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: INTEGRATING SPECIES AND INTERACTIONS INTO SIMILARITY METRICS: A GRAPH THEORY-BASED APPROACH TO UNDERSTANDING COMMUNITY SIMILARITY
Review round: 1
Reviewer: 2

Output:
Basic reporting: In general, the manuscript is well written and clear. The introduction places the study in an appropriated context and the structure of the ms is adequate.
Experimental design: The research seems to be within the Scope of the journal and it is clearly identified how the study fills a methodological gap. The methods are clearly described, although some points could be further developed (see below).
Validity of the findings: The results appear to be robust, and the conclusions seems to be coherent with presented findings.
Additional comments: Measuring community similarity beyond species composition can provided valuable information but it is challenging. A network-based tool that considers dissimilarities in species interactions and composition is a step-on into these difficulties. In this regard, the manuscript may constitute a notable contribution.  However, there were many aspects in which the study could be strengthened and/or improved.

Suggestions
A more detailed explanation of the methods would help to a better understanding. In particular, there were four points that I think would benefit from a clearer description:
1-	If I understand properly the GED is based on the minimum edit path cost. Here, the cost of the edit path is the summation of costs assigned to different network transformations. If this is true, the GED should range between 0 and some number (depending on costs and number of links and nodes), and yet it seems to have an upper bound of one as if it was normalised. To a full understanding of the index, I think this normalisation should be explained.  Although it might appear to be unimportant, this may help the reader to interpret the index. For instance, depending on this normalisation the index might be symmetric (i.e. equal dissimilarity when comparing network A with B than B with A) or not. Moreover, this normalization may also influence the comparison of GED values generated under different cost scenarios.

2-	When describing the AMI, an explanation on how the intersection between Jaccard and GED values was calculated would help.  Moreover, I think that using a commonly used correlation coefficient (e.g. Spearman’s one, if non-linear relationships) would make the results more accessible to most readers (including myself).

3-	Perhaps, it would be worthy to more clearly explain the reasons behind the choice of the costs presented in Table 1, and not others.

4-	An explanation on how the Jaccard index was calculated would also help. Notice that the “Jaccard index” normally refers to a similarity (intersection over union), but it seems the authors were calculating a dissimilarity index (lines 161-162).

Beyond AMI scores, it could be important a more careful interpretation of the Jaccard vs GED relationship. For instance, In Figure 1. it can be appreciated a similar shape in this relationship across scenarios. Here, it seems that at low compositional similarities (as measured by the Jaccard index), GED values present the highest variability; whereas from moderate to high Jaccard values, GED values are always high, showing little variation. The first interpretation that come to my mind is that when species composition is relatively similar, species interactions are also so and, hence, the GED “saturate”. In other words, dissimilarity due to interactions and composition may be to some extend related. Support for this interpretation come from that the relationship between GED and Jaccard appears to be stronger in the scenario 9, where GED values should be dominated by interaction editions (according to Table 1, this scenario has the lowest species/node editing costs). This interpretation might be wrong (and it is certainly contrary to the current one) but I think it exemplifies the need for a clearer and more detailed interpretation of the results.

 The fact that GED values depend on arbitrary costs might be seen as a drawback. Yet, as the authors already noticed in lines 248-251, it is also one of the main advantages of the method. That is, by assigning different costs to different editions (e.g. the inclusion of nodes, or links of any type), one could “partition” the contribution of different “elements” (i.e. species, positive or negative interactions etc.) in the dissimilarity between communities.  This is a very interesting idea that seems to be easy to demonstrate using the presented dataset (forgive me if I’m wrong). I think the research would largely benefit from including such demonstration and focus on it rather than on the comparison with Jaccard values. That is, I would only compare the Jaccard with GED with costs related to interaction editions equal to zero, so that I can demonstrate that the “compositional GED” is analogous to a widely used and robust index of compositional similarity. Then, I would add costs to editing interactions and compare “compositional GED” values with “compositional + interaction GED” values. This should better demonstrate the influence of changes in interactions while partially solving the issue of choosing arbitrary cost values.

A better structuration of the discussion could be also important. From the title, abstract and introduction, the ms seems to be mostly methodological and, yet, in the discussion little attention is paid to explain the advantages and disadvantages of this method compared to similar ones (e.g. the already cited Poisot et al. 2012 Ecol. Lett. 15). Providing the differences and similarities (as well as pros and cons) with other methods would help the readers (and potential users) to know when to use them. In this line, some recommendations for setting proper cost values would also help. Finally, I think that the conclusions would benefit from sticking closer to the main results of the ms and not too much to almost epistemological stuffs (i.e. what an adequate replicate of a community is?).

Minor suggestions:
-	Since the ms appears to be methodological, I would first present the method and, then the dataset in the methods section.
-	Figure 1 might look better if scatterplots are arranged in a 3 X 3 panel.  Moreover, for an easier interpretation I would use the same values in y axes across all plots.
-	In lines 148-149 it should be explained why one need several iterations (likely because GED is approached through a heuristic algorithm).
-	In lines 180 to 181 it is stated that interaction editions received a low cost in scenario 9. While this is true, this scenario is the only one where species editions have lower costs than interaction editions (according to Table 1). Would this be important to the interpretation of the results?


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: INTEGRATING SPECIES AND INTERACTIONS INTO SIMILARITY METRICS: A GRAPH THEORY-BASED APPROACH TO UNDERSTANDING COMMUNITY SIMILARITY
Review round: 2
Reviewer: 1

Output:
Basic reporting: Dear authors,

I am glad to read the new version of your manuscript.
My previous set of comments were carefully addressed but I still have several ones that you must address before publication.

Hope this new set of comments would be helpful as you revised your manuscript.

Kevin Cazelles
Experimental design: Three comments here:

First, I think including 49 scenarios in the study is way better than 9 but I do not understand the choice of the values. Why only use 0.25 and .75 for the interactions? To me, 27 scenarios would be enough, testing 0/.5/1 for the three kind of costs.

Second, I am not satisfied by the comparison with the work of Poisot et al. (2012). I think it should be a part of the study not only "a complement". I think the AMIs values used to compare GED and the Poisot's approach should be provided for all scenarios. Alternatively, the choice of the scenarios selected must be carefully justified, which is not currently the case. I am quite surprise that the authors "found a weak relationship between BST /BWN and GED across all scenarios" and I wonder if this is only true for the selected scenarios though I hope the authors will prove me wrong.

Third, I think the authors should better explain why they have decided to use the Adjusted Mutual Information (AMI). Several questions/comments on this point:

- If I understand correctly the work of Vinh et al. (2010), the AMI is based on a contingency table based on which I(U,V) and H(U,V) are computed but I cannot figure out what is the contingency table in your approach?

- For most ecologists it may not be clear what a AMI of 0.134 (maximum value in your study) means. I understand that AHI ranges from 0 to 1 but this does not imply that 0.134 is a low value. I think this should be crystal clear as it crucial to interpret your results. One way is to create a box with a simple and well detailed example the authors could make up.

- Looks like AMI is related to a correlation coefficient, why not use a correlation coefficient instead of AMI? I have nothing against AMI but it should be better explained why you have decided to use AMI.
Validity of the findings: See point 2 above.
Additional comments: - l.95: a datasets => a dataset

- l.130: I would make explicit the three different kind of costs in this equation.

- l.145-146: implements a evolutionary => implements an evolutionary

- I think l.176-189 must come before l.166-175.


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: INTEGRATING SPECIES AND INTERACTIONS INTO SIMILARITY METRICS: A GRAPH THEORY-BASED APPROACH TO UNDERSTANDING COMMUNITY SIMILARITY
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors have done a great job answering my previous comments. I only have some minor suggestions/questions.
Experimental design: It is not clear to me why Poisot et al.’ beta-diversity measures are only compared with GED for a small subset of scenarios and not with all of them.
Validity of the findings: The AMI index is well explained now. However, it is still unclear to me how the mutual information is computed. That is, as far as I know measures based on mutual information are normally applied to categorical variables (e.g. cluster affiliations) and I not sure how can this be applied to quantitative variables. Perhaps it is my fault and I apologise for insisting on this, but I think this is an important detail to make clear. For instance, if one has a vector X (let’s say Jaccard values) with values 1, 2, 3, 4 and 5 and Y vector (e.g. GED) with values 1.1, 2.1, 3.1, 4.1 and 5.1; what would the AMI score be? If I understand right, both vectors are not sharing information, thus the mutual information would be 0 and so would be the AMI. Yet, both vectors are highly correlated. I think a clarification about this would help to better understand the results.
Additional comments: In lines 157-159 and 197-199 you referred to the importance of “interactions”, but it is not clear whether you meant flipping of interactions, deleting/adding interactions or both.

I found few spelling typos (but please take this carefully since I am not an English native speaker):
Line 186: “represent to” should be “represents the”
Line 247: “allow” should be “allows”
Line 291: “reduce” should be “reduced”; and “a” should be “an”.


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: INTEGRATING SPECIES AND INTERACTIONS INTO SIMILARITY METRICS: A GRAPH THEORY-BASED APPROACH TO UNDERSTANDING COMMUNITY SIMILARITY
Review round: 2
Reviewer: 3

Output:
Basic reporting: The authors present a methodological framework to compare how similar to ecological communities (food webs) are. To my opinion the paper is nice and the topic interesting. However, there are some parts that would need to be improved before publication.

