFFERENTIATION BETWEEN MAXILLARY AND MALAR MIDFACE POSITION WITHIN THE FACIAL PROFILE
Review round: 1
Reviewer: 1

Output:
Basic reporting: This manuscript is written in a clear and unambiguous style. It is well structured, with clear objectives. The literature review and the references are sufficient and pertinent to the subject of research. The conclusions are drawn from the data.
Experimental design: Appropriate
Validity of the findings: Novel and valid
Additional comments: Theodore Eliades
Academic Editor, PeerJ

Dear Dr. Eliades:
Thank you for considering me to review the article entitled “Differentiation between maxillary and malar midface position within the facial profile"
General comments:
In general, this is an interesting, well conducted and written study. It describes a novel approach (through new cephalometric landmarks and measurements) to evaluate/differentiate between fullness and flatness. In other terms, it is a 2D cephalometric study that has a 3D perspective on differentiating between the malar and the maxillary position. The study design, sample, and the performed statistical analyses were conducted in an appropriate way.
I would like the authors to consider the following comments and requests to buttress their conclusions.
1- The authors are basically evaluating flatness/fullness in adults. We do not know why they excluded growing individuals. Are there any evidence on midface flatness changing with growth? I would like the authors to add a sentence on effect of growth on midface flatness if there is any.
2- In table 1, it would be interesting to report the matching percentage among the different groups for NJ/SN and NG/SN. As an example: flat group: 90% match (only 3 individuals did not match); Normal toward flat group: 100% match..etc. And how the patients that did not match were grouped? Where did the 3 individuals in the NJ/SN flat group (that did not match with the NG/SN group) fit…etc?
Authors can report this, either in the text or in table 1 by adding one row below.
3- An interesting finding that the authors may highlight is the no difference in the maxillary length ANS-PNS between the different groups, whether categorized on NJ/SN or NG/SN. This finding implicates that the length/size of the maxilla does not play a role in the presence or not of flatness. The evaluation of flatness/fullness in this study is based on landmarks and measurement that they are higher than the palatal plane. Tis may explain why a small maxilla (in length) does not associate with flatness and a bigger maxilla with fullness.
4- Did the authors check gender differences among the different parameters? And if not, do they assume that results will not be different?
5- I suggest adding points J and G in Figures 1b-c and Figures 2A-B-C-D for better visualization.

Thank you and best regards,


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: DIFFERENTIATION BETWEEN MAXILLARY AND MALAR MIDFACE POSITION WITHIN THE FACIAL PROFILE
Review round: 1
Reviewer: 2

Output:
Basic reporting: I appreciate the courage shown by the author in conducting this research, including the time and effort they have put in. However there are certain shortcomings in this paper.
Experimental design: The authors evaluate the relative position of two geometric landmarks in the anteroposterior plane, regarding the length of the anterior cranial base and the points S and N. However midfacial flatness or fullness cannot be absolutely evaluated, i.e. without considering the position of the mandible and the forehead. The use of G point (“a more lateral landmark” according to authors) to evaluate the anteroposterior position of malar area is problematic.

Patients with craniofacial anomalies were excluded: this needs further clarification. How were the asymmetries in the Orbitale position or the zygomatic regions excluded?
Validity of the findings: Several contemporary 2- or 3D techniques are available for craniofacial shape analysis, in order to overcome the limitations of the conventional cephalometric analysis.
Additional comments: No additional comments


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

Input:
Title: CONCENTRATION-DEPENDENT POLYMORPHISM OF INSULIN AMYLOID FIBRILS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The article is clearly written, but in places features a somewhat clumsy language. For example I would advise to avoid phrases like "When the 0.2 mM-formed fibril conformation is added" and replace them by something like "When the LCF are added", whereby LCF stands for low concentration fibrils.
Also, the language should be proof read for general grammar, mainly missing articles. Example: “Ability of the same protein to adopt distinct pathogenic conformations” should read “The ability of the same protein to adopt distinct pathogenic conformations”

The structure of the article is clear, there is no superfluous material, and the literature citations are mostly adequate. I would recommend adding one or two references that discuss the existence of secondary nucleation pathways in the case of insulin aggregation, e.g. the work by Fodera et al., J. Phys. Chem. B 2008.

Raw data is provided in an easily accessible form.
Experimental design: The experiments are well described and designed. The research question is also well-defined and a very important one. Secondary nucleation of amyloid fibrils is receiving increased attention in recent years, as the potentially dominant route towards the generation of new aggregates. One of the key questions is whether secondary nucleation is able to propagate structural characteristics of the seed fibrils, as well as in the case of fibril growth, where this has already been demonstrated in multiple studies. The present study addresses this question in a clever way, by exploiting the concentration-dependent polymorphism of insulin aggregation. Despite the fact that the amyloid fibril formation experiments in this study are performed under highly non-physiological conditions, the study is highly relevant for the field of amyloid fibril formation and the link with disease. This is because very little is known about the molecular mechanisms and determinants of fibril surface-catalysed secondary nucleation, and therefore any mechanistic insight gained for any amyloid system is a valuable contribution at the current state of knowledge.
The conclusions of the study are based on the results of three types of experimental characterisations of fibrils: ThT fluorescence intensity (normalised by fibril mass), FT-IR spectroscopic signatures of fibrils and AFM analysis. All three techniques lead to the same conclusions, which makes the results very robust.
Validity of the findings: Overall, the study leaves no significant doubts as to the validity of the findings. I just have a few minor suggestions that might improve it further:

1) It would help to have  also the unseeded data plotted in Figure 3, in order to help visualise the degree of seeding achieved by the lowest employed seed concentrations.

2) For the FT-IR and ThT experiments, it support the conclusions if the authors measured the degree of completion of the aggregation reaction at the plateau, at least in some select cases. With that I mean that the authors should centrifuge the samples and quantify the concentration of soluble insulin in the supernatant. This is in principle necessary before the authors can normalise the ThT signal by fibril mass. It needs to be shown that the sample converts (near) quantitatively into fibrils.

3) It would also help to show an FT-IR spectrum of monomer, such that it can be established that the differences in the observed FT-IR spectra between high and low concentration fibrils do not stem from different monomer contributions.
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: CONCENTRATION-DEPENDENT POLYMORPHISM OF INSULIN AMYLOID FIBRILS
Review round: 1
Reviewer: 2

Output:
Basic reporting: No comment.
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The manuscript is clearly written and the authors findings are well explained


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

Input:
Title: CONCENTRATION-DEPENDENT POLYMORPHISM OF INSULIN AMYLOID FIBRILS
Review round: 1
Reviewer: 3

Output:
Basic reporting: Some references should be cited:

On line 32-33: Doig AJ et al.  ACS Chem Neurosci. 2017:1435-1437;  Nasica-Labouze J et al. Chem Rev. 2015 May 13;115(9):3518-63;  Man VH et al. J Phys Chem B. 2017 Jun 22;121(24):5977-5987.

