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: GENETIC INACTIVATION OF ALPHA-SYNUCLEIN AFFECTS EMBRYONIC DEVELOPMENT OF DOPAMINERGIC NEURONS OF THE SUBSTANTIA NIGRA, BUT NOT THE VENTRAL TEGMENTAL AREA, IN MOUSE BRAIN
Review round: 1
Reviewer: 3

Output:
Basic reporting: This interesting and carefully executed study shows that the pattern of development of DA neurons in the Substantia Nigra (SN), but not in the Ventral Tegmental Area (VTA) is altered in alpha-synuclein knockout mice. Previous studies in alpha-synuclein knockout mice have shown that inactivation of this gene can lead to the reduction in number of DA neurons in the SN. The brain area known to be the most affected in PD patients.  In the present study the dynamics of changes in TH-positive cell numbers in the SN and VTA during a critical period of their embryonic development was evaluated in alpha-synuclein knockout mice.
Experimental design: The authors report that mice deficient in alpha-synuclein have altered number of DA neurons in the SN and have no differences in the formation of DA neurons in VTA, the structure that is much less susceptible to degeneration in PD brain.
Validity of the findings: The manuscript is well written and methods are described with all the necessary details. The observations reported here are novel and provocative.
Additional comments: The only suggestion for authors is to correct in Figure 2 legend – Substantia Nigra.


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: GENETIC INACTIVATION OF ALPHA-SYNUCLEIN AFFECTS EMBRYONIC DEVELOPMENT OF DOPAMINERGIC NEURONS OF THE SUBSTANTIA NIGRA, BUT NOT THE VENTRAL TEGMENTAL AREA, IN MOUSE BRAIN
Review round: 1
Reviewer: 4

Output:
Basic reporting: Sufficient introduction and background context are provided and the relevant prior literature is cited and clearly referenced.

In general, the text is technically correct and conforms to professional standards; however the article does suffer from a number of typographical errors and grammatical mistakes and minor corrections are needed.

The article is structured appropriately and the figures are relevant to the articles’ content. Fig. 1 is clearly described, appears statistically correct (in concert with raw data) and does not present any concerns.

As presented, Fig. 2 provides only two images, one at E11.5 and one at E13.5.  These figures are not both labeled, with the VTA and SN shown only in the E13.5 image.  No indication is provided whether these are from WT or aKO sections (presumably the former), nor is there clear indication of which neurons are TH+ve.

With regard to Fig. 2 it is suggested that the authors provide additional, clearly labeled images describing WT versus aKO sections at all relevant ages. It should not present a significant challenge to provide this additional data and would greatly strengthen the article and provide further clarity for the reader.

All appropriate raw (statistical) data has been made available in accordance with the PeerJ Data Sharing policy.
Experimental design: All raw data appear to be statistically sound and proper controls have been employed. Imaging data would be greatly improved by addition of side-by-side comparisons of sections from WT and aKO animals. Additional, more detailed labeling of figures is warranted.

Conclusions are concise, appropriately stated and limited only to results and original research question.

Overall, no concerns.
Validity of the findings: All raw data appear to be statistically sound and proper controls have been employed. Imaging data would be greatly improved by addition of side-by-side comparisons of sections from WT and aKO animals. Additional, more detailed labeling of figures is warranted.

Conclusions are concise, appropriately stated and limited only to results and original research question.
Additional comments: Some improvements are suggested, by the inclusion of additional imaging, showing WT vs aKO sections that indicate which neuron populations are labeled as TH+ve.

Some grammatical and typographical errors are present and it would improve the article if these were corrected (a minor concern).


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

Input:
Title: NOVEL, NON-SYMBIOTIC ISOLATES OF NEORHIZOBIUM FROM A DRYLAND AGRICULTURAL SOIL
Review round: 1
Reviewer: 1

Output:
Basic reporting: The references by Segovia et al., 1991; Sullivan et al., 1996; Van Insberghe et al., 2015; Jones et al., 2016, are cited referring to "isolation of non-symbiotic Rhizobia from plant material". However, these papers are about the isolation of non-symbiotic rhizobia from rhizospheric soil. Only in the work by Sullivan bacteria were also isolated from the surface of roots. I think this should be clarified.
Experimental design: No comment
Validity of the findings: My only concern is that if megaplasmids are present in the described strains, the genomic analysis could be biased by not separating chromosomal from plasmid sequences.
Additional comments: In this paper the authors report the finding of non-symbiotic Neorhizobium strains in a soil of a region in Spain. This finding was serendipitous, because the selection procedure was directed to the identification of rhizobium species. The authors analyzed different aspects of the strains, including a partial sequence of some representatives. This analysis showed that the strains belong to a specific group, close to but different from N galegae. The possible presence of plasmids is suggested by the presence of repABC regions. This paper contributes to the scarce knowledge on the composition of rhizosphere populations.