In practical terms, one big objective of similarity measures in ecology should to allow obtaining information, in whatever means, of one ecological system given a known observed one. Take as example system stability or persistence. To illustrate this potential functionality of the framework, the paper should include experiments or solid discussion in this direction. An specific question that could be addressed is: two similar networks with respect to GED have dynamical behavior closer than two similar networks in terms of Jaccard (or $B_{WN}$, $B_{ST}$)?

After reading the paper I think it is not clear the role taxonomic identities (or the taxonomic tree) have with respect to the GED. As far as I understood, species within the different ecological systems are group according to its taxonomic similarity. In this way, two different species can be considered equivalent in the author’s representation of the ecological system. This has strong implications in the GED and in particular in its minimization. Thus, besides deciding GED costs, the main problem authors face in obtaining the minimum GED is to find which nodes of the first graph correspond to which nodes of the second graph. That is, the graph matching problem with the particularity that only taxonomic identic species can be matched. These needs to be explained in detail within the paper.

In section 2.3, line 145, authors use the word flipping a link. Later on, line 297, they explain that this refers to the change of the relation between a consumer and a resource. The mining of flipping an edge should be described already at section 2.3 otherwise the paper is not comprehensible.

I do not like the Discussion section in general and in particular the first paragraph which I consider to be misleading. Although they do not specifically say so, authors seem to indicate that the difference between the GED and Jaccard could be related to some physical or mediated factors. This is not the case for the experiments performed in the paper. The observed differences are mainly due to the fact that the different metrics measure different, probably orthogonal, aspects of the system and include different levels of information. Am I right? With respect to the rest of the discussion, I am not sure that its length cannot be reduced. Probably some parts can be merged, or moved, to the introduction to easy readability. Specifically from lines 236 to 271 which seem to be somehow describing the state of the art. This is just a proposal.

I do not like how the conclusions section is organized, to me the term summary should include the most important aspects of the paper and I probably find more interesting what is said in the second paragraph. I would consequently integrate both paragraph. Besides, I would replace item “b)” by saying “the non-relationship between the measures evidence the crucial requirement of adding structural relationships to the similarity measure” or something like that. Does it make sense?

Line 302: “variables DOES not show robust”
Experimental design: no comment
Validity of the findings: Comment on this aspect are included in the main text of the review.
Additional comments: Within the basic review text.


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

Input:
Title: INTEGRATING SPECIES AND INTERACTIONS INTO SIMILARITY METRICS: A GRAPH THEORY-BASED APPROACH TO UNDERSTANDING COMMUNITY SIMILARITY
Review round: 3
Reviewer: 1

Output:
Basic reporting: 'no comment' - see previous rouds of review.
Experimental design: 'no comment'
Validity of the findings: 'no comment'
Additional comments: This is my third review for the manuscript of this manuscript. The authors have answered
all my comments and I believe that the manuscript is now ready for publication in PeerJ.
I hope this publication will lead network ecologists to further investigate the potential
of the combination GED + AMI.

Below, I would like to mention a few points regarding the authors' answers to
the reviewers' comments that should not lead to any revision.

> "the reviewer 1 in the first round required to increase the number of scenarios to hundreds of parameter combinations, but now he said that 27 scenarios is enough (we performed 49 scenarios)""

I do not think that this is confusing. I just thought the 27 scenarios would have been slightly clearer, but
it is just nitpicking, I admit. 27 scenarios and above is good (again better than 9 as in the first
version of the manuscript).

> "In another example, the same reviewer suggested the use of correlation coefficient, but in a previous version another reviewer asked for removing it. In other example, reviewer 3 asked for experiments to analyze the dynamics of ecological communities with high or low similarity which is not the topic of the study at all."

My personal opinion is that when reviewers' comments are going is different directions
it often means that some parts of the manuscript lack clarity. I think if a subset of your
readership (in this case, the reviewers) are confused, it is likely that other readers
will be and so I think that instead of simply pointing out the confusion, it is better to
find out where the confusion stems from. In all fairness, such situation frequently happens when bringing news tools into a discipline.

That being said, I do think that the new version of the manuscript is clearer:
GED and AMI are well described and I think this manuscript now needs to find
its own place in the body of literature of network ecology.


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: INTEGRATING SPECIES AND INTERACTIONS INTO SIMILARITY METRICS: A GRAPH THEORY-BASED APPROACH TO UNDERSTANDING COMMUNITY SIMILARITY
Review round: 3
Reviewer: 2

Output:
Basic reporting: The authors have done a great job addressing all my questions. I also appreciate they provided R code to calculate AMI. This, together with their explanation, clarified how AMI was calculated. I have just one minor suggestion that might be somehow irrelevant. During the AMI calculation it is necessary to discretise the variables and how to conduct such discretisation (i.e. number of bins and discretisation method) may affect AMI scores. The authors chose default parameters of the function “discretize” from the R package “infotheo”. That is, the number of bins was equal to the cube root of the number of observations and bins have the same frequency of observations. I was playing around with these parameters and they seem to be very robust to their inferences (i.e. I got the highest AMI scores when using them, which further supports the low AMI scores they found). Thus, I think explaining that a discretisation was performed and why these parameters were chosen might potentially make clearer the AMI calculations. Nevertheless, this may turn to be somehow far from the main scope of the ms, so it is up to the authors to follow this suggestion.
Experimental design: -
Validity of the findings: -
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: INTEGRATING SPECIES AND INTERACTIONS INTO SIMILARITY METRICS: A GRAPH THEORY-BASED APPROACH TO UNDERSTANDING COMMUNITY SIMILARITY
Review round: 3
Reviewer: 3

Output:
Basic reporting: The authors have successfully addressed the concerns I had with the paper.
Experimental design: The authors have successfully addressed the concerns I had with the paper.
Validity of the findings: The authors have successfully addressed the concerns I had with the paper.
Additional comments: The authors have successfully addressed the concerns I had with the paper.


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

Input:
Title: LAND COVER DRIVES LARGE SCALE PRODUCTIVITY-DIVERSITY RELATIONSHIPS IN IRISH VASCULAR PLANTS
Review round: 1
Reviewer: 1

Output:
Basic reporting: This article meets the standards in every way.

The text is clearly and professionally written, with excellent organization. The paper as a whole is easy to follow and to understand.

The introduction provides a comprehensive review of patterns and hypotheses linking productivity and diversity. This section is well-organized and thorough. My initial thought was that references to the Nutrient Network experiments (Grace, Adler) would be appropriate, but I see that they are included in the discussion. Otherwise, the relevant literature is cited, and it provides a context for the current study.

The paper is structured with appropriate sections. The figures are relevant and are labelled and described well.

The raw data in this study came from publicly-available sources, rather than being generated by the authors. The original sources of the data are provided and the analysis code is available.

The results are relevant to the questions raised in the introduction, and the paper is a self-contained unit.
Experimental design: This article meets the standards in every way.

This paper begins by reviewing what is known about patterns linking productivity and biodiversity of plants, and reviewing the proposed mechanisms that create those links. Although it is no longer the “hot topic” it used to be, the question of how productivity influences diversity remains unsettled. The authors identify a few important gaps that their study can address. First, the effect of anthropogenic nutrient additions may differ from the effect of naturally high productivity. Second, much of the work on this question has been done at very large or fairly small scales, whereas a regional scale allows for better understanding of the role of heterogeneity in land use. This study covers a regional area at a large grain size (10 km square units), and includes a large proportion of pasture land, so it is well suited to address these two gaps.

The methods for quantifying land use and productivity appear to be logical and sound. The data analysis methods are complex and unfamiliar to me (and probably to many readers) but are described clearly and justified well. I would like to see a little more description of the plant data. What are the records that were used – species lists for certain areas, maps of species distributions?
Validity of the findings: This article meets the standards in every way.

The authors are able to draw sound conclusions from their analysis. By comparing pasture, non-pasture, and heterogeneous land cover, they can address questions about effects of human impacts and land use. The use of various measures of spatial and temporal heterogeneity in productivity makes it possible to draw some conclusions about mechanisms causing the observed patterns. Those conclusions are limited by the available data, and the authors don’t overstep those limits.
Additional comments: This is a very nice contribution to the field!


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

Input:
Title: LAND COVER DRIVES LARGE SCALE PRODUCTIVITY-DIVERSITY RELATIONSHIPS IN IRISH VASCULAR PLANTS
Review round: 1
Reviewer: 2

Output:
Basic reporting: White and others report the results of a really interesting study. Their approach of using 10x10km hectads covering the whole island of Ireland is certainly novel for studying productivity-diversity relationship in plants. The idea is good and the results are interesting. However the method section has some gaps, which do not allow to assees the validity of the analysis. And there are also some issues with the theoretical aspects of the study that might need a bit more work. Overall, though, the manuscript is very promising.

The manuscript is written in very good English. The text is clear and unambiguous. Confusing passages are not obscure because of the language, but because of the content that is not fully described, etc.