On line 37: Nguyen PH et al. J Phys Chem B. 2014 Jan 16;118(2):501-10; Pellarin R et al. J Am Chem Soc. 2010 Oct 27;132(42):14960-70.

Later in the discussion: Scheidt T et al. Sci Adv. 2019 Apr 17;5(4):eaau3112; Törnquist M,
et al. Chem Commun (Camb). 2018 Aug 2;54(63):8667-8684; Bunce SJ et al.
Sci Adv. 2019 Jun 21;5(6):eaav8216.
Experimental design: well done
Validity of the findings: interesting and novel results are well identified.
Additional comments: The authors report a nice experimental study on the aggregation kinetics of insulin.
My only comments is to cite the references of litterature that I have reported.


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: ICARITIN, AN INHIBITOR OF BETA-SITE AMYLOID CLEAVING ENZYME-1, INHIBITS SECRETION OF AMYLOID PRECURSOR PROTEIN IN APP-PS1-HEK293 CELLS BY IMPEDING THE AMYLOIDOGENIC PATHWAY
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1. title editing from "Icaritin, a beta-secretase 1 inhibitor, impedes amyloidogenic pathway of amyloid precursor protein secretion in APP-PS1-HEK293 cell" to " Icaritin, a beta-secretase 1 inhibitor, inhibits  amyloid precursor protein secretion in APP-PS1-HEK293 cels by impeding amyloidogenic pathway ";

2. Does APP-PS1-HEK293 cells purchased? then from where? What is the nature of this cell line? Is APP-PS1 overexpressed in HEK293 cels? Please give some background information about the cells;

3. Line 168, "the Aβ1-40 content is decreased", please specify where? the Aβ1-40 both intracellualr and extracelluar or ...?

Please see all other suggestions as following:

Line 20, BACE1 ---à beta-secretase 1 (BACE1);
Line 28, delet “And”;
Line 29, change “when” to “ at”; add “and “ between 5um and 1o um.
Line 30, add “and “ between 5um and 1o um.
Line 30, “ lower”, please specify intracellular or extracellular or both?
Line 47, “problem facing China” ---à “problem China facing”
Line 38-39: same change as the title
Line 52: first letter should be in capital for “nobel prize”

Line 53, “his” ---à “her”
Line 57: delet “ and it”

Line 60: “besides” replaced by “furthermore “

Line 63: “model rats through” replaced by “ rats model by”

Line 64: abeta generation? May abeta accumulation better?

Line 65: “its neuroprotective effection” replaced by “ possibly neuroprotective effects in vitro”

Line 102: “.” Replaced by “,”, add”then” in front of “ cells”

Line 103: replace “and” with a “,”

Line 110: add “ content” before“ following”

Line 114: change “cells” to “cell “

Line 124: add a subtitle “ Western Blots”;
“ describe ahead” replaced by “ following procedures”; add” Briefly” in front of “After”

Line 133: “during” replaced by “ with”; “ with” replaced by “ by”

Line 134: “antibody” replaced by “ secondary antibodies”

Line 137: add a subtitle “ Immunofluroscence”

Line 138: “ -20 degree “ or “4 degree “? Please confirm

Line 141 and 142: please confirm “ 37 degree” or “ room temperature “? After fixation, why need to be done at 37 degree? It is not live cells anymore, correct?
Line 155: replace “.” By “ ;” ; then add “ Just” before “like”; add “ are” before “both”

Line 160 and 161: “ when” replaced by “at”

Line 161 and 162: replace “,” with “and” between “ 5uM and 10uM”

Line162: add “ that” after “ suggest”; delet”,””

Line 163: replace “ and” by “ at”

Line 166 and 167: move “ culture media obtained from APP-PS1-HEK293 cells” to the “,”, in front of “ the contents”

Line 168: please specify the “decreased” both intracellular and extracellular or ...?

Line 176 ,177 and 180: “with control” replaced by “ with the control group”
Line183: “with control” replaced by “ with that in control group”

Line 186: “ with” replaced by” from”; also please specify what is the difference? Components or functions?

Line 191: where are the data of increased expression of a-secretase?

Line 192: “ this is “ replaced by “ which are”; “ has” replaced by “ have”

Line 196: delet”we”

Line197: “didn’t expect” replaced by “ unexpected “; delet” role”; replaced “ when” with”at”

Line 198: add “ and” in front of “ maybe”

Line200: “problems” replaced with” points”?

Line 201: “patient” replaced by “ patients”

Line204: “ do “ replaced by “ does”

Line 216: “ rat” replaced by “ rats”; add “ and” before“ these”

Line 220 and 221: the same change as title
Experimental design: Q1: Some background information of APP-PS1-HEK293 cells needed; for APP-PS1-HEK293 cells, non Aβ-42 could be available or produced? If yes, why measure Aβ-40 but not Aβ-42 when exposed APP-PS1-HEK293 cells to ICAT? Since the amyloid plaques in Alzheimer’s brains consist of mostly Aβ42 and some plaques contain only Aβ42, even though Aβ40 concentration is several-fold more than Aβ42.
ICAT could decrease Aβ-40 both intracellular and extracellular, but doesn’t mean Aβ-42 (amyloid plaques consisting mostly or only) accordingly decreased as well, since the concentration of Aβ-40 in CSF has been found to be several fold more than that of Aβ-42. Of course, high concentration of Aβ-40 doesn’t mean Aβ-42 must be high either, but depends how much (or the ratio) Aβ-40 transformed to Aβ-42 or even the degradation ratio of Aβ-42 to Aβ-40.
Q2: Did authors by any chance test α-secretase pathway about APPsα or AICD ( APP intracellular domain)changing?
In many instances, an increase in non-amyloidogenic APP metabolism is coupled to a reciprocal decrease in the amyloidogenic processing pathway, and vice-versa, as the α- and β-secretase moieties compete for APP substrate. As a result, if tested a-secretase pathway, even if it a non-amyloidogenic pathway, but could support amyloidogeic BACE1 pathway; if tested AICD, which play a role in transcriptional transactivation, may provide a clue for mRNA and protein expression changing.
Validity of the findings: Some expressions are not accurate, and rewording needed.
Please see the edited file uploaded.
Additional comments: Amyloid plaques, extracellular deposits primarily composed of the 4 kDa, 40–42 amino acid Aβ peptide, a product of amyloid precursor protein (APP) proteolysis, and neurofibrillary tangles (NFTs), intracellular aggregates of the microtubule associated protein tau are two characteristic brain lesions at the microscopic level to define Alzheimers’ Disease (AD). Aβ peptide is generated following the sequential cleavage of APP by β- and γ-secretase in the amyloidogenic pathway. A beta-secrtase1 (BACE1) exhibits all the properties of the β-secretase, and as the key rate-limiting enzyme that initiates the formation of Aβ, BACE1 is an attractive drug target for AD. This study explore the Icaritin, (BACE1) inhibitor, inhibits the amyloid precursor protein secretion by impeding the amyloidogenic pathway.