Some points that in my opinion would contribute to the paper are:
1 - Authors comment that the soil used has no history of legume cultivation, but, have other plants been cultivated in that soil?
2 - The identification of repABC regions suggests the presence of plasmids. These could be easily visualized by using the approppriate electrophoretic techniques.
3 - If megaplasmids are present, the genomic analysis could be biased by not separating chromosomal from plasmid sequences.

Minor points:
Line 44: change to: ..they have rarely been isolated..

Line 81 - 82: "isolation of non-symbiotic Rhizobia from plant material (Segovia et al., 1991; Sullivan et al., 1996; Van Insberghe et al., 2015; Jones et al., 2016)."
In the references cited, non-symbiotic rhizobia were isolated from rhizospheric soil. Only in the work by Sullivan bacteria were also isolated from the surface of roots. I think this should be clarified.

Line 102: See general comment Nª1

Line 116: "..100 µl of each dilution was plated.." change to: 100 µl of each dilution were plated

Line 131: "primners" change to primers

Lines 135 - 136: "10mM of each dNTP" Shouldn't this be 10 micromolar?

Lines 192 - 195: "while the remaining 53, surprisingly, clustered with sequences from genus Neorhizobium (Fig. 1B). These sequences were compared and classified into groups (twenty in total) if they differed in at least one nucleotide. In order to facilitate further studies, a representative strain of each these rpoB sequence types was chosen"
If I understand correctly, the 20 branches shown in Fig 1B represent the different groups. Table 1 lists 55 strains isolated in this study, but it is not clear which strains belong to each group. Please clarify.
Also, it would be interesting to see which are the variations in the rpoB sequence, maybe  in a Supplementary figure?

Lines 206 - 207: "with legume plants were hampered by the fact that the known hosts of Neorhizobium spp. are very diverse and are not found in the Tomejil area"
Which are plants that can be nodulated by Neorhizobium?

Lines 223 - 226: "This gene, together with an upstream ORF, was present in genome assemblies of the Tomejil strains and contained sequences partially complementary to those of fnrN primers that may explain the successful amplifications observed (Fig. S2B).
Was it absent in the Neorhizobium spp. genomic sequences available in databanks, N. galegae and N huautlense?

Lines 231 - 232: "All five genomes contained a set of repABC sequences characteristic of rhizobial plasmids"
See general comment Nª 2

Line 279: "to enrich in soybean-specific" change to: to enrich soybean-specific

Line 291: "we had been able" change to: we were able


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: NOVEL, NON-SYMBIOTIC ISOLATES OF NEORHIZOBIUM FROM A DRYLAND AGRICULTURAL SOIL
Review round: 1
Reviewer: 2

Output:
Basic reporting: In this manuscript Soenens and Imperial describe the isolation and genomic sequences of Rhizobiaceae belonging to a novel non-symbiotic Neorhizobium genospecies. Overall, the reported original research work is sound and the conclusions are supported by the presented data. Below, some points are raised that should be considered in a revised version.
Experimental design: 1) The method used to isolate the genetically characterized Neorhizobium strain(s), semi-selective enrichment needs to be clarified for the reader who is not familiar with growth requirements of Rhizobiaceae. I therefore suggest to include the complete recipe of the semi-selective MNBP medium together with the relevant references.
2) For a better understanding of the isolation method the authors may consider to provide Fig.S1 in the main part of the manuscript, not in the supplement. This could also be complemented by indicating numbers (28 isolations, 342 Rhizobium, 99 fnrN positive, 46 Rhizobium, 53 Neorhizobium etc.) to give an overview of how, in the end, 5 draft genome sequences were obtained.
Validity of the findings: 1) There is an issue with the PCR amplification using the fnrN primers. A product of the correct size would indicate the presence of the fnrN gene which is indicative of Rhizobium spp. or Agrobacterium spp. However, as shown in Fig.S2, the fragment sizes are clearly different (between the positive control and the isolates). Therefore, the authors erroneously assumed to have isolated fnrN+ strains (53 as the rpoB sequences revealed). That should be clearly stated in the results section (lines 186-193; lines 297-298) with an indication of whether the remaining 46 isolates resulted in a fnrN band of the expected (correct) size.
2) To better understand the false positive fnrN reaction of the Tomejil strains it should be noted how similar the sequences of the primers with the identified target sequence in the sequenced genomes are. Lines 224 to 226 states a „partial complementarity“ which does not explain the positive results in the PCR reaction.
3) The authors report the presence of highly similar repABC sequences in all sequenced Tomejil strains. However, no information is given on other (cargo) genes that are located on the same contigs as the repABC genes. Such an information would provide additional valuable information on the transferability of those plasmids or chromids. At least a scan for DNA transfer genes should be done and the results indicated. Also the possibility that those large elements could represent chromids (see doi:  10.1093/gbe/evv227 and references cited therein) should be considered or discussed.
Additional comments: Minor points:
line 58: …study, Sullivan and colleagues (Ref) were able…..
line 131: …primers…
line 151: …were assembled ….
lines 327-328: please specify what is meant by: …a large diversity of Rhizobial diversity…