It is difficult to give a thorough overview of all the studies related to vegetation productivity-diversity relationship (PDR). And it is impossible to do it within one journal paper. There is just not enough space. So the authors have to come up with an angle that covers the topic from the aspects that they consider the most novel and interesting in their analysis. White et al focus on mechanisms behind PDR, like heterogeneity, fertilization, ecosystem stability, historical and evolutionary background. However, most of the mechanisms discussed are operating within ecosystems, and it remains unclear from the Introduction how, for example, ecosystem stability or fertilizing work on the hectad scale. Also, in the literature, most studies that have found positive relationship between heterogeneity and diversity have been conducted within an ecosystem, and not between them on such an understudied scale as 10x10 km grids. The Introduction does not really exaplain how the described mechanisms could work on hectad scale, which a) has a different spatial resolution, and b) cuts different ecosystems into random fragments and puts them together in artificial grid squares. I do not want to say that it´s either bad or wrong, but I´d like to see at least some attempts to try to translate the mechanisms on this novel spactial scale.

This translation/transition has been attempted with historical and evolutionary history mechanism (lines 96-109), but it is not very convincing, because while Pärtel et al 2007 analysis dealt with biogeographical-scale processes, claiming that the PDR in temperate zone habitats, especially grasslands are affected significantly by the last Ice Age (which obliterated high productive habitats from temperate zones, while tropical habitats have enjoyed relatively stabile conditions for the past couple of million of years), White and others assume or expect the same kind of outcome from human activity ("Frequently, highly productive areas such as farmland are more recent additions to the landscape compared to natural grasslands or woodlands. Consequently, there has not been a sufficiently long evolutionary time for the species within these areas to undergo significant speciation; older, but often less productive, regions are, therefore, more likely to have a higher species richness, suggesting that human activity might impact macroecological relationships."). So, assumedly the authors try to replace the effect of the Ice Age with human influence on habitats, because Ireland is largely covered by pastures, which are artificial (? - more about that later) habitats, and therefore the negative PDR is expected mainly because of human influence, but not because of geological history. Just that both processes kind of have the same outcome. It´s and interesting idea, but based on the analysis and data in this study, it is impossible to separate these effects as Ireland has both a lot of pastures, but at the same time it was also covered with the permanent ice during the last glacial maximum. In order to understand the role of evolutionary processes, like spaciation, in generating habitat-specific diversity patterns, it is necessary to include some kind of assessment of past habitats and land cover (eg Zobel et al 2011 in GEB for Canary islands). There seem to be studies that have mapped the potential natural habitat cover for Ireland (e.g. Cross 2006 in Biology and Environment: Proceedings of the Royal Irish Academy "The potential natural vegetation of Ireland"), and by dividing species pool according to habitat preferences, it would be possible to separate these effects. But not based on the analysis presented in this manuscript.

So, in the current form, the Introduction is not very well integrated with the rest of the manuscript, and is a bit misleading about the constraints of the potential interpretation of the analysis.
Experimental design: The issue of promising a bit too much comes up again when the authors describe the objectives of the study. The main research goal is expressed in lines 131-4: "Using both temporal and spatial measures of EVI (plant productivity) in conjunction with land cover data will allow us to investigate the productivity-diversity relationship at large spatial scales, and how human modified landscapes might modulate any established macroecological relationship through landscape scale impacts on biodiversity."; the authors add later (L 139-42), that: "As the Irish landscape has been heavily modified through agriculture (Aalen, Whelan and Stout, 2011), we hypothesise that this impact will have left its signature on spatial patterns of biodiversity beyond the field level, and that including land cover will improve models of the relationship between productivity and diversity." Additionally, I will deal with the "temperate and spatial measures of EVI" below.

Now, the Methods section. There are couple of issues that needs to be pointed out.

1) Vascular plant data or Species occurrence data (lines 145-55). The authors do not describe the data at all. Is it occurrence data like in GBIF? Or vegetation survey´s data? Or some combination of different data types? What are the sources of this data - NBDC has not probably recorded all the data by themselves? How many species there were in the dataset? How many records? What was the location precision threshold? Very important is also the timeframe of data. Assumedly only records from certain time period were used? Otherwise the diversity data would not fit with the land use data. In lines 290-1 the authors say that "The island of Ireland has undergone a large transformation in land use from its natural state (Aalen et al. 1997), with most of the landscape dominated by pasture.", but I could not find a timeframe during which this transformantion happened.

In lines 153-4 the authors say that "Species occurrence data was aggregated to the hectad level (10 km square) in order to retain a maximum number of observations for analyses." First of all - a hectad should be 100 km square, not 10 km? But secondly - Ireland has several vegetation maps dating from different time periods. The authors also cite this: Online Atlas of British and Irish Flora. And this atlas has been using the same 10x10 km hectad scale. My first assumption, when agreeing to review this manuscript, was that the authors have used vegetation atlas data for this PDR. Because the spatial scale is exactly the same, and the vegetation survey´s that are the basis of vegetation atlases have been done really systematically and by using the same methodology in all the hectads. Plus, the data is freely available. But after going through the "Data" section it seems that the authors have not used the vegetation atlas data, but instead something else. Why?

2) Irish landscapes. For a person not very familiar with the nature in Ireland, it remains realtively vague, what exactly is a pasture. The passage that provides some description is in the Discussion (lines 290-303). Something like that should be in the Introduction or at least in Methods. And it should give a bit more precise overview of that particular habitat  type. For example, in lines 290-1 it says that "The island of Ireland has undergone a large transformation in land use from its natural state (Aalen et al. 1997), with most of the landscape dominated by pasture." the authors do not provide the timeframe of this transformation. Nor the range of it. What were the habitats that were replaced by pastures? When did the massive fertilization with the nitrogen started? Or the sowing and reseeding? Are these processes still going on, or have they intensified? As the authors core idea is to find signs of human influence on PDR on macroecological scale, knowing the duration of these processes is crucial information.

3) Hectad grouping. The analyzed hectads are divided into three groups, based on the land use dominance (hectad is 50% or more covered by one specific of habitat or type of land use):pasture-dominated, heterogeneous, non-pasture dominated. More than half of the hectads are pasture-dominated, while there seems to be altogether two hectads dominated by urban land use; zero hectads by industrial, quarry and artificial vegetation; 4 by arable land; 6 by heterogenous agriculture (btw, where these hectads classified as heterogeneous or non-pasture dominated?); one by forest; 9 by scrubs; 61 by inland wetlands; and zero hectads where both inland and marine waters were dominating. So, 61 hectads dominated by inland wetlands vs 22 that are dominated by all other habitats and land use types.

The long list extracted from the GitHub repository data matrix is meant to show what a strange construction this non-pasture dominated hectad group is. It feels like more adequate title for it would be "inland wetlands" (after removing the few non-wetland hectads). That would make things much clearer. Because when you look at figure 3, it´s striking how pasture hectads are the most productive, then come the heterogeneous ones, and the non-pasture dominated hectads are the least productive. One would expect that both heterogeneous and non-pasture dominated terrestrial habitats would be on average more productive than species poor grasslands, because other habitat types would typically contain woody species, which have significantly more aboveground biomass than pasture vegetation, which has more or less only herbacous layer.

Ireland´s land cover is what it is, and I don´t want  to blame authors for not having more forest hectads etc in the dataset. I just wanted to show how the explanation of why pastures are the most productive hectad type in this data is buried too deep. That information of the non-pasture dominated hectads comprise mainly of wetlands should be included in the main text (or as a table or sth). The fact that forests and woodlands seem to be cut down and the remnants are very fragmented, which has generated a situation where the most productive land use type is pasture, which is heavily influenced by human activities, is in my mind a very convincing argument supporting what the authors are claiming - that human influence affects PDR in macroecological scale.

4) Hectad filtering. Going back to the same GitHub repository data matrix I was surprised to see how there were no hectads dominated by waters. Because of some big lakes (e.g. Lough Neagh that covers nearly 400 sq km) and very rugged coastline I was expecting to find hectads dominated by waters and only including a tip of a peninsula etc. But no. Did you remove any of the hectads from the dataset? The island area is 84421 sq km, but there are only 830 square shaped hectads in the dataset. Something seems to be missing, but the Methods section does not mention anything about it.

5) EVI calculations. MODIS satellite data covers the years 2000-2017 (2003-2017 for aquatic systems), and if I understood correctly, you used the data available within this timeframe. Then you scaled the 250 m pixel scale into hectad scale, but instead of calculating the total EVI for the hectad, you used median value (lines 162-4). Why? Why not calculate the total EVI for each hectad in each time point? Or at least have the mean value - that should contain less noise in representing the productivity of an hectad. Using median means that it´s impossible to know which land use type represents the productivity value in heterogeneous hectads, which in turn makes interpreting the results much more difficult.

Another question regarding EVI calculations is the "time point". You never define in the text, what is one time point in your study. Are these daily measurements like MODIS takes, or mean values for some time period? Did you use EVI data for the whole year, or only for vegetation period? This last question is also significant when it comes to comparing the results of this study with the other PDR studies, because the latter ones almost always use peak aboveground biomass as the proxy for productivity. Using data for all seasons and for different land use types dominated by different vegetation (e.g. evergreen vs deciduous/herbaceous vegetation) could have "side-effects" to the productivity estimations that are difficult to predict. So, the process of how staellite data was transformed into EVI values per one hectad needs to be described in more detail.