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: ICARITIN, AN INHIBITOR OF BETA-SITE AMYLOID CLEAVING ENZYME-1, INHIBITS SECRETION OF AMYLOID PRECURSOR PROTEIN IN APP-PS1-HEK293 CELLS BY IMPEDING THE AMYLOIDOGENIC PATHWAY
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1. The ms needs to be run by a native speaker to improve the structure and clarity of the sentences.
2. Not all relevant literature is cited (please see below). A similar compound has been investigated recently in an AD model, this needs to be mentioned in the discussion.
Choice of a cell model not explained.
3. Article structure is OK, figures are of acceptable quality. More details about experimental design need to be provided in figure legends which are currently too short to be informative.
4. Data don't fully support the conclusions. Other APP fragments (sAPPbeta, sAPP alpha, CTF) need to be analysed as per an extended comment below.
Experimental design: 1. Research is original.
2. Research question is not very clearly defined (please see comment below).
3. Data are preliminary and don't fully support the conclusion.
4. Methods need more detail, e.g. how intracellular beta was measured? what BACE1 substrate was used? (please see comment below)
Validity of the findings: The data present in this report do not fully support the conclusion that icaritin impedes amyloidogenic pathway. More proteolytic fragments of APP processing need to be measured.
Additional comments: Here, Feng et al describe anti-amyloidogenic effects of icaritin, a flavonoid compound from a Chinese medical herb. The work is mostly done in APP-PS1-HEK293 cells and the only data that report direct effect of icaritin on BACE1 activity is coming from a BACE1 activity kit (substrate used in this assay was not indicated).

Icaritin has some documented neuroprotective properties in vitro and the authors hypothesize that it should also have an effect on BACE1 activity based on its chemistry. Their previous work show neuroprotective effects on icaritin on rats memory in vitro. It would be good to change the word “secretion” to “processing” in the title because here the authors don’t investigate APP protein secretion.

To analyse the effect of icaritin on amyloidogenic processing, the authors looked at mRNA levels and protein levels of PS1, ADAM10 and BACE1 and abeta40 and saw that these amyloidogenic components were downregulated while ADAM10 was upregulated upon treatment (Fig.5). In order to know whether proteolytic activities of these enzymes in cells also change upon treatment with icaritin, sAPPalpha, sAPPbeta and CTF need to be measured by Western blotting or ELISA.

The compound was tested on cells at 0.5, 5 and 10uM (Fig.5). In terms of LDH leakage rate (Fig. 3 B), it is not clear why there was low level of leakage (=good viability) at 5 and 10uM but not at 0.1-1uM of icaritin. The LDH data make it difficult to interpret western blot data on Fig.5 and cell viability data (Fig.3 A).

Fig.5A: what is the molecular weight of the fragments shown on Western blots? Did the authors detect other long and short abeta species? Why abeta42 was not measured alongside abeta40 to see if ratio of 42:40 is not shifted towards generation of longer beta species?
Fig.4: how was the intracellular Abeta extracted and measured? More details on cell lysate processing need to be provided.

In the introduction, the authors state that genetic evidence demonstrated that abeta plays a pivotal role in AD pathogenesis (lines 47-50). It would be more cautious to say that genetic evidence indicates that dysregulated gamma-secretase-mediated processing of APP is involved in familial AD. Abeta is just one of the proteolytic fragments that happens to accumulate in AD brain. There is also accumulation of misfolded tau which may have a significant role in AD pathogenesis, too.

There is a recent study (doi:10.7150/ijbs.6232) exploring anti-amyloidogenic effects of “icariin” which is a compound obtained from the same plant (Epimedium) as icaritin but with a higher molecular weight. They observed a reduction in APP and BACE1 expression in APPV717I Tg mouse model of AD. It would be helpful to discuss structural similarities/ differences between icaritin and icariin.

Overall, the data present in this report are preliminary and do not fully support the conclusion that icaritin impedes amyloidogenic pathway. More proteolytic fragments of APP processing need to be measured.


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: ICARITIN, AN INHIBITOR OF BETA-SITE AMYLOID CLEAVING ENZYME-1, INHIBITS SECRETION OF AMYLOID PRECURSOR PROTEIN IN APP-PS1-HEK293 CELLS BY IMPEDING THE AMYLOIDOGENIC PATHWAY
Review round: 2
Reviewer: 1

Output:
Basic reporting: 1. Please confirm Line136-137 about portein lysate procedure. Does really need lysis buffer incubating with cell line for 30min at 4 degree? But not as usual, adding lysis buffer to the cells in the plate wells, a couple of minutes late, scraping them off on ice? Also, usually 12,000rpm, 30 min in centrifuge, should be done at 4 degree but not room temperature?

2. You don't really try many concentration, why emphasize so much " concentration depended"? Suggest don't use these words, 5uM and10uM work, that is it.
Experimental design: This is a original primary research with aims and scope of the journal, and research questions are well defined, relevant and meaningful. Methods described with detail information for replication, and investigation performed with a high technique and ethical standard as well.
Validity of the findings: All the data provided are relatively sound, and conclusions are well stated and supported by results.
Additional comments: It is much better for writing after edited, but always keep in mind this is a scientific report. Any conclusion you draw based on the data you got, don't say more than what your data could tell. More accurate, better.


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: ICARITIN, AN INHIBITOR OF BETA-SITE AMYLOID CLEAVING ENZYME-1, INHIBITS SECRETION OF AMYLOID PRECURSOR PROTEIN IN APP-PS1-HEK293 CELLS BY IMPEDING THE AMYLOIDOGENIC PATHWAY
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors have done a great job revising the manuscript which reads much better now.
Experimental design: All questions have been addressed and improvements have been made accordingly.
Validity of the findings: I have a few minor remarks about the current version of the manuscript:

1. There is a typo in Abeta molecular mass indicated on western blot in Fig.5A. Abeta40 should be around 4.3kDa (not 32kDa) and Abeta42 around 4.5kDa (not 35kDa).

2. Raw data for Abeta42 is missing from supplementary data (uncropped blots from Fig.5).

3. From the raw data on uncropped blots (panel PS1-G) it looks like upon icaritin treatment PS1 expression goes up  in 2 experiments out of 3, which probably warrants correction.