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: NOVEL, NON-SYMBIOTIC ISOLATES OF NEORHIZOBIUM FROM A DRYLAND AGRICULTURAL SOIL
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: In line 282, the sentence should say: "... replication of a large...."


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: NOVEL, NON-SYMBIOTIC ISOLATES OF NEORHIZOBIUM FROM A DRYLAND AGRICULTURAL SOIL
Review round: 2
Reviewer: 2

Output:
Basic reporting: No further comment
Experimental design: No further comment
Validity of the findings: No further comment
Additional comments: No further 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: OCCURRENCE OF TERMITES (ISOPTERA) ON LIVING AND STANDING DEAD TREES IN A TROPICAL DRY FOREST IN MEXICO
Review round: 1
Reviewer: 1

Output:
Basic reporting: The English language in this submission needs considerable improvement before it is suitable for publication in PeerJ.  I would encourage the authors to find a qualified English Language editing service that is familiar with science writing to edit the entire manuscript.  The current version has numerous - too many to point out - grammatical errors.
Listed below are some examples but the only remedy is to have someone edit the entire submission prior to returning this manuscript for consideration;
Lines 14-16 - "As ecosystem engineers, termites play a key role in ecological processes, although its functional role in the dynamics of woody material degradation in tropical dry forests has been poorly studied, particularly at canopy level." Should read more like…  'Termites play a key role as ecosystem engineers in numerous ecological processes although their role in the dynamics of wood degradation in tropical dry forests, particularly at the level of the crown canopy, has been little studied.'
Lines 22-25 - "We also found a high density of trees associated with termites in trees with diameters at breast height fewer than 15 cm. Interestingly, 72 % of variation in the density of trees associated with termites is explained by the density of standing dead trees." We found that a high density (I AM NOT CERTAIN WHAT THE AUTHORS MEAN BY HIGH DENSITY Figure 3 does not address plot density only density of living and dead trees with and without termites BUT THAT TERM SHOULD BE FOLLOWED BY A VALUE OR RANGE OF VALUES such as… 0.03-0.08 trees/m2 ) of trees with a Diameter at Breast Height (DBH) less than 15-cm in a plot was associated with the presence of termites in those trees.
Lines 49-51 - "Dead woody material also known as coarse woody debris is an important component of decomposition process since it represent as much as 42% of above-ground biomass in tropical forests" Should read more like… Coarse woody debris (CWD) is an important dead-wood component of tropical forests, and therefore the decomposition process, that can represent as much as 42% of the above-ground biomass (citations)
Lines 66-68 - "In this study we analyzed the interaction of termites with living and standing dead trees of the tropical dry forest at Chamela, Halisco, in order to understand the role of termites in the decomposition dead woody material in the canopy of a tropical dry forest of Mexico."  Should read more like…   In this study we recorded the presence of termites in and on living and standing dead trees in a tropical dry forest at Chamela, Halisco, in order to illuminate the role termites could play in the decomposition of dead woody material in the crown canopy of a tropical dry forest of Mexico.

Lines 72-75 - the authors must qualify this statement because standing dead wood has a lifespan - it does not remain standing forever.

The font size and character changes several times in the manuscript with the first incident occurring in Line 78.  This must be corrected.

Line 111 - the authors must describe the method they used to "estimate the density" which eventually is reported as the number of trees per square meter.

Lines199-201 - could that difference be explained by periodic inundation during flooding events that would kill the termites?

Lines 236-244 - The main idea posited in these sentences should probably be placed in the Introduction to justify the authors concentration on above ground necromass.  The question I had throughout reading this submission was how much of the wood in Chamela was on the ground? until I got to those lines near the end of the manuscript.