The matter of how EVI was calculated affects also the measures of productivity. In lines 179-197 the authors describe 4 productivity measures that they used for the analysis. First of all, are the measurements correlated with each other? But more importantly - how can the authors be sure that the second measure "Spatial standard deviation" (lines 184-6) indeed measures spatial variation of EVI? Because they define the measure as: "mean across all time points of a hectad’s spatial variation in EVI (standard deviation across pixels).". But like I described in the previous paragraph, the variability of productivity in one hectad could also be the result of seasonal variation, and differences in seasonal productivity dynamics between different land use types. And as the hectad´s productivity is calculated based on the median EVI value on all the 1600 pixels in each hectad - does that mean that the basis of productivity variability in each hectad could be calculated based on different pixel values? Because the median-valued pixel is probably different in different time points. That could really make the producitivty variability measurements just random numbers.
Validity of the findings: As I´ve had quite a few critical things to point out for the Methods, I will not focus too deeply in the Discussion section in this round of reviews. I just want to point out one thing. The Discussion starts with summarizing the main results: "The relationship between productivity (mean EVI) and vascular plant species richness in Ireland does not follow the typical hump-back model identified in many previous investigations of productivity-diversity relationships in plants below the continental scale (Mittelbach et al. 2001)." (lines 279-281). Are hump-back and negative PDRs really a different thing? If we would sample the entire productivity gradient, negative PDR would be impossible - if there is 0 species there is also 0 productivity and vice versa. So, from that point of view it is very difficult to oppose hump-back PDR with negative PDR. Statistically these relationships are different, but from the ecological point of view not so much. (See also Pärtel et al 2010 Ecology "The productivity–diversity relationship: varying aims and approaches" for more discussion about that.) I would be more careful in building up the excitement in the Discussion by confronting these results.
Additional comments: In conclusion - the study is based on a very interesting idea. Methods section has some gaps at the moment that obstruct comprehensive assessment of the scientific outcome of this study.


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

Input:
Title: LAND COVER DRIVES LARGE SCALE PRODUCTIVITY-DIVERSITY RELATIONSHIPS IN IRISH VASCULAR PLANTS
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The improved version of the manuscript is very good. All the theoretical gaps pointed out in the review are now filled, also the methods are now sufficiently explained. It´s a very interesting study and I would recommend to publish it.


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: ACCELERATED CONSTRUCTION OF AN IN VITRO MODEL OF HUMAN PERIODONTAL LIGAMENT TISSUE: VACUUM PLASMA COMBINED WITH FIBRONECTIN COATING AND A POLYDIMETHYLSILOXANE MATRIX
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript shows clear and professional organization and sufficient reference and background. All the data were analyzed and interpreted appropriately. The figures and tables are shown with good quality and support the hypotheses.
Experimental design: Methods described in the manuscript is sufficient to follow. Research question well defined, relevant and meaningful.
Validity of the findings: After systematic evaluation, the authors successfully developed novel construction of an in vitro model of human periodontal ligament tissue by using vacuum plasma combined with fibronectin coating and a polydimethylsiloxane matrix. This new method is absolutely suitable for the publication. Data was interpreted completely and supported the conclusion.
Additional comments: Overall, the manuscript contains sufficient data to support its conclusion. The authors created a new in vitro model of human periodontal ligament tissue successfully. The organization of the full text is scientific and professional for publication. The authors may need to consider polishing the language for a better understanding of international researchers.


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: ACCELERATED CONSTRUCTION OF AN IN VITRO MODEL OF HUMAN PERIODONTAL LIGAMENT TISSUE: VACUUM PLASMA COMBINED WITH FIBRONECTIN COATING AND A POLYDIMETHYLSILOXANE MATRIX
Review round: 1
Reviewer: 2

Output:
Basic reporting: The authors have examined the role of the combination of fibronectin coating and
vacuum plasma treatment with polymethylsiloxane cell culture in achieving
accelerated osteogenic gene expression and construction of an in vitro human
periodontal ligament fibroblast stretching model.

General comments:

1.	The manuscript is well written with a clear introduction and extensive review of the published literature.
2.	The authors have brought out the research question into purview in a lucid manner.
3.	The correct provenance of the cell line has been described. The methods and the technical details have been well described.
Experimental design: No comment
Validity of the findings: My major concern is with the data analysis and its representation

1.	The statistics section in this MS seems to be highly inadequate where there is no clarity on the statistical tests used (paired or unpaired tests), the distribution of data and whether a post hoc analysis was done or not.
2.	There is no mention of the statistical platform (Stata, R, SPSS etc.) used for the statistical analysis. The statistical paltform should be mentioned in the method section.
3.	The data has been inadequately represented in the MS with the use of only p-values for statistical significance ignoring the importance of confidence intervals.  Author should include confidence intervals
4.	Tables for the results need to be added and the quality of the graphs should be improved.
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: ACCELERATED CONSTRUCTION OF AN IN VITRO MODEL OF HUMAN PERIODONTAL LIGAMENT TISSUE: VACUUM PLASMA COMBINED WITH FIBRONECTIN COATING AND A POLYDIMETHYLSILOXANE MATRIX
Review round: 1
Reviewer: 3

Output:
Basic reporting: A.	The English language should be improved for a clearer understanding of the international audience. Manuscript revision by native English-speaking colleagues could be helpful in this regard. Some examples where languages could be improved: line396-398, 400-401, 409-410, etc. Other minor corrections are below,
-	Line 62-65: Two subsequent sentences were started with “During orthodontic treatment,…”. Please consider rewriting these sentences.
-	Line 80: “Chemical and physical treatment techniques…” - - > “Several chemical and physical treatment techniques…”
-	Line 82: “It enhances binding of the …” - - > “It enhances the binding of the …”
-	Line 84: “physical treatment technique.” - - > “physical treatment technique to enhance hydrophilicity.”
-	Line 85-86: “followed by acrylic acid…” - - > “followed by treatment with acrylic acid…”
-	Line 254-255: “Cell nuclei stained blue and the cytoplasm (cytoskeleton) stained green.” - - > “Cell nuclei were stained blue and the cytoplasm (cytoskeleton) were stained green.”
-	Line 303-304: “PDMS is a hydrophilic material, and therefore treatment devised to improve its surface wettability is usually considered conducive to cell and extracellular matrix growth.” - - > “PDMS is a hydrophobic material, and therefore it’s chemical and physical treatments were devised to improve its surface wettability which is usually considered conducive to cell and extracellular matrix growth.”
-	Line 390: “…of surface modification methods,” - - > “…of surface modification methods reported,”
-	Line 397: “…cellular polarity appearance indicated accelerated model construction ability,” - - > “…cellular polarity appearance indicated the accelerated model construction ability,”

B.	The introduction could be improved as suggested below,
-	The main theme of the paper is the accelerated construction of an in vitro model of human periodontal ligament tissue. Please discuss the significance of developing in vitro model of human periodontal ligament tissue at the end of first paragraph (line 73) or the beginning of second (line 75) paragraph. As these types of models were reported previously (Liao et al 2013, Liao et al 2013,), please explain the context that the current model is addressing. A brief discussion about the previous models will be appropriate.
-	Line 34: Sentence “Orthodontic treatment makes one beautiful.” Orthodontic treatment is used for prevention of teeth disease and beautification. As this is the introductory sentence of the abstract, a comprehensive introduction of orthodontics will be more suitable here.
-	Line 62: “….tissues of tooth and bone.” Addition of a brief introduction (1-2 line) of orthodontic treatment will be helpful to the reader.
-	Line 61: “….tooth roots to the alveolar bone.” Please cite the reference.
-	Line 63: “….stretching and compression forces.” Please cite the reference.
-	Line 72-73: “The difference between clinical observation and biochemical responses inspired us to further examine the bone metabolism mechanism in PDL tissues.” It will be clearer to the reader if rewritten as “Similar biochemical responses for directional and compression forces inspired us ….” Please elaborate on how is that observation relevant to the study presented here.
-	Line 78: “…a low Young's modulus value.” Please cite the reference.
-	Line 87-90: “A combination of chemical….of ECM proteins.” For clarification to the reader, please indicate that combined use of chemical and physical techniques to modify surface properties was successful before as reported in [ref].

C.	Please structure the abstract as background/methods/results or any format of your choice for clarity.

D.	There are few minor corrections for figures.
-	Figure 4: Please provide sufficient space between E & G and F & H, respectively for more clarity. The ‘NN’ of figure E and ‘FN’ of figure F appear to be in figure G and H, respectively.
-	Figure 6: “Note that cells from group PF had significantly higher Runx-2 gene expression than group PN on day 1 and day 3, and OCN gene expression on day 1.” - - > “Note that cells from group PF had significantly higher Runx-2 and OCN gene expression than group PN on day 3.” Please mention what the ‘*’ are indicating and remove the ‘*’ from day 1 (Runx-2).