4. Raw western blot data for Fig.5E: the label should read "ADAM10" not "AMDM10".
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: EFFECTS OF MAXIMUM RESIDUE LIMIT OF TRIFLUMEZOPYRIM EXPOSURE ON FITNESS OF THE RED IMPORTED FIRE ANT SOLENOPSIS INVICTA
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript needs a thorough editorial review to eliminate awkward phrases, subject-verb tenses, and singular and plural subjects. The similarity of this paper and Pan et al. (2017) is pretty noticeable. The only significant difference is that triflumezopyrim was tested instead of sulfoxaflor. I am not sure it is really telling us something new about these new neonicotinoids.

Maybe if the authors could explain why and how ants are important nontarget insects of neonicotinoids. I thought the issue with neonicotinoids was with nectar, pollen and honeybees and maybe parasitoids of some plant insects.  I don't see what the results of this study tell us about these new neonicotinoids.

"The objectives of the study were to determine the effect of field residues of triflumezopyrim on colony fitness and the behavior of workers." Field residues are actually food tolerance in rice grains and hulls. Are the ants actually feeding on the rice and hulls?

How does RIFA enter into the rice ecosystem? Maybe this could be better explain why they are being tested.
Experimental design: I am having difficulty understanding how these studies tell us anything about the risks of triflumezopyrim in the environment. I don’t see the connection between red imported fire ants (RIFA) survival and non-targets, especially pests. The two concentrations tested had no effect on RIFA. So!!!!

The food tolerances of triflumezopyrin in rice grains and hulls are actually 1.0 μg/ml and 0.4 μg/ml, respectively, according to the Federal Registry as of 2017.  I am not sure how this relates to what is actually being tested. In the first and third study the ants are fed sugar water solutions containing 0.5 μg/ml and 0.2 μg/ml triflumezopyrin.  How much a.i. do the ants actually consume? It isn’t clear in the methods how Figure 2A and 2B are generated. In a second study, I think the filter paper is treated with these solutions, but it is not clear.

How does this relate to the food tolerances of rice? The methods and material section as written are totally inadequate. It is necessary for the reader to look up each reference, Pan et al. (2017), Huang et al. (2016) and Wang et al. (2015), to be able to understand how the tests and data being generated.

Is any concentration of triflumezopyrim in sugar water toxic to the fire ants? You mention on line 121 that these are sublethal doses. I think the toxicity of triflumezopyrin must be established if you want to actually claim them as sublethal doses.
Validity of the findings: The title is misleading by claiming the effect of residues. At least 2 of studies involved ants consuming sugar water baits.

In the conclusion the authors state that "further studies using field-realistic concentrations of triflumezopyrim are needed in order to support these findings." What are realistic concentrations?  They should have been tested in this study.
Additional comments: Title and Line 19: In several of the experiments the ants are feeding on sugar water solutions containing triflumezopyrin. These are not residues.
Line 25-26. Ok. So what does that mean.
Line 51-52. What nontargets insect are impaired?
Line 66. Topical, contact or oral imidacloprid treatment?
Line 166. I really don’t understand this result at all. If triflumezopyrim were toxic, then I could see a decrease in the ability to hold on to a substrate. I don’t see why it should increase.
Lines 191-192. Are RIFA eating the treated rice? I don’t understand what the logic behind these statements.
Line 200. I don’t think paradigms is the correct word here.
Line 213. Why not test the toxicity of triflumezopyrim instead of just assuming that it is less toxic?
Line 236. This study should have used “field-realistic concentrations.”


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: EFFECTS OF MAXIMUM RESIDUE LIMIT OF TRIFLUMEZOPYRIM EXPOSURE ON FITNESS OF THE RED IMPORTED FIRE ANT SOLENOPSIS INVICTA
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The manuscript “Effects of residues of triflumezopyrim exposure on fitness of red imported fire ant Solenopsis invicta” by Li et al, investigated the chronic impacts of triflumezopyrim to red imported fire ant, Solenopsis invicta after 56 days of exposure from free feeding. The three experimental paradigms which include food consumption and colony growth; active behavior (grasping ability and walking speed) and the aggressiveness were monitored in a 56-day treatment. Their results indicated that 0.5 ug/ml and 0.2ug/ml triflumezopyrin increased individual aggressive index, and not in group aggressive encounters; 0.5 ug/ml triflumezopyrin increased moving speed and grasping ability, while 0.2ug/ml triflumezopyrin only increased grasping ability alone. However, neither 0.5 ug/ml nor 0.2 ug/ml triflumezopyrin had significant impact on S.invicta colony growth and food consumption. For the manuscript, I have several major and minor comments.
Major comments:
1.	The red imported fire ant, Solenopsis invict, is a well-known invasive ant species that was introduced into China since 2003. As an eusocial insect, the physiology and behavior of S. invicta in subcolony could change in a 56 day treatment, so it is very important to use the entire colony, not subcolony, for long-term experiments in social insect study. If only workers in the subcolony, growth and food consumption bioassay and active assay were not comparable to the whole colony (include workers, brood, larva, pupa, female, male alate and queen) in natural condition in 56 days treatment.

2.	Make sure your result descriptions are consistent:

Abstract L21, Our results showed 0.5 ug/ml not 0.2ug/ml triflumezppyrin increased moving speed and grasping ability of S. invicta.
Result, L165, Grasping rate of workers in triflumezopyrim treatments is higher than that in control.
Result, L173, 0.2ug/ml triflumezopyrim treatment is not significant from either that in 0.5 ug/ml triflumezopyrim or control.
Discussion, L194, Although 0.2 and 0.5 ug/ml triflumezopyrin treatment increased grasping ability, walking speed …..,
Discussion, L205, Our results showed 0.2 and 0.5 ug/ml triflumezopyrin treatment increased grasping ability, walking speed …..,

3.	How did you conclude that triflumezopyrin did not have any negative impact on the fitness level of S. invicta at 0.5 ug/ml and 0.2 ug/ml exposure regimes? Three paradigms which related to the fitness of a fire ant colony were chosen in this study, however, two paradigms have significant impacts on S.invicta subcolony.

4.	It is the best to generate a separate table for the results and statistical analyses for food consumption result.

5.	What is the social form of the fire ant, Solenopsis invict, in your study, which needs to be added in MS.

6.	Paper needs to go over thoroughly to optimize sentence structure
Minor comments:

Spelling errors in L101, L129 and L134.


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: EFFECTS OF MAXIMUM RESIDUE LIMIT OF TRIFLUMEZOPYRIM EXPOSURE ON FITNESS OF THE RED IMPORTED FIRE ANT SOLENOPSIS INVICTA
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The authors have done a nice job of revising the manuscript. I have made a few minor comments along the margin of the revised draft.