Likewise, the authors also should clearly define the term "canopy" early in the manuscript.
Experimental design: The design and statistical analysis was appropriate and the aims are within the scope of the journal.
Validity of the findings: The information provided by this work is very interesting but would have benefited from some measure of active feeding rather than the simple 'signs' of activity. It also goes without mention that some description of the termites involved - was it only one of the 30+ species found in the forest or all 30+? - would have been a major contribution.

The work is, however, important because it draws much needed attention to above ground necromass in forests and the route(s) by which that significant source of carbon is moved through the forest ecosystem.
Additional comments: This is important work that was conducted in a scientifically sound manner.  Please take the time to have this work presented in an English Language journal.


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: OCCURRENCE OF TERMITES (ISOPTERA) ON LIVING AND STANDING DEAD TREES IN A TROPICAL DRY FOREST IN MEXICO
Review round: 1
Reviewer: 2

Output:
Basic reporting: Reporting is clear throughout. There are a few minor grammatical errors (e.g. Line 78) that should be corrected. There is sufficient coverage of the relevant literature and background literature provided. The overall structure of the article is professional, although there are numerous cases of changes in font type and size that should be changed (e.g. Lines 78-88) as these are not necessary or helpful. Raw data are shared. This is a small, correlational study and thus the questions, study design, and conclusions are self-contained and relevant to the methods and results.
Experimental design: This is original, primary research. The research questions are straight-forward and do address a research gap in termite ecology and deadwood processes that are currently largely unknown. Results are preliminary. That is, the small scope of the sampling design and the limited data collected (occurrence of termite galleries on live and dead trees) provide little information beyond whether or not termites are prevalent in standing live or dead wood in this ecosystem. That said, the results are nevertheless an interesting contribution as so little is known about termite occupancy of standing dead wood in ecosystems throughout the world. Methods are sufficiently described for replication although limited in scale and scope.
Validity of the findings: Results are narrowly novel, but nevertheless valuable to the field. This study can certainly serve as a starting point for asking broader questions about termite/standing dead wood interactions. Data are generally robust, although lacking in important details, such as type/species of trees, species of termite and preference for live/dead wood. The limited spatial scope of the sampling and lack of sampling in any other habitats other than “riparian” and “deciduous forest” suggests caution in generalizing results. Furthermore, limited description of any other important habitat impacts such as human activity levels, suggests caution in generalizing results. Conclusions are clearly stated and linked to results. I would caution against speculating too much and trying to test new hypotheses in the Discussion (Lines 212-215), but this is a minor criticism.
Additional comments: Lacking termite species, tree species, and other relevant habitat variables (e.g. land use history and role of human activity), this study is nevertheless an interesting, preliminary, contribution.


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: OCCURRENCE OF TERMITES (ISOPTERA) ON LIVING AND STANDING DEAD TREES IN A TROPICAL DRY FOREST IN MEXICO
Review round: 1
Reviewer: 3

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: The authors need to answer: what was the interaction studied in the manuscript? It is not clear throughout the text. The presence of termite galleries on wood can not be freely interpreted as consumption. In general, the galleries on the wood usually only connect the colony to the resource source. In other words, the frequency of termite galleries in trees is different from the rate of wood consumption and also does not represent an indication of the abundance of these insects in the studied ecosystems. Therefore, the discussion is overly speculative and few supported by the results. Part of the conclusions are also not supported by the results (more details see "general comments for the author").

I suggest that the discussion and conclusion be rewritten and more focused on the results found. Speculations are welcome, but need to be made with caution because they can lead the reader to misinterpretations of the results.
Additional comments: The manuscript "Interaction of termites (Isoptera) with living and standing dead trees in a tropical dry forest in Mexico" is well written and has interesting aims.

It analysed the "interaction" (density and proposition of living and standing dead tree associate with termites) of termites with the trees in the forest canopy in two ecosystems from Mexico.

The manuscript sought to answer six questions (lines 82-88). In my opinion, all the questions were answered. However, the authors need to answer: what was the interaction studied in the manuscript? It is not clear throughout the text.

The methods and statistical analysis were appropriately used in the manuscript.

The results were well presented, using tables and figures appropriately. On the other hand, it was frustrating not to see a termite species checklist present in the trees, making it difficult to interpret the type of interaction between these insects and the vegetation. For example, because the colonies of the Kalotermitdae species live within the wood, the occurrence of these insects would undoubtedly indicate the consumption of wood in the ecosystems. On the other hand, only the presence of galleries of species of the subfamilies Nasutitermitinae and Termitinae does not make clear the type and the intensity of the existent interactions.