E.	There are few minor corrections for raw data.
-	Some of the values in the raw data for Fig. 2 (AFM) are written in Chinese or Japanese. Translating these values into English will help the international audience.
-	Consistent naming of the images or documents in the raw data as followed in the manuscript will improve clarity.
Experimental design: A.	The current work fits well with the scope of the journal.
B.	The addressed research question was well defined. As mentioned previously, the introduction needs to elaborate on how the present work will address the identified knowledge gap.
C.	Experiments were performed with a high technical standard.
D.	The methods are described in sufficient details to replicate the results.
Validity of the findings: A.	Data is robust.
B.	The conclusions are clear and adequately address the original research questions.
C.	Could you please elaborate in the discussion section how the acceleration of the gene expression and cellular polarity in the current model compares with the previous models?
Additional comments: No comment.


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

Input:
Title: CLIMATIC NICHE COMPARISON ACROSS A CRYPTIC SPECIES COMPLEX
Review round: 1
Reviewer: 1

Output:
Basic reporting: This manuscript is very clear and mostly well structured, necessary data is publicly available. However, some methods are included in the results (line 175-178). I suggest some further literature review in the niche modeling area especially regarding methods and the history of the field which they keep presenting as new although it has a history of more than 20 years. Results are relevant although some methodological concern will be presented later.

Some small typos have to be fixed including the confusion between allopatry and allotropic in the abstract. A small wording concern regarding the use of the word niche to refer to the climatic niche should be assessed. This becomes particularly relevant when reaching the discussion/conclusions where other niche axes are discussed such as host plants but are to considered part of the niche.
Experimental design: The research is within the scope of the journal. The research question is well defined and relevant to several biology fields including biogeography and climate change research. Methods are replicable although not state of the art as stated by the authors in line 231. Also in the methods description line 111 authors state ENM's "simulate suitable environments" I urge them to re-write this as ENM constructions does not involve any simulation of data but rather construction of models that are interpolated and extrapolated in space.

I have some detailed concerns below:

1-  Maxent is known to produce overly complex models that contain more parameters than observations. This is particularly true in cases of low sample sizes like this one and directly affects the estimation of species distributions and in this case all the conclusions regarding climatic niches. For example, with overfit models it is to be expected that niches of geographically separate species will be different as they are geographically overfit. This problem is easily to overcome by not using defaults in the modeling process but rather comparing different combinations of feature classes and regularization multipliers. This can of course be done manually but also using freely available software. Here are two possibilities the authors should read and consider:
    a- Brown JL (2014) SDMtoolbox: a python-based GIS toolkit for landscape genetic, biogeographic and species distribution model analyses. Methods in Ecology and Evolution, 5, 694–700.
    b- Muscarella R, Galante PJ, Soley-Guardia M et al. (2014) ENMeval: An R package for conducting spatially independent evaluations and estimating optimal model complexity for Maxent ecological niche models. Methods in Ecology and Evolution, 5, 1198–1205.

2- Why choose a 5km grid for small insects? Is there some estimate of their dispersal capabilities that lead to this choice? It appears to me that at a 5km grid scale (about 25 square km) it is to be expected that sympatric species are experiencing the same average climate but probably not at a more microclimatic scale.

3- Several papers have stated problems with using AUC as a metric of model performance  and suggested many alternatives including Omission rates and partial ROCs. However, the authors chose to ignore these criticisms and use AUC with no mention of the problems. I believe they have to use a different measure or at least mention the potential shortcomings  and why they think they are not important. I am suggesting some relevant literature but this is by no means comprehensive:
   a.Peterson AT, Papeş M, Soberón J (2008) Rethinking receiver operating characteristic analysis applications in ecological niche modeling. Ecological Modelling, 213, 63–72.
  b. Lobo JM, Jiménez-valverde A, Real R (2008) AUC : a misleading measure of the performance of predictive distribution models. , 145–151.

4- Why using the "maximum training sensitivity combined with specificity" this is not justified here. Conclusions of this paper are heavily dependent on this choice since it determines the area for which the environmental variables are extracted to compare the climatic niches. Probably it would be better to try several thresholds that are used in the literature and see how robust the results are. Especially it strikes me as odd that they are not using the very widely accepted T10 and LPT thresholds. For example see:
   Pearson RG, Raxworthy CJ, Nakamura M, Peterson AT (2007) Predicting species distributions from small numbers of occurrence records: a test case using cryptic geckos in Madagascar. Journal of Biogeography, 34, 102–117.

5- Related to point 4. Lines 175-178 they authors chose an arbitrary division of the suitability for their comparisons. This is wrong. The interpretation of suitability is species dependent and what is suitable for one is not necessarily suitable for the other. This would be easier to understand when using LPT as it shows how the lowest presence threshold can vary widely between  models from things as low as 0.06 to anything and so 0.4 can be high suitability for certain species.

6- Why test all possible pairs for niche equivalency? The niche conservatism hypothesis refers to sister species and so that conclusion will only make sense when testing sisters and having them be equivalent/divergent.
Validity of the findings: There is a lot of speculation not stated as so. Specially when dealing with how these finding support the molecular division of the species. These conclusions are very circular and should be acknowledged as so. Indeed, models were built based on the molecular subdivision and so the fact that the models differ only allows for speculation of how this supports the molecular hypothesis. If the authors want a real test they would have to creat a "null" model in which the combined points for the complex are divided in different species and modeled and those models compared. If observed results are different than the randomization the they could draw conclusions on the support for this taxonomic hypothesis.
Additional comments: This paper has very interesting questions and the conclusions could be relevant to different fields. However, current methodology brings the results into question and do not allow for robust answers to the questions. I believe methodological fixes are easy to implement and conclusions will likely change and bring more light into the history of this very interesting species complex.


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: CLIMATIC NICHE COMPARISON ACROSS A CRYPTIC SPECIES COMPLEX
Review round: 1
Reviewer: 2

Output:
Basic reporting: Overall the text is clean, easy to read, and unambiguous
Experimental design: The research questions are well defined and appropriately test.  One exception regards the parameterization of the ENMs. It much better use omission error rate vs. AUC for model parameterization. AUC alone tends to result in overfit models  (see Radosavljevic & Anderson,2014).  For example, looking at your response curves for s10, they clearly overfit
as presented and do not have any biological realism.  Further, in the future, its best to use spatial jackknifing, tests of multiple regularization parameters, and independent tests of feature classes for model parameterization. These factors are particularly useful for reducing model overfitting. Spatial jackknifing has demonstrated clear advantages over a random sampling of test/training occurrence data. In experiments on the effects of these two treatments, randomly partitioned occurrence datasets produced inflated estimates of performance and led to over-fit models (Radosavljevic & Anderson,2014).

 For future research please consider using SDMtoolbox (my own software, cited below) or ENMeval for evaluating these factors.

See: Brown JL, Bennett JR, French CM (2017). SDMtoolbox 2.0: the next generation Python-based GIS toolkit for landscape genetic, biogeographic and species distribution model analyses. PeerJ

Radosavljevic, Aleksandar, and Robert P. Anderson. "Making better Maxent models of species distributions: complexity, overfitting and evaluation." Journal of biogeography 41.4 (2014): 629-643.
Validity of the findings: My main critique regards the author's interpretations of their niche equivalence and similarity tests. In 2008, Warren et al. (2008) proposed a pair quantitative tests of niche similarity: 1. an Identity Test that tested if two niches are equivalent (based on correlative distribution models) and, 2. a Background Test, that tested when Identity tests were significant, whether they are simply more similar than expected by chance. The Background test was aimed to test the power of the Identity Test, asking the question if the two distribution models are equivalent due to matching environments available in the habitat. If habitats contain identical environments, then species’ niches might be statistically equivalent solely due to the lack of different environments between distributions of the species. In 2012, Broennimann et al. introduced two complementary tests in Environmental space (vs. Geographic space of Warren et al. 2008, 2010). Though the two tests were conceptually similar, they renamed their tests to Equivalence and Similarity tests, corresponding to the Identity and Background Tests of Warren et al. 2008, respectively.  However, in the manuscript Broennimann et al. they did a poor job explaining the exact purpose of each statistical test, so much so, the similarity test is widely misused or misinterpreted.  Like Warren’s Identity tests, the Similarity test of Broennimann should only be used to detect the power of the Equivalence test to detect a difference. Thus, it only provides context to a non-significant Equivalence test and it should not be a separate test to detect ‘similarity’ among species.   Please re-read Warren et al 2008 and in that context reread Broennimann et al 2012.

*Please change your citation from Broennimann et al. 2016 to Di Cola et al 2017

Under my interpretation, their results provide strong evidence of niche divergence (as all equivalence tests are all significant which equals non-equivalent niches), not niche conservatism. Further, given all significant equivalence tests, there is no need to interpret similarity test results.

One important critique that needs to be clarified.  In the supplementary plots of niche overlap - all equivalence test show that only 1 iteration has been performed.  Is this an error?   Please confirm that you did perform 100 iterations of each equivalence test.