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: EFFECTS OF MAXIMUM RESIDUE LIMIT OF TRIFLUMEZOPYRIM EXPOSURE ON FITNESS OF THE RED IMPORTED FIRE ANT SOLENOPSIS INVICTA
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The authors followed the reviewer’s comments, made most of changes basically. Especially, they used language editing services to improve the English languages of the manuscripts. Therefore, the current version is suitable for publication with minor revision. I have two major and two minor comments.
Major comments
1.	Make sure your result description is correct
Abstract L23-24  Our results showed 0.5 μg/ml not 0.2 μg/ml triflumezopyrim improved the grasping ability of S. invicta, (Line 167-168, your statistical analyses result  showed 0.2 μg/ml triflumezopyrim and control,  Mann-Whitney test, U=18, P=0.002) and 0.5 μg/ml triflumezopyrim increased their moving speed.

 Please Change to “Whiles both 0.5 μg/ml and 0.2 μg/ml triflumezopyrim improved the grasping ability of S. invicta, and 0.5 μg/ml not 0.2 μg/ml triflumezopyrim increased their moving speed. In addition, although ….. ”

2.	Statistical analyses result letter was showed in Table 1, but not in table 2, please keep consistent.

Minor comments:
1.	Line 190 10 ng or 1.25?? /insect
2.	Spelling errors in L67, L128, L133 and L139.


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: EXPRESSION AND PROGNOSTIC ANALYSES OF EARLY GROWTH RESPONSE PROTEINS (EGRS) IN HUMAN BREAST CARCINOMA BASED ON DATABASE ANALYSIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: By using publicly available datasets, Fei et al. analyzed the mRNA expression of the Early Growth Factors (EGR) 1 to 4 in breast cancer samples and their correlation with survival in those patients.

•	Professional English and proper punctuation should be used through out the text.
•	The acronym “BC” for Breast Cancer should be spell it out the first time the term is used in the text (abstract).
•	Briefly clarify in the text the meaning of SBR grading 1-3 and include a reference that includes more details about it.
•	Parts of the discussion section are introducing the EGRs, which should be in the introduction section.
•	Figures 4 and 5 are poorly explained in the results and the legends do not describe the figures. Overall proper figure legends should be added.
•	Lines 37, 51: please add referring literature supporting the data provided.
•	Include citation of Oncomine: Rhodes DR, Yu J, Shanker K, Deshpande N, Varambally R, Ghosh D, Barrette T, Pandey A, Chinnaiyan AM. 2004. Oncomine: a cancer microarray database and integrated data-mining platform. Neoplasia 6:1–6
•	Include citation of kmplot: Nagy A, Lánczky A, Menyhárt O, Győrffy B. Validation of miRNA prognostic power in hepatocellular carcinoma using expression data of independent datasets, Scientific Reports, 2018;8:9227
Experimental design: •	Oncomine data needs to be re-analyzed to contain the most up-to-date datasets or add the date this analysis was last performed (I performed an Oncomine analysis with EGR1-4, which showed different results compared to the one presented on Fig. 1.)
•	Threshold Fold Change used for Oncomine analysis should be included in text (line 72)
•	Line 106, please add ratio or percentage of studies. For example: EGR1 was downregulated in 17 out of 48 Unique Analysis (~35%)
•	Include more details on how SBR grading and EGR expression was calculated.
•	Data from figures 4 and 5 are not explained in the text.
Validity of the findings: There seems to be a correlation between low EGRs in breast cancer tumors compared to normal tissues. However, based on this metanalyses, EGR mRNA levels does not seem to be consistent to be used for prognostics as suggested by the authors.
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: EXPRESSION AND PROGNOSTIC ANALYSES OF EARLY GROWTH RESPONSE PROTEINS (EGRS) IN HUMAN BREAST CARCINOMA BASED ON DATABASE ANALYSIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: * Abstract: "the survival data of breast cancer patients were compared to the transcriptional expression levels of the four proteins": The authors mean transcription exp levels were compared in BC patients ...... the structure of the sentence is wrong and can be rephrased to "the association between the survival data and tc levels were investigated". Also, the mentioned four proteins were not identified.

* Abstract: "In addition, The mRNA expression level of EGR1/2/3 were related to MRFS in BC patients with metastatic recurrence". What does M in MRFS mean and "related": What is the direction of the p value?

* Abstract: "EGR4 may have played a role in the development of BC". However, in the results section, the authors mentioned "However, EGR4 has no relevant dataset to indicate a difference in expression level". This is confusing.

* Introduction: The authors reviewed some literature of EGR proteins; however, they did not clarify the gaps in the literature and build the rationale for their study.

* Discussion: Although this section is more organized than the results section, it did not discuss in depth the authors' results and talked a lot about the classification grade and different types of survival. Also, they mentioned up and down regulation in many subtypes without adequate discussion.
Experimental design: - The authors should further describe the TCGA database: how they retrieve BC samples, how many studies they retrieve, any special filtration parameters they used, all alive or including dead, certain race, certain histopathology, certain types of studies, certain gender, etc, they removed patients with unknown or missing data? ... BC has the largest no of samples in tcga and hence has a lot of variations.

- Define the abbreviation the first time they are mentioned: OS, RFS, DMFS, PPS

- Line 89: Put a link for bcGenExMiner v4.2

- Line 97: "or 1.5-fold changes of the difference was statistically significant" Reference?

- Lines 102 and 103 of the results section should be moved to the methods section. Same for lines 128-133.
Validity of the findings: - The results section: should be divided into sections e.g (Expression level in BC tissues, then Association with pathological features, then association with survival) to be more clear.

- You should organize your results by expression levels, not by database. This will enable you to compare expression levels for the same receptor from different databases.

- I did not see classifications by grade except in pathological grade (intermediate and poor differentiation grade) and this could be classified within all 4 molecular subtypes mentioned: luminal A, B, TNBC, and Her2. Also, the results structure is confusing and should be divided and structured better.

- Line 108: In the TCGA dataset which contains 593 EGR1 genetic samples of patients with BC": remove EGR1 from this sentence.

- Lines 146 and 147: "to analyze the EGRs and BC of SBR" should be "to analyze the relation between EGR expression levels and SBR grading of BC patients"

- Line 147: "box-type graph results": It is called "box plot"

- Line 154: This should be a new section with a new title "EGR mutation in BC"

- Table 1: Solie breast, Ma breast, curtis breast: Add in the footer the explanation of these names of reference.
Additional comments: - Although the overall language is good, the manuscript needs revision by an expert language editing service. Also, lack or excess of spacing sometimes should be fixed (line 69, 79, and 239).

- Some sections in the manuscript need better organization, especially the results section.

- The figures are good illustrations, but some has low resolution.