A considerable part of the discussion suggests a link between frequency of occurrence of termite galleries on trees with the importance of these insects in the decomposition of plant necromass. The presence of termite galleries on wood can not be freely interpreted as consumption. In general, the galleries on the wood usually only connect the colony to the resource source. In other words, the frequency of termite galleries in trees is different from the rate of wood consumption and also does not represent an indication of the abundance of these insects in the studied ecosystems. Therefore, the discussion is overly speculative and few supported by the results.

The focus of the discussion should be just the frequency of occurrence of termite galleries on trees. Any link between the gallery frequencies with the abundance or rate of wood consumption by termites needs to be done with great caution, because it may induce readers to think that galleries occurrence is directly linked to the importance of termites in the process of decomposition of plant necromass.

The same happens with the conclusions of the manuscript (see lines 266-269): "as well as the positive relationship between the density of trees associated with termites and the density of standing dead trees demonstrate that termites are important agents in wood decomposition at canopy level in this forest".

Finally, I suggest that the discussion and conclusion be rewritten and more focused on the results found. Speculations are welcome, but need to be made with caution because they can lead the reader to misinterpretations 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: OCCURRENCE OF TERMITES (ISOPTERA) ON LIVING AND STANDING DEAD TREES IN A TROPICAL DRY FOREST IN MEXICO
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have addressed the requested changes by the reviewers and I have no further substantive criticisms.
Experimental design: The authors have addressed the requested changes by the reviewers and I have no further substantive criticisms.
Validity of the findings: The authors have addressed the requested changes by the reviewers and I have no further substantive criticisms.
Additional comments: The authors have addressed the requested changes by the reviewers and I have no further substantive criticisms.


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: MODELLING THE GROWTH OF THE BROWN FROG (RANA DYBOWSKII)
Review round: 1
Reviewer: 1

Output:
Basic reporting: The article is valid and meets the scope of the journal; however before acceptance and publication, it is necessary to improve the manuscript structure. As English is not the mother tongue of the authors, Thus, I suggest improving the language of the article. The literature cited in the article is sufficient to support the hypothesis but I suggest the citation of more studies on growth models in amphibians. The figures should be corrected and if possible reduce the amount of them. The results are interesting; however authors need to improve the discussion.
Suggestions for improving the article are mentioned in the attached file.
Experimental design: The experimental design needs to be improved. In my opinion it is not interesting to produce models for frogs at different ages, since for recommendation of producers and technicians, it will be necessary to apply only one model that corresponds to the whole life of the animal. The application of polynomial and linear models to describe the growth of frogs, I believe is not valid.
Validity of the findings: The findings are valid for publication in the journal, and important for describing the growth of amphibians, since there is very little material for this type of animal production.
Additional comments: The article is valuable and should be published, but I suggest that the authors spend some more time in thoroughly review of the article.
Some basic comments regarding the article structure are mentioned in the attached document.


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: MODELLING THE GROWTH OF THE BROWN FROG (RANA DYBOWSKII)
Review round: 1
Reviewer: 2

Output:
Basic reporting: I think that authors should read more references of the relationship between body mass/body length and age, especially in Chinese frogs in recent years. For instance, Lu Xin’lab in Wuhan University has published some papers about life-history traits in frogs in recent years. I have added some references related to age-body relationship in the text in attached file.
Experimental design: there are relative small samplings
Validity of the findings: no comment
Additional comments: I have now read and reviewed manuscript entitled “Modelling the growth of the brown frog (Rana dybowskii)”. Authors establish a working model to examine the growth of one-year-old and two-year-old (age 1 and age 2) brown frogs (Rana dybowskii) from metamorphosis or out-hibernation to hibernation under the same environmental conditions. They found that the third and fourth-order polynomial models provided the most consistent predictions of body weight for age 1 and age 2 brown frogs, respectively. Their findings will not only be useful for amphibian research but also for frog farming management strategies and decisions. It is interesting. However, we believe that, if the concerns outlined below can be addressed.
1. I think that authors should read more references of the relationship between body mass/body length and age, especially in Chinese frogs in recent years. For instance, Lu Xin’lab in Wuhan University has published some papers about life-history traits in frogs in recent years. I have added some references related to age-body relationship in the text in attached file.
2. The English expression need improve and some sentences I can not understand means and I can only guess.
3. In the paper, some minor errors in references need corrected and I have marked in text.
Totally, I like this paper and hope that it can be published when my comments have been addressed.
Some questions in attached file nee be also addressed.