Warren, D.L., Glor, R.E. & Turelli, M. (2008) Environmental niche equivalency versus conservatism: quantitative approaches to niche evolution. Evolution, 62, 2868-2883.
Warren, D.L., Glor, R.E. & Turelli, M. (2010) ENMTools: a toolbox for comparative studies of environmental niche models. Ecography, 33, 607-611.
Di Cola, V., Broennimann, O., Petitpierre, B., Breiner, F.T., D'amen, M., Randin, C., Engler, R., Pottier, J., Pio, D. & Dubuis, A. (2017) ecospat: an R package to support spatial analyses and modeling of species niches and distributions. Ecography, 40, 774-787.
Broennimann, O., Fitzpatrick, M.C., Pearman, P.B., Petitpierre, B., Pellissier, L., Yoccoz, N.G., Thuiller, W., Fortin, M.J., Randin, C. & Zimmermann, N.E. (2012) Measuring ecological niche overlap from occurrence and spatial environmental data. Global ecology and biogeography, 21, 481-497.
Additional comments: Great job and cool study.  I look forward to your future research.  Do remember to properly tune your models in the future.


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: CLIMATIC NICHE COMPARISON ACROSS A CRYPTIC SPECIES COMPLEX
Review round: 2
Reviewer: 1

Output:
Basic reporting: Overall the text needs some english revision mainly in the newly added parts.
Experimental design: I thank the authors for thoroughly addressing my methodological concerns.
Validity of the findings: I thank the authors for thoroughly addressing my previous concerns with this manuscript I believe the new analyses help make this manuscript stronger. One of my comments remains to be addressed, as stated in my previous review:
"6- Why test all possible pairs for niche equivalency? The niche conservatism hypothesis refers to sister species and so that conclusion will only make sense when testing sisters and having them be equivalent/divergent."
In this same direction I believe their new results give support to niche divergence and not niche conservatism as stated in the abstract section.
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: RESILIENCE OF BST-2/TETHERIN STRUCTURE TO SINGLE AMINO ACID SUBSTITUTIONS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript is written in a clear and comprehensible manner. Professional English is used throughout (with a few minor typos). The authors provide all background information that is required to understand the research question and the methods used. Figures are clearly labeled. As outlined in more detail below, the manuscript may benefit from a table summarizing all SNPs that were analyzed and their effects on structure/function of tetherin.
Experimental design: The research question is well defined. The methods are suitable to model the effects of SNPs on tetherin structure. One limitation of the present study is the lack of a cell biological assay analyzing the effects of the predicted structural changes on tetherin function. For example, it remains unclear whether the Ile120Phe mutation affects the antiviral activity of tetherin by disrupting the (local) secondary structure.
Validity of the findings: In most cases, the conclusions drawn by the authors are justified. The authors nicely discuss their findings in the context of previous publications that analyzed the structure-function relationship of tetherin. In some cases, the exact identity of the SNPs that were analyzed remains unclear (see below).
Additional comments: In their present study, Roy and colleagues model the effects of naturally occurring polymorphisms on the (coiled-coil) structure of the host restriction factor tetherin. They show that missense mutations are absent from a region in the N-terminal part of the coiled-coil domain. Furthermore, they convincingly demonstrate that none of the missense mutations alters the global structure of tetherin, while some mutations have (minor) effects on local tetherin structure (e.g. G64P, A100P, I120F). Unfortunately, it remains unclear whether these mutations abrogate the antiviral activity of tetherin as the study is limited to in silico analyses. I summarized a few more specific points below that should be addressed to improve the quality of the study.

Major points:
(1)	A table summarizing all SNPs in the tetherin ectodomain and their effects on structure (this study) and function (restriction of virion release, NF-κB activation) as well as their frequency in the population may be helpful.

(2)	Lines 109-113, 221/222, Fig. 2B: The authors highlight that “11 of 78 mutations appear to occur on the exterior” of tetherin. Fig. 2B, however, suggests that the majority of all mutation can be found on the outside (blue). Fig. 3 shows 12 residues on the inside of the dimer. Which information is correct?
What percentage of all amino acids in the ectodomain can be found on the inside/outside? Is the enrichment for mutations on the inside/outside significant?

(3)	Lines 130-132: How do the authors define global disruption of the coiled coil? Can they include a mutant disrupting the structure as positive control in Fig. 3? For example, would (non-natural) mutations between residues 93 and 117 induce global changes?

(4)	Fig. 3: Which mutants were selected for analysis in Fig. 3? Fig. 2A shows several missense mutations close to the C-terminus of the coiled-coil (positions 170-180), while these mutations were obviously not tested in Fig. 3. Why not?

Minor points:
(1)	lines 24-26: “most mutations have little effect on structure, even those that have been previously shown to affect function. Thus, Tetherin sequence is likely less important than maintenance of the coiled-coil structure.” I fully agree with the authors that the absence of any marked effects of SNPs on tetherin structure suggests that maintenance of the coiled coil is important for tetherin function. However, this statement may also be misunderstood: the observation that previously described mutations affecting tetherin function do not alter structure suggests that sequence rather than structure is important. I suggest to rephrase these sentences to clarify this point.

(2)	line 35: “developed numerous methods to evade viral Tetherin” should be rephrased as Tetherin is not viral.

(3)	In Fig. 1, the C‐terminal anchor is modeled as an alpha-helix rather than a GPI anchor. The authors should clarify that there is evidence for both types of membrane anchor.

(4)	Line 66: The authors state that “82 nucleotide polymorphisms” are listed in the Ensembl database. However, the total number of polymorphisms illustrated in Fig. 2A seems to be higher. What is the reason for this discrepancy?

(5)	A few additional studies may be cited:
- lines 31/32: Discovery of the antiviral activity of tetherin:  PMID 18200009, 18342597
- lines 36-38: Discovery of the signaling activity of tetherin: PMID 23159053
- lines 98/99: Lack of sequence conservation across species: PMID 26401043

(6)	Lines 103/104: Why were the analyses limited to SNPs in the ectodomain?

(7)	Lines 111-113: A study by Sakuma et al. suggests that dimerization is not essential for the antiviral activity of tetherin (PMID 19742323). This should be mentioned.

(8)	Fig. 2: Do any of the missense mutations affect known glycosylation sites or disulfide bridges? An alignment illustrating SNPs and functional motifs may be helpful.
The color-coding may be misleading as blue and red illustrate synonymous/non-synonymous mutations in (A), but location on the outside/inside in (B).

(9)	Fig. 3: The color-coding may again be misleading and suggest that the blue and red lines indicate the mean of mutations on the outside and inside of the dimer, respectively.

(10)	Line 163: “over a region 18 amino acids in length”. The region seems to be even larger according to Fig. 5B.

(11)	 Lines 189-194: In their publication, Yang et al. suggest that “the clusters of charged residues, particularly those in the tetherin head domain, contribute to tetherin function, perhaps through opposing interactions with the negatively charged phospholipids of cellular and virion membranes or by maintaining specific structural properties of the tetherin head.” Even if the charged residues are not on a single face of the coiled-coil, these hypotheses may still be true. The authors should change their statement accordingly.

(12)	Lines 234-236: The authors suggest that the proximity to glycosylation sites and a trafficking motif may explain the functional defects of the charge swap mutants. Are some of the SNPs also located in that region?

(13)	Line 259: “most mutations had no effect on functions”. Which mutations had an effect on function? Were they also analyzed in the present study?

(14)	Figure 4: Some of the data is covered by yellow boxes.

(15)	Typos/minor corrections:
- line 39: “an N-terminal transmembrane helix”
- line 132: “(Figure 3)”
- line 139: “shown in Figure 4A”
- line 147: “(Figure 4B)”
- Fig. 4: “E for beta bridge” is not shown in the figure.
- Line 194: “Figure 7B-C”
- References/citations are not always in the correct format (e.g. line 236)


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: RESILIENCE OF BST-2/TETHERIN STRUCTURE TO SINGLE AMINO ACID SUBSTITUTIONS
Review round: 1
Reviewer: 2

Output:
Basic reporting: the manuscript is clear
Experimental design: experimental design is rational and relative complete
Validity of the findings: since most of the  evidences are computational simulation, I suggest to add some experimental confirmation data.
Additional comments: Human Tetherin, also known as BST-2 plays important role in NF-kB signaling by binding enveloped viruses thus inhibiting viral release. The authors here aim to study the structure-function of Tetherin by screening several human genome mutations and estimate the effect on structure, they found that all the mutations did not affect the global structure of Tetherin but affected local structure changes. They also used the known functional defects to match their system, the conclusion is consistent. This work is interesting, but, for final publication, the manuscript needs some revisions.

Major:
1) Firstly, I suggest the author can pick up several mutants (Ala100Pro and Ile120Phe), which are not investigated before, and then do the functional assay to validate the conclusion. This will be more supportive for the paper publication.
2) The authors mentioned that the loss of the activity of the reversal of the charge in the N-terminal may not due to the proposed reason by Yang and coworkers. And the authors propose that this loss of activity may be because the mutation site is close to the trafficking region. Is there some data to support the hypothesis?

Minor:
1) in Figure 2A, residues 93-117 should be labeled, otherwise, it is hard to tell where this region locates;
2) the main text should be more carefully edited. For example, in line 231, malleability was mistakenly spelled as “malleablility” ; in line 256, the reference should be [Pace and Scholtz, 1998] instead of Pace and Scholtz [1998].