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: EXPRESSION AND PROGNOSTIC ANALYSES OF EARLY GROWTH RESPONSE PROTEINS (EGRS) IN HUMAN BREAST CARCINOMA BASED ON DATABASE ANALYSIS
Review round: 1
Reviewer: 3

Output:
Basic reporting: English style needs to be improved. Some sentences need to be rephrased.
References are fine, background is ok.
Article is soundly structured.
Experimental design: Research questions need to be further investigated.
Specific comments:
1) Metabric database needs to be included in the analyses.
2) EGR transcription factors expression needs to be stratified following the main subtypes of BC.
3) Since EGR are transcription factors with well known target genes what about the expression of EGR gene signatures in BC subtypes
Validity of the findings: Findings are valid but insufficient to support authors conclusions
Additional comments: The manuscript in its present form is not suitable for publication, it needs further experimental work as previously mentioned.


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: EXPRESSION AND PROGNOSTIC ANALYSES OF EARLY GROWTH RESPONSE PROTEINS (EGRS) IN HUMAN BREAST CARCINOMA BASED ON DATABASE ANALYSIS
Review round: 1
Reviewer: 4

Output:
Basic reporting: In this manuscript, the authors combine the data from previous studies on early growth response proteins (EGRs) in breast cancer, by taking advantage of the analysis tools incorporated in the selected databases and draw their conclusions.

- Unfortunately, the manuscript is written in poor English and needs extensive proofreading before it may be published as a review.

- Additionally, the manuscript lacks the professional article structure of the subheadings. Very crowded data presented as a single paragraph with an extensive number of abbreviations which the authors did not spell out at their first mention in the text or spelled them incorrectly (e.g. "RFS: completion free survival, DMFS: completion free survival" among others).
All these add difficulty to follow the authors in their elaborations
Experimental design: - Overall, the study does neither report any new findings (not provided to be novel) nor independently validate any of the previously published findings by wet-lab results.

- The authors should pay attention that the applied databases have different depth of coverage and pipelines for data calling with feature different sensitivity and specificity.

- Overall the paper qualifies as a review rather than an original research article.
Validity of the findings: - Not tested in the current work as mentioned above. The work is lacking a validation step in their lab to add to the previous knowledge.
Additional comments: In addition to the above comments:
- By the end of the introduction, the authors stated in their aim that “Additionally, its expression in breast cancer was analyzed to determine its value in the treatment and diagnosis of breast cancer”. There was no evaluating analysis for diagnostic value assessment (e.g. ROC curve analysis) or the treatment response all over the manuscript.

- The title of the manuscript is not precisely describing the real work by the authors.


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: EXPRESSION AND PROGNOSTIC ANALYSES OF EARLY GROWTH RESPONSE PROTEINS (EGRS) IN HUMAN BREAST CARCINOMA BASED ON DATABASE ANALYSIS
Review round: 2
Reviewer: 1

Output:
Basic reporting: English was clearly improved and proper literature was included. Tables and graphs were modified to be clear. The tracked changes manuscript does not contain all the modifications made, only the major additions.
Experimental design: The authors covered most of the reviewers points.
Validity of the findings: The authors explicitly included in title and text this work limitations (metanalyses only without experimental data).
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: EXPRESSION AND PROGNOSTIC ANALYSES OF EARLY GROWTH RESPONSE PROTEINS (EGRS) IN HUMAN BREAST CARCINOMA BASED ON DATABASE ANALYSIS
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors have adequately addressed my concerns in this section.
Experimental design: The authors have adequately addressed my concerns in this section.
Validity of the findings: The authors have adequately addressed my concerns in this section.
Additional comments: The authors have adequately addressed my concerns in this section.


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

Input:
Title: EXPRESSION AND PROGNOSTIC ANALYSES OF EARLY GROWTH RESPONSE PROTEINS (EGRS) IN HUMAN BREAST CARCINOMA BASED ON DATABASE ANALYSIS
Review round: 2
Reviewer: 3

Output:
Basic reporting: - Clear with great improvement in the language.
- The text was enriched with the appropriate citations.
- The structure has been improved all over the manuscript
Experimental design: - It is now clear how this work fills an identified knowledge gap with some limitations that have been mentioned by the end of the manuscript.

- The description of the methods has been improved with sufficient details and information to replicate.
Validity of the findings: - Detailed work has been provided.
- Conclusions are well stated and linked to the original research question.
Additional comments: - The authors have adequately addressed the concerns raised by the reviewer. Thank you


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: PELVIC FLOOR PRESSURE DISTRIBUTION PROFILE IN URINARY INCONTINENCE: A CLASSIFICATION STUDY WITH FEATURE SELECTION
Review round: 1
Reviewer: 1

Output:
Basic reporting: In this paper, they establish the group of the features that provides the greatest capability between continent and incontinent women. To achieve this, they combined a ranking method and a feature subset method. Overall, I didn’t see much novelty of this paper. Moreover, the other critical problem is that the dataset is too small to build a reliable model. Using such small dataset for performance evaluation, I doubt the results they presented are not reliable either. In section of results, they presented the feature comparison between before and after feature selection. This is not enough. What they should do more is to compare other feature selection algorithms.
Experimental design: More comparison is needed.
Validity of the findings: No much novelty is found in the paper.
Additional comments: In this paper, they establish the group of the features that provides the greatest capability between continent and incontinent women. To achieve this, they combined a ranking method and a feature subset method. Overall, I didn’t see much novelty of this paper. Moreover, the other critical problem is that the dataset is too small to build a reliable model. Using such small dataset for performance evaluation, I doubt the results they presented are not reliable either. In section of results, they presented the feature comparison between before and after feature selection. This is not enough. What they should do more is to compare other feature selection algorithms. I have some concerns as follows.

Major comments:

In abstract, it is not clear to see the contribution of this paper. I suggest them to rewrite this section and highlight the contribution of this paper.

In Feature extraction, rather than describing how to compute the features, the authors should clarify why they choose those features. Is there any evidence to explain the importance of the features for classification in this study?

The result comparison between before feature selection and after the feature selection is not enough. To explain the superiority of their feature selection method, they should compare their feature selection method
with different feature selection methods, like using a combination of different ranking (like and feature subset searching methods.

In dataset, I doubt the reliability of the results they presented, because the datasets are too small to build a reliable model and evaluate the classification performance.

For performance evaluation, the accuracy is an important metric for evaluating the overall performance of a model, but they still need to include more other evaluation metrics, like sensitivity, specificity, or auROC, which are all important to comprehensively evaluate the model’s performance .

Minor comments:

It’s not clear why they choose the Mahalanobis distance as the subset evaluation metric, rather than others, like typical Euler distance.

Why fixing the kernel type of SVM to linear.