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

Input:
Title: MODELLING THE GROWTH OF THE BROWN FROG (RANA DYBOWSKII)
Review round: 2
Reviewer: 1

Output:
Basic reporting: I agree with publishing the paper because it follow my suggestions. There are also some minor questions in references.  Please revise carefully them.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: I agree with publishing the paper because it follow my suggestions. There are also some minor questions in references.  Please revise carefully them.


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

Input:
Title: BREAST FIBROBLASTS IN BOTH CANCER AND NORMAL TISSUES INDUCE PHENOTYPIC TRANSFORMATION OF BREAST CANCER STEM CELLS: A PRELIMINARY STUDY
Review round: 1
Reviewer: 1

Output:
Basic reporting: Comments
The manuscript by Bixiao wang et.al aimed to address the role and effect of NFs and CAFs in the phenotypic transformation of BCSCs. There are some technical concerns which limit the originality of the data presented.
	The authors mentioned about the use of CD44+CD24- BCSCs and association of BRCA1 mutational status. There are several controversies regarding this. It has already been reported by Somasundaram et al., 2016 that the best stem cell marker for BRCA1 mutated cancer would be ALDH1 and not CD44+CD24-
	The authors failed to put the reference appropriately in the Materials and Methods (line 141) for mammosphere culture and dissociation.
	Moreover, the histological type of the tissue was shown infiltrating ductal carcinoma initially and later on in discussion part the authors mentioned that the samples were collected from breast cancer patients of mammary gland hyperplasia which actually lacks the clarity about the type of tumor analyzed and creates confusion.

Experimental design: There is a procedural problem in preparation of Condition Media (CM) as the authors are adding 10% FBS to the CM before use which actually dilutes the properties of CM and the addition of growth factors seem to be unnecessary.

The methods described were not listed properly and they did not to specify which is the control medium used.

Table-1 regarding the clinical information described in Materials and Methods shows the uneven hormonal status of the samples used for the study. All the samples should be of same kind eg. either triple negative or triple positive etc. This is because only a few samples are analyzed in the study.
Validity of the findings: In the preliminary experiment, to distinguish between CAFs and NFs, the authors have analysed the expression status of αSMA. Since αSMA is not a conclusive marker to differentiate both, more marker genes should be included and analysed for a better confirmation. Also the IHC of αSMA shows non-specificity which could be a procedural problem (Figure -1).

The validation of heterogeneity among CAFs and NFs has already done and reported, so further validation of the same in the study is not relevant in both results and discussion.
Discussion is too long. It can be summarized with more clarity and should be precise.
Additional comments: As indicated above


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

Input:
Title: BREAST FIBROBLASTS IN BOTH CANCER AND NORMAL TISSUES INDUCE PHENOTYPIC TRANSFORMATION OF BREAST CANCER STEM CELLS: A PRELIMINARY STUDY
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript describes interesting approach in the study of the role of CAFs and NFs in the breast cancer stem cells phenotype. Article structure, language and figures are of good level.
According to the authors, the experiments aimed “to explore the similarities and differences between BCSCs that are induced by CAFs and NFs from a functional and observational perspective to lay the foundation for further mechanistic exploration”.  The main questionable points are:
 -What is the direct evidence of BCSCs induction by CAFs and NFs?
- The effects of conditioned medium from CAFs and FNs on MCF-7 showed increased number of mammospheres compared with controls. What is the meaning of a high number of mammospheres? Since the initial cell number is the same, did CM induce cell proliferation or did inhibit cell dead?
Experimental design: They used cultures of cells from 6 breast cancer patients and only in the protein expression analysis they compared the CAFs and FNs from all six. KEGG pathway analysis identified the major biological pathways in the 2 types of cells, showing that they were different in spite of both being positive for α-SMA. It would be very interesting to discuss in deep the meaning of protein expression in relation to cell phenotypes. For example, why the pathways up-regulated in CAFs were associated with intercellular adhesion and connection? Are these pathways associated with the formation of the cytoskeleton only in CAFs?
Minor observation:
Fig 1B- Are they both CAFs and NFs in same magnification?
Line 402- “Fibroblasts are the predominant cell type in the extracellular matrix.” Please correct to: Fibroblasts are the predominant cell type in the connective tissue or in stroma
Validity of the findings: no comment
Additional comments: No additional comments


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

Input:
Title: BREAST FIBROBLASTS IN BOTH CANCER AND NORMAL TISSUES INDUCE PHENOTYPIC TRANSFORMATION OF BREAST CANCER STEM CELLS: A PRELIMINARY STUDY
Review round: 2
Reviewer: 1

Output:
Basic reporting: The Authors have incorporated the recommendations by the reviewer in the revised manuscript.
Therefore the manuscript may be accepted in the journal Peer J.
Experimental design: I appreciate for the authors for properly designing the experiments.
Validity of the findings: Revised manuscript has all the recommendations incorporated in their finding.
Additional comments: The manuscript may be accepted in the journal Peer J.