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: RESILIENCE OF BST-2/TETHERIN STRUCTURE TO SINGLE AMINO ACID SUBSTITUTIONS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The manuscript is written in a clear and comprehensible manner. Professional English is used throughout (with still a few typos). The authors provide all background information that is required to understand the research question and the methods used. As suggested, additional literature has been cited in the revised version of the manuscript.
Experimental design: The research question is well defined. The methods are suitable to model the effects of SNPs on tetherin structure. One limitation of the present study is the lack of a cell biological assay analyzing the effects of the predicted structural changes on tetherin function. Although suggested by both reviewers, it still remains unclear whether mutations such as Ile120Phe affect the antiviral activity of tetherin by disrupting its (local) secondary structure.
Validity of the findings: The conclusions drawn by the authors are justified. The authors nicely discuss their findings in the context of previous publications that analyzed the structure-function relationship of tetherin.
Additional comments: In response to my previous comments, the authors have made several important changes that improved the quality of their manuscript. Among other things, they included two supplemental tables summarizing all mutations that were analyzed. Furthermore, they have cited additional studies and improved/corrected some of their figures.

Nevertheless, I encourage the authors to address a few remaining minor points:

1)	Lines 213/214: The authors state that “the tetra-serine substitution showed large but localized changes in structure”. In Supplemental Table 2, however, local structural defects of this mutant are described with “NA”. Why?
2)	Similarly, the authors highlight that “Arg64Pro and Ala100Pro induced alterations in secondary structure” (line 168). However, this defect is not indicated for the Arg64Pro mutant in Supplemental Table 1.
3)	In line 167, the authors mention “five mutations” that disrupted helices. Which mutations are they referring to in this case?
4)	Supplemental Table 1 lists 78 SNPs, while 77 are mentioned in the text (e.g. lines 69, 115, 119, …).
5)	It still remains unclear whether the enrichment for missense mutations on the outside of tetherin is significant (see major point 2 of the initial review).
6)	Lines 155/156: “Position 64 is the most frequently mutated position”. This is not entirely correct. Although five different missense mutations have been described at this position, they are very rare. Other positions are mutated more frequently.
7)	Lines 163/165: Refer to Figure 4B.
8)	Legend of Figure 7: “Overlap is shown in grey A yellow box indicates the area where these mutants are within the sequence.” The color of the box has been changed to blue.


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: RESILIENCE OF BST-2/TETHERIN STRUCTURE TO SINGLE AMINO ACID SUBSTITUTIONS
Review round: 2
Reviewer: 2

Output:
Basic reporting: the article is well organized
Experimental design: the experimental design is  proper
Validity of the findings: because they basically focus on computational simulation, so they barely used the experimental data to support the conclusion
Additional comments: I still think it is important to pick up several mutants (Ala100Pro and Ile120Phe), which are not investigated before, and then do the functional assay to validate the conclusion.


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: INFLUENCE OF FOETAL INFLAMMATION ON THE DEVELOPMENT OF MECONIUM ASPIRATION SYNDROME IN TERM NEONATES WITH MECONIUM-STAINED AMNIOTIC FLUID
Review round: 1
Reviewer: 1

Output:
Basic reporting: Basically the manuscript is relatively well written, but the result section is rather too brief. For example, there are no mention about acute-phase inflammatory reaction score in the text, even though the score 3 seems to have statistical significance in the table. It is better to describe it in detail.  Please see a reference below;
Hofer N, Jank K, Strenger V, Pansy J, Resch B. Inflammatory indices in meconium aspiration syndrome. Pediatr Pulmonol. 2016 Jun;51(6):601-6. doi: 10.1002/ppul.23349. Epub 2015 Dec 10. PubMed PMID: 26663621.
Experimental design: 1. Line 53-54 “funistis and chorioamnionitis”: Based on their definition in the Methods section, the terms seem to mean acute inflammation in this study. Because there is another entity, chronic chorioamnionitis, which is not related to amniotic infection syndrome, the term “acute chorioamnionitis and funisitis” should be used through the whole text.
2. Line 117: the definition of term (neonate) is required, for example born at >=37 weeks of gestation.
3. Line 117-118 “Level II neonatal intensive care unit”: the “Level II NICU” is not familiar to non-pediatric people. Please describe it briefly. Is there any chance term neonates with meconium stained amniotic fluid do not admit to level II NICU? maybe the neonates with more severe symptoms only could be enrolled. If so, it should be described as another limitation.
4. Line 131-132 ”chorioamnionitis was defined as infiltration of neutrophils identified in chorionic plate…”: What is the reason the location of inflammation is limited to the “chorionic plate”? In general, acute chorioamnionitis is defined as neutrophil infiltration in the chorioamniotic membranes, even though acute inflammation in the chorionic plate is also considered as acute chorioamnionitis. Please confirm the diagnostic definition of acute chorioamnionitis from the pathologist of your institution.
Validity of the findings: Line 52-54: the authors said their aim was to investigate whether the incidence of MAS is associated with the presence of funisitis and chorioamnionitis, but the conclusion said some clinical variables (male sex, antenatal asphyxia) were also identified as independent variables. It is better to amend the aim or conclusion appropriately.
Additional comments: The results in this study supports the previous reports on MAS, however there are no definite novel findings. If clinical progression and/or treatment outcomes in detail are added in the results, readers would be more interested in the study.

Line 165 “Of 99 neonates...”: it is better to describe it as “ of 99 term neonates with meconium-stained fluid…”

Table footnote line 4: the details of the acute-phase inflammatory response score is not in “Table S1” but in the supplementary information. Why don’t you put it in the Method 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: INFLUENCE OF FOETAL INFLAMMATION ON THE DEVELOPMENT OF MECONIUM ASPIRATION SYNDROME IN TERM NEONATES WITH MECONIUM-STAINED AMNIOTIC FLUID
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript has lots of grammatical errors which need to be corrected.
Line 49 - “ Although asphyxia” - Asphyxia is not the sole cause for meconium passage in utero and other factors such as in utero infections, placental insufficiency, post maturity, IUGR etc are involved in MSAF/ MAS. So the sentence could be rephrased as   “fetal distress and subsequent…”
Line 50 - “stimulation have” - Should be changed to “stimulation has”
Line 51, 52 - “occurs in the absence of asphyxia, and the presence of additional causatives such as intraamniotic inflammation has been extrapolated” -change to  “  occurs due to other pathologies such as intraamniotic inflammation ”
Line 57, 58 - “Dependence of MAS development on clinical variables and histopathological findings were assessed”. Modification - “ Clinical variables and histopathological findings associated with the incidence of MAS were studied”
Line 64, 65 -  “In neonates with meconium stained amniotic fluid, male sex and funisitis, as well as evidence of antenatal asphyxia, were identified as independent variables for MAS development” - Modification -  “In neonates with meconium stained amniotic fluid, male sex ,funisitis and low 1-minute APGAR score were identified as independent variables for MAS development”. 1 minute APGAR is not a definitive indicator of antenatal asphyxia and such an inference is not appropriate
Line 75 - 78 - “ In addition, advanced therapeutic regimens for severe respiratory failure using surfactant replacement, nitric oxide  inhalation, and extracorporeal membrane oxygenation, led to a marked decrease in mortality and morbidity associated with MAS”  - Modify - The use of surfactant replacement, nitric oxide therapy and extracorporeal membrane oxygenation has lead to a marked decrease in mortality and morbidity associated with MAS in the recent times”
Line 79 - “ newborn born at a “ modify - “ newborn born in a”. “Even at a” - Modify - “ Even in a “
Line 81 - “ do not provide early, precise discrimination of neonates who are likely to develop severe respiratory failure, from their peers, who only experience transient respiratory failure”
Modify -“ do not provide any insights as to who are likely to develop severe respiratory failure ”
Line 141 - “In our unit, routine blood tests are scheduled at admission and on day 1 for neonates with meconium- stained amniotic fluid, where blood gas analysis” modify - “In our unit, routine blood tests that are done for neonates born through MSAF are blood gas analysis, cell counts, blood sugar level, and serum inflammatory markers and these tests were done in the study subjects”
Line 142 - 14 8 -“ For the inflammatory markers, elevation of C-reactive  protein (CRP;>0.3 mg/dL), α1-acid glycoprotein (α1AG; >20 mg/dL), and haptoglobin (>13 mg/dl) have been routinely assessed since 1983 using turbidimetric immunoassay (Quick Turbo, Shino-Test Corporation, Japan), based on previous reports, which found early diagnostic  property of these biomarkers and composite scores calculated from these values, or acute-phase  inflammatory reaction score, for severe neonatal infection (Speer et al., 1983; Ipek et al., 2010;  Nakamura et al., 2015) (see Article S1 for details of this composite score)”. modify - “ An acute-phase inflammatory reaction score predicting severe neonatal infection was calculated based on the values of CRP, Alpha1-acid glycoprotein and haptoglobin”
Line 151 - “ First,…” modify - no need for using first. Start the sentence with “ The clinical variables were …”
Line 152 - 154 - Needs to be changed. What the authors intend to say is not clear.
Line 168 - “statistically invariant” modify into - “statistically not significant”
Line 182 - 183 - “suggesting the importance of inflammatory and genetic factors in investigating the mechanism of MAS”. Modify - “ pointing towards the role of inflammatory and genetic factors in the pathophysiology of MAS”
Line 195 - “ additional control variable” - change to “ an additional factor involved in the development of MAS”
Line 229 - “ Instead, CRP, α1AG and haptoglobin were monitored at admission and on day 1, which are known to increase plasma IL- 1βand IL-6 levels.” This sentence is confusing. It is IL 6 secreted from the neutrophils that will result in the increase in CRP production in the liver”
232 - “Were not identified “ modify to “ were identified”
234 - “ at the timing of admission” - modify - “ at the time of admission”