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: PELVIC FLOOR PRESSURE DISTRIBUTION PROFILE IN URINARY INCONTINENCE: A CLASSIFICATION STUDY WITH FEATURE SELECTION
Review round: 1
Reviewer: 2

Output:
Basic reporting: This report is the first attempt to establish the group of features that provides the greatest discrimination capability between continent and incontinent women in order to use intravaginal pressure data to automatically diagnose female UI.
Although the methods of extracting data from four clinically used maneuvers and analyzing three classifiers with and without FSS are interesting and unique, it is necessary to discuss whether this system can be put to practical use in urology outpatients. In general, by asking the patient's complaints, it is almost clear whether the patient has urinary incontinence or continence.
The following points should be considered and revised.
Experimental design: 1.	The diagnosis of urinary incontinence in women begins with questions and questionnaires such as when and how much urine leaks. In fact, the diagnoses of the urinary continence group, UI(stress UI, urge UI, and mixed UI) are almost possible at this point. Moreover, the urodynamics study that is often used clinically as an objective test can also assess the degree of intravesical pressure and involuntary contraction, and diagnose the severity of UI. The authors should describe the potential benefits for outpatient clinics in the future, if applying this methodology and device used in this study.
2.	The factor that determines the degree of urinary incontinence are considered simply the balance of intravesical pressure to urinate and urethral sphincter pressure to retain it. Although urinary sphincter pressure and intravaginal pressure may be correlated, the data measured on the patient side are only vaginal pressure by four maneuvers. Please provide some rationales that the authors did not need to consider the other measurable elements, directly intravesical pressure and abdominal pressure (rectal pressure).
Validity of the findings: 3.	Although the six extracted features effective were selected in Fig. 4, it should be explained and considered from the clinical significance why they are significantly different between continent and incontinent.
4.	It is difficult to understand how 97.9% and 95.8% are calculated in the lines 313-314 of Results. Please explain this way of calculation.
5.	Please mention some of the limitations in this study in a separate paragraph of Discussion.
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: PELVIC FLOOR PRESSURE DISTRIBUTION PROFILE IN URINARY INCONTINENCE: A CLASSIFICATION STUDY WITH FEATURE SELECTION
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: All my concerns have been roughly addressed.


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: PELVIC FLOOR PRESSURE DISTRIBUTION PROFILE IN URINARY INCONTINENCE: A CLASSIFICATION STUDY WITH FEATURE SELECTION
Review round: 2
Reviewer: 2

Output:
Basic reporting: From the medical point of view, I think that this study is not immediately usable in clinical practice, but it has been appropriately commented and revised, including the points pointed out by other reviewers.
Experimental design: Because of the fundamentals of this study, although not necessarily a complete revision, the two points I pointed out have been appropriately commented and revised.
Validity of the findings: The three points I pointed out are appropriately commented and revised.
Additional comments: From the medical point of view, I think that this study is not immediately usable in clinical practice, but it has been appropriately commented and revised, including the points pointed out by other reviewers.


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

Input:
Title: SYSTEMATIC ANALYSIS AND PREDICTION MODEL CONSTRUCTION OF ALTERNATIVE SPLICING EVENTS IN HEPATOCELLULAR CARCINOMA: A STUDY ON THE BASIS OF LARGE-SCALE SPLICESEQ DATA FROM THE CANCER GENOME ATLAS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Not all critical literature was cited and commented.
Experimental design: The study is purely theoretical and incomplete. Experimental confirmation of the propossed model is required.
Validity of the findings: I have doubts regarding the originality of essential aspects of this study.
Additional comments: In this study, the authors have used data available at The Cancer Genome Atlas to analyze the existence of patterns of alternative splicing events characteristics of hepatocellular carcinoma (HCC) and have propossed to use them to predict cancer behavior and patients outcome. The study is interesting but, to justify the originality of their work, the authors must clearly define the differences with a similar paper published three years ago (*) that has not even cited or included in the list of references. The valuable results from the present study require confirmation in a validation cohort.

*Global profiling of alternative RNA splicing events provides insights into molecular differences between various types of hepatocellular carcinoma.
Tremblay MP, Armero VE, Allaire A, Boudreault S, Martenon-Brodeur C, Durand M, Lapointe E, Thibault P, Tremblay-Létourneau M, Perreault JP, Scott MS, Bisaillon M.
BMC Genomics. 2016 Aug 26;17:683.


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

Input:
Title: SYSTEMATIC ANALYSIS AND PREDICTION MODEL CONSTRUCTION OF ALTERNATIVE SPLICING EVENTS IN HEPATOCELLULAR CARCINOMA: A STUDY ON THE BASIS OF LARGE-SCALE SPLICESEQ DATA FROM THE CANCER GENOME ATLAS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Alternative splicing events have been popularly recognized in recent years to contribute towards tumorigenesis. The authors in this study have generated a predictive model to assess patient overall survival in hepatocellular carcinoma based on splicing events. Although, this is not the first study to have used predictive modeling on RNAseq patient database, it is the first to do so more effectively (AUC for ALL events 5 years ROC curve of  0.821). However, there are few points for the authors to consider to make their study clearer and better:

1) Minor grammatical errors throughout the manuscript. Authors are advised to proofread it thoroughly. For example- 'protein' in Lines 37 and 38 should be replaced by 'proteins' and the references in parentheses should come before full stop at the end of a sentence.

2) Figure 5 needs to be in high resolution. It is hard to read the labels for heatmaps.

3) For better understanding of the readers, kindly label the x-axis in Figure 8 well. Is the hazard ratio predicting increase and decrease in overall survival of the patients here?

4) A flowchart describing the process of data processing will be useful for the readers.
Experimental design: 1) In the UpSet plot, authors show how different AS events interact, but the predicted model for patient outcomes only uses single or ALL events for OS risk estimate. It will be good to see and understand how and to what extent different AS event types when interacting contribute to the patient outcome in HCC.

2) authors are requested to delineate how the events per AS types were selected for LASSO regression.
Validity of the findings: No comment.
Additional comments: No additional comments


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

Input:
Title: SYSTEMATIC ANALYSIS AND PREDICTION MODEL CONSTRUCTION OF ALTERNATIVE SPLICING EVENTS IN HEPATOCELLULAR CARCINOMA: A STUDY ON THE BASIS OF LARGE-SCALE SPLICESEQ DATA FROM THE CANCER GENOME ATLAS
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript passed all the basic reporting requirements
Experimental design: The manuscript passed all the experimental design requirements
Validity of the findings: The manuscript passed all the validity of the findings requirements
Additional comments: Alternative splicing events (ASEs) play a role in cancer development and progression. The authors herein investigated a systematic analysis of ASE and constructed a robust prediction model of hepatocellular carcinoma. The authors claim that the final prediction model performs well in predicting the prognosis of HCC patients and the findings in this study improves understanding of the association between AS events and HCC. I support publishing this manuscript with some major and minor revisions:
1)	As the authors mentioned the biggest drawback of the manuscript is lack of experimental verification for understanding the mechanism between AS events and SFs in the HCC patients. Are the authors planning to do any invitro and in vivo assessments as a future work? Can the authors mention in their final conclusions?
2) I recommend the authors to add a flow chart for their study design
3) In the TCGA- based data collection, patients with a survival time less than 90 days were excluded, I assume clinical follow up data can be available? can the authors add a reason why it was excluded for 90 days instead of 30 days or lesser?