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: BREAST FIBROBLASTS IN BOTH CANCER AND NORMAL TISSUES INDUCE PHENOTYPIC TRANSFORMATION OF BREAST CANCER STEM CELLS: A PRELIMINARY STUDY
Review round: 2
Reviewer: 2

Output:
Basic reporting: The revised version of manuscript is improved by the inclusion of some missed informations, mainly in M&M, and by the discussion reformulation, as well. Detailed comments on the reviewer’s questions are in the rebuttal letter, however some of them are are not clear enough. In replying to my questions, they claimed that “….the number of mammospheres in the groups with CM-CAFs and CM-NFs were obviously higher”. Sorry, these data are not a direct evidence of BCSC number. The efficiency of mammosphere formation is a complex index resulting from several factors. For example, an increasing in mammosphere formation may represent  low apoptosis ration,  increased proliferation of early progenitor cells or stem cells, or  an increase in stem cell self-renewal.
Absolutely dispensable the inclusion of Figure 1S.
Please, check the English .
Experimental design: no comment to add
Validity of the findings: no comment to add
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: LATENT PROFILES OF ELITE MALAYSIAN ATHLETES’ USE OF PSYCHOLOGICAL SKILLS AND TECHNIQUES AND RELATIONS WITH MENTAL TOUGHNESS
Review round: 1
Reviewer: 1

Output:
Basic reporting: There are some theoretical backgrounds are not so clear as follow.

First, I am confused by authors’ definitions of psychological skills and techniques on line 58-61. The authors’ defined psychological skills as “...encompass developmental targets or desired attributes that are central to high performance...” I don’t understand what this means. According to Oxford dictionary, skill is defined as “the ability to do something well.” The Collins Dictionary, skill refers to “... a type of work or activity which requires special training and knowledge...”

As to technique you defined as “... capture the methods or processes by which individuals attain and sustain such outcomes (e.g., self-talk, imagery). Again, the Oxford dictionary defined it as “A way of carrying out a particular task, especially the execution or performance of an artistic work or a scientific procedure.” The Collins Dictionary defined technique as “... a particular method of doing an activity, usually a method that involves practical skills...”

By your definition of skills and techniques, I don’t understand what is (are) your research questions. Also, from your methods and materials section, you used “TOPS” to measures athletes PSTs in training (i.e., goal setting, self-talk, imagery, attention control, emotional control, activation, relaxation, automaticity) and competition (goal setting, self-talk, imagery, negative thinking, emotional control.) Are these psychological skills or techniques?

Second, you have a false citation. On line 62-63, you cited Van Yperen and colleagues (2014) support the importance of psychological skills on high performance. How come this is true because Van Yperen et al., 2014 talked about achievement goals.

Third, you said that mental toughness is “state-like in that it has properties that can vary or endure across situations and time...” If this is true, it seems contradictory against Jones’ definition (2002) that mental toughness is “the natural or developed psychological edge...” Also, by your methods and materials section, you used Gucciardi and colleagues (2015) measure to assess participants’ mental toughness. The items you demonstrated (e.g., I strive for continued success and I am able to regulate my focus when performing tasks) are trait or disposition.

Fourth, the link between PST and mental toughness needs to be a further explanation on line 116-117. You simply stated that “...one or two PST are most salient to mental toughness...” By what? Why? Please explain more.
Experimental design: 1.	You said you sampled 285 Malaysian elite athletes participated in your study. How elite are they? What are their elite records? Please explain more.

2.	Your participant's age ranged from 15-44 years. How come 44 years are still engaged in competitive sports. How many percentages they are represented in the whole sample. Please show the describe it.

3.	Before doing your main statistical analyses, did you find any outliers, skewness, the kurtosis of your study? What was the normality of your data?

4.	I suggest you use traditional format to present your methods and material, such as participants, measurements, procedures, statistical analyses...
Validity of the findings: The general findings are significant and meaningful. But I found on line 213 you mentioned: “...high adaptive PSTs use athletes reported significantly higher levels of mental toughness...” What means to high adaptive PSTs stands for? Please explain it.
Additional comments: The paper is thoughtful but needs lots of revisions to make this paper acceptable.