- References - Needs major modification. Doi is usually not included in prescribed referencing standards such as Vancouver standards.
Experimental design: The incidence of MAS is very high in the study group. The authors need to state the possible reason for this. Was it because MAS was over diagnosed?
APGAR at 1 of <7 is not considered a low APGAR. It is not indicative of intrapartum asphyxia. Many guidelines such as ACOG take APGAR values that are much lower and at a later time as being significant.
The authors have specified “high birth weight” as  associated with MAS. But the difference of birth weight between the groups is only 200 - 250 grams. Though it is statistically significant, what is the clinical significance of this?
Inflammatory markers are invariably elevated in MAS even without any underlying infection. Did authors look at the blood culture? That is the gold standard to diagnose sepsis.
What is the utility of histopathological funisitis? It will not help in predicting MAS earlier as HP Examination will take time.
Validity of the findings: Did the authors look at the requirement of resuscitation between the two groups? Along with cord blood gas, it would have been an important variable
The Wiswell definition is used to classify the severity of MAS. <40% FiO2 and less than 48 hours duration of respiratory support requirement comes under mild MAS. The authors have not included mild MAS here. Is there any specific reason why mild MAS was excluded?
Was thin vs Thick meconium studied?
What was the weight for gestational age status? - SGA/ IUGR Vs AGA. SGA s are a very important risk factor for MAS
Why preterms were excluded? MAS does occur in preterms and may be chorioamnionitis/ funisitis play a more important role in preterm with MAS than term with MAS.
Additional comments: The manuscript has a lot of grammatical errors and need to be revised.


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: INFLUENCE OF FOETAL INFLAMMATION ON THE DEVELOPMENT OF MECONIUM ASPIRATION SYNDROME IN TERM NEONATES WITH MECONIUM-STAINED AMNIOTIC FLUID
Review round: 2
Reviewer: 1

Output:
Basic reporting: The manuscript is well-written with the appropriate responses for the reviewer's suggestions.
Experimental design: experimental design is OK with the clear description of the limitations.
Validity of the findings: The results are not quite novel, but they support the previous findings well.
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: COMPARISON OF HEPARINIZED SALINE AND 0.9% SODIUM CHLORIDE FOR MAINTAINING CENTRAL VENOUS CATHETER PATENCY IN HEALTHY DOGS
Review round: 1
Reviewer: 1

Output:
Basic reporting: - Please consider shortening the length of the Introduction to focus on topics pertinent to this study including catheter complications and NS versus HS pros / cons.
- Please ensure that all studies are clearly indicated as human or veterinary to avoid confusion.
- Please note that one of the references is listed twice (López‐Briz E, Ruiz Garcia V, Cabello JB, Bort‐Marti S, Carbonell Sanchis R, Burls A. 2014. Heparin versus 0.9% sodium chloride intermittent flushing for prevention of occlusion in central venous catheters in adults. Cochrane Database of Systematic Reviews. 10.1002/14651858.CD008462.pub2)
Experimental design: - Please expand on the justification behind the power analysis: what was the difference that you were assessing for between the control and HS groups (i.e., lack of patency?
prolongation on clotting times? incidence of phlebitis?). Considering that human meta-analyses including >2000 human patients are insufficiently powered to determine a difference in HS versus saline for maintaining catheter patency, it seems unlikely that 24 healthy dogs would be enough to determine this.
- Please specify the type and size of CVC placed. The two incidents of ventricular arrhythmias suggest that the CVC may have been placed too far distal.
- Please explain how proper placement (position of distal end of the CVC) was determined.
Validity of the findings: - While many studies in the human literature suggest that NS and HS flushes are of equal efficacy for preserving CVC patency, recent meta-analyses suggest that the studies are largely underpowered and the conclusions remain uncertain (https://www.ncbi.nlm.nih.gov/pubmed/30058070). Please adjust your Discussion and Conclusions accordingly.
Additional comments: - Please avoid use of first-person language such as “we” or “our” throughout the manuscript.
- Please avoid statements suggesting novelty of research, since it is not possible to know what similar studies other researchers may have completed and not yet published.
- The second paragraph of the Discussion seems slightly out of place or irrelevant to this study’s objectives. Please consider revising to focus on the study objectives (i.e., maintenance of catheter patency / reduction of complications rather than to reduce systemic thromboembolic complications [not assessed by the present study]).
- In the third paragraph of the Discussion, it could be reiterated that advanced imaging was not used to determine the cause of the inability to aspirate blood from the catheter.
- Please move the discussion re: the statistical differences in temperature over time further to the 4th paragraph.
- Line 331: Would APCs be a more logical consequence of distal guidewire placement? Please suggest other possible causes of VPCs in these dogs.
- Line 343: This sentence requires re-wording for clarity since numerous limitations are mentioned.
- Please clarify that the results are applicable to healthy dogs and that a similar investigation is required in critically ill dogs before clinical recommendations can be made.
- Table 1: Please consider removing this table as it does not add anything to the manuscript that is not already stated in the text.
- Tables 2 and 3: Please specify “healthy” dogs.


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: COMPARISON OF HEPARINIZED SALINE AND 0.9% SODIUM CHLORIDE FOR MAINTAINING CENTRAL VENOUS CATHETER PATENCY IN HEALTHY DOGS
Review round: 1
Reviewer: 2

Output:
Basic reporting: This is a well done study and provides an answer to a long sought after question. Thanks!

1.  Consider changing purpose to "comparing two flush solutions" instead of determining whether HS was "more effective".
2. Be more consistent with 0.9% sodium chloride vs. normal saline.
3. Line 122: consider deleting "as determined by prolongation of the aPTT" - unnecessary
4. Line 128-129: This sentence is too vague. The references cited simply shows similar CVC patency comparing HS and S.
5. Line 236: "regardless of the treatment each patient received" is confusing. Change to "in both groups" or "for all dogs"
6. Line 257: Can you find a more recent reference than Fleeman, 2001?
7. Line 308: "no evidence of systemic infection" is too broad of a statement since the study only took into account temperature and clinical signs.
8. I do not know the mechanism for VPCs due to the guide wire tickling the atrium although I agree it is seen. Any references you can cite?
Experimental design: 1. Since reflux may contribute to CVC thrombosis formation, please specify what  type of catheter plug/connector was used such as needle-free connectors.
2. As mentioned in the discussion, flushing technique may impact CVC patency. Please specify method used.
3. Very minor, but please specify if CVC lumens were clamped between aspirations/flushes.
4. couldn't find the footnote for the exact type of catheter placed.
5. Would strengthen results to give priming volume of catheter and comment on chosen volume of flush.
Validity of the findings: no comment
Additional comments: Great job! Thanks for doing this study.


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

Input:
Title: COMPARISON OF HEPARINIZED SALINE AND 0.9% SODIUM CHLORIDE FOR MAINTAINING CENTRAL VENOUS CATHETER PATENCY IN HEALTHY DOGS
Review round: 2
Reviewer: 1

Output:
Basic reporting: No Comment
Experimental design: No Comment
Validity of the findings: No Comment
Additional comments: Dear Authors - Thank you for adequately addressing the concerns raised during review of your initial manuscript. All of my concerns have been addressed and incorporated into your revised manuscript. A remaining edit, however, is at Line 360 where it has yet to be specified (currently listed as XXX) what number of dogs would be required to assess the difference in catheter occlusion between HS versus NS in dogs. Further to that, there are no outstanding concerns. Thank you and well done!


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: COMPARISON OF HEPARINIZED SALINE AND 0.9% SODIUM CHLORIDE FOR MAINTAINING CENTRAL VENOUS CATHETER PATENCY IN HEALTHY DOGS
Review round: 2
Reviewer: 2

Output:
Basic reporting: Thank you for the edits. The paper is stronger as a result.

1. Line 340: Consider adding "(post-hoc)" in between "observed power" for clarity.
1. Line 344: "XXX" should be filled in
2. Line 438: All CAPS of authors should be changed.
Experimental design: Thank you for providing further clarification regarding your methods.

1. Although there is a lack of clinical evidence, guidelines usually recommend a flush volume of at least 2x the priming volume.
-Royal College of Nursing. (2010) Standards for infusion therapy. London, Royal College of Nursing.
-Infusion Nurses Society. Infusion nursing standards of practice. Journal of Infusion Nursing. 2011;34(1, supplement):S1–S110. doi: 10.1097/01.nan.0000393791.46613.51.
Validity of the findings: no comment
Additional comments: no comment