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: SYSTEMATIC ANALYSIS AND PREDICTION MODEL CONSTRUCTION OF ALTERNATIVE SPLICING EVENTS IN HEPATOCELLULAR CARCINOMA: A STUDY ON THE BASIS OF LARGE-SCALE SPLICESEQ DATA FROM THE CANCER GENOME ATLAS
Review round: 2
Reviewer: 1

Output:
Basic reporting: No additional comments
Experimental design: No additional comments
Validity of the findings: No additional comments
Additional comments: The authors have responded to my comments in a nonconvincing way.


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: SYSTEMATIC ANALYSIS AND PREDICTION MODEL CONSTRUCTION OF ALTERNATIVE SPLICING EVENTS IN HEPATOCELLULAR CARCINOMA: A STUDY ON THE BASIS OF LARGE-SCALE SPLICESEQ DATA FROM THE CANCER GENOME ATLAS
Review round: 2
Reviewer: 2

Output:
Basic reporting: The manuscript passed all the criteria.
Experimental design: The manuscript passed all the criteria.
Validity of the findings: The manuscript passed all the 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: ASSESSING THE EFFECT OF FISH SIZE ON SPECIES DISTRIBUTION MODEL PERFORMANCE IN SOUTHERN CHILEAN RIVERS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The text is well structured however the language needs improvements in several aspects. Throughout the whole text (especially in the discussion) a lot of misspellings, wrong use of adverbs and articles or weird use of singular & plural forms occur, some examples can be found in L97, L241, L283, L304, L338, L340, L351. Furthermore, there are a lot of unclearly formulated sentences that need more clearness. This unconcise formulation leads to a lot of questions for the reader, so it is highly important to clear them out to enable a clear understanding, a good example here is L63 where the authors mention that ‘anthropogenic variables represent the main group of threats to river fishes’ – it is simply unclear what is meant by ‘anthropogenic variables’ and how variables can represent threats. Also, the following conclusion that the role of body size on SDM performance is therefore highly relevant for conservation is simply not following a logical line of argumentation. If there is a specific aspect related to body size that goes beyond the general effect of human-induced pressures, the authors should clearly explain it but this is lacking. This would definitely give additional strength to the manuscript. Another example is in L97, where the size gradient of ‘this species’ is mentioned. Probably the authors mean the size gradient of ‘these species’ which completely changes the meaning of the sentence.
The manuscript only contains 2 tables. However, the authors decided to place the important information about the fish size that was used in the modelling into the supplemental material. (L96). Layout of table 2 can be improved
Data: The authors only provided coordinates of species occurrences. Further evaluation of results was not possible. It is also surprising that the authors used two versions 2.3.3 (L146) and 3.3.2 (L204) of R.
Experimental design: The description of the materials do not provide sufficient detail to appropriately evaluate the quality of the data. Firstly, there is no further information on the quality of the data in the database of the environment ministry that builds an important basis for the analyses. It stays unclear which time period is covered and which methods were used to detect the species. Also, the field sampling is not sufficiently respectively unclearly described. A sampling time of 45-60 minutes seems rather long.
There is no clear justification why the river segments without presences of the fish species of interest can be regarded as true absences. I think it would be much more reliable to use a pseudo-absence approach in this case as probably the information of absences is not available from the ministry database.
Please provide information about the correlations between predictor variables before using them in a GLM method that is sensitive to correlations.
The models are based, in relation to the number of variables, on few observations. Creating a distribution model on 9 observation is inappropriate. Furthermore, model performances are weak (below 0.5) in a lot of cases – see Table 1. These models must be discarded as their predictions are not reliable and not useful for any kind of interpretations. From the results it seems that some species are positively related to human pressures. Hence, doubts are raising that the environmental gradients of the species are fully covered by the data that is used for the modelling. I also have to disagree with the interpretation that the results indicate any impact of anthropogenic uses. Probably, the data mainly covers the human uses and misses the natural environmental gradient but to proof this the amount of data is probably to less.
Please provide information about the size of the area that is covered by the investigation area (estimated from Fig1 it seems rather small). This would be important to judge about the general validity of the results.
The authors did some evaluations in Google Earth. It is not clear how this data was processed to derive land use information. Furthermore, there are global datasets available that describe land cover (e.g. Globcover) and can be also used for the evaluation of land use in Chilean rivers.
Validity of the findings: Data: The authors only provided coordinates of species occurrences. Further evaluation of results was not possible based on the submitted data.
Unfortunately, the discussion represents the weakest part of the manuscript.  This section needs a thorough shaping towards the main aim of the manuscript. One example is that the hierarchical framework of habitats sensu Frissell et al, 1986, is firstly mentioned in L312. It makes sense to refer to this concept but this information should be provided to the reader already in the introduction. Another example, in L334ff you somehow contradict yourself by first stating that there is no broad agreement on predictor variables. I would disagree here and somehow you also list then common factors that are used by modellers, such as temperature, discharge, flow velocity. So, I would not say that there is no broad agreement how river ecosystems should be described and several modellers describe the river systems in similar ways but the specific variables they use differ. In a lot of cases this makes sense but of course the direct comparability suffers. However, the biggest issue with this part of the discussion is that it rather stays unclear how the research presented in the manuscript contributes to solve this issue.
A direct comparison of Radinger et al. (2017) and Markovich et al. (2012) is not applicable. The-former is based on field-sampling data, the latter is based on distribution grids so completely different information basis.
There are several issues more in the discussion but as long as the methodogical issues are not solved it may be not useful to elaborate on these issues as the results will look very different after an implementation of the changes.
Additional comments: Although I like the idea of the paper there arise too much issues in the manuscript that have to be solved to provide a solid piece of research. Firstly, try to establish a clear terminology throughout the manuscript in alignment with the recent literature and perform a thorough proof reading of the text. A mixture of misspellings and weak formulations makes it hard to follow your ideas, e.g. in several parts of the manuscript the term ‘turbulence’ is used I assume the authors basically mean flow velocity. I would recommend to fully replace the term turbulence. Furthermore, clearly indicate the limitations of your analyses. You used a small number of occurrences and the quality of the models is not very good in a lot of cases. At some point it is necessary to discard models when there quality drops beneath certain thresholds. Finally, streamline the discussion. You have some statements where the presented research does not really provide any contribution (or you have to better explain how the research contributes)