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: LATENT PROFILES OF ELITE MALAYSIAN ATHLETES’ USE OF PSYCHOLOGICAL SKILLS AND TECHNIQUES AND RELATIONS WITH MENTAL TOUGHNESS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Comments provided in text.
Experimental design: Comments provided in text.
Validity of the findings: Comments provided in text.
Additional comments: This article used LPA to identify underlying classes of athletes who vary in their use of PSTs. These profiles were then used to predict mental toughness. The manuscript is well written and tackles a novel area of the literature using an analytical technique that is quite popular at the moment. I do, however, have some reservations about the paper in its current format and have made comments/suggestions for consideration.

Introduction
1. Lines 71 to 73: Could the authors clarify what they mean by “whereas the skills and strategies of self-talk and emotional control distinguished these two groups of performers in terms of practice settings (Taylor, Gould, & Rolo, 2008).”
2. Lines 76 to 78: Similar to the above, it’s not clear what is meant by “in contrast, goal setting, imagery and relaxation contributed most to these distinction between these two groups in practice contexts (Tanaka & Gould, 2015).” Details on the above would help to support the statement in lines 79 to 80 suggesting that athletes who are more successful tend to use PSTs more frequently.
3. Line 77 to 78: Consider rephrasing the sentence.
4. Lines 88 to 90: It would be useful to see this statement expanded upon “However, this assumption may be erroneous as the development and maintenance of PSTs typically incorporates both group-based and individualized components (for examples, see Dosil, 2006).” Can the authors elaborate on an example of what they’re referring to?
5. Lines 100 to 114: More detail needs to be provided about mental toughness, including the importance of the construct for sport (e.g., performance), perspectives on dimensionality, major developments, etc. There have been several reviews published in 2017 that the authors could draw on in this regard.
6. Some detail on the types of psychological skills and techniques associated with mental toughness would be useful in the section on PSTs and mental toughness.
7. Lines 126 to 129: I disagree that omitting hypotheses is the best approach. Based on the literature you present and what we know about mental toughness, surely an argument could be made about the types of PSTs you would expect mental toughness to correspond with?

Method
1. Lines 137 to 151: Evidence on the reliability and validity of the measures that were used needs to be included, especially with respect to cross-cultural applicability.
2. It’s difficult to see how a person with a year of experience in their sport could be part of a “national squad.” While this may be true, the authors should elaborate on the criteria that athletes needed to meet in order to be included on these national squads.
3. What about parental consent for participants that are not legally considered adults? Was this part of the ethical clearance submission (i.e., did the authors get ethical clearance to collect data from minors?)? Did parents’ consent to their children participating in the study (based on the IC document attached, it doesn't seem that parental consent was obtained)?
4. In the informed consent document that outlines the measures participants completed, there is no reference to the TOPS in the alphabetized list. Did the authors obtain clearance to administer this inventory?

Results
1. Lines 192 to 193: What specifically are the authors referring to when they state “…. the degree of improvement plateaued (Morin et al., 2011)?” This is quite a subjective statement and requires further clarification (perhaps more detail would be useful).
2. Lines 196 to 198: The authors make the following statement: “Finally, the average probability that participants were correctly classified in the given latent profile or misclassified provided additional evidence for the superiority of the 3-class solution in practice (>.85) and competition settings (>.92),” but this is only interpretable alongside the average probabilities associated with other class solutions. I suggest including these in the table along with the model fit indices.
3. It would be useful to have confidence intervals reported for all zero-order correlations.
4. Some of the internal consistency estimates are low (< .70). Could the authors elaborate on how they determined the internal consistency of these scales to be adequate, particularly in light of the fact that information about the reliability and validity of the instruments is not reported in the method section?
5. Why did the authors not test for potential confounds (such as age, playing experience, sex) and control for them where relevant? This is particularly relevant considering the extensive age range and length of participation range quantity discrepancy in the current sample, which seems an important issue to elaborate on when reporting the results.

Discussion
1. The authors report only one study has looked at mental toughness and PSTs. While this may be accurate, the discussion could elaborate on broader research that has indirectly found mental toughness is related to various types of PSTs.
2. Lines 269 to 271: This statement is quite speculative; the cross-sectional nature of the data cannot definitively imply PSTs as precursors of mental toughness (they could be outcomes too). Some revision to the phrasing here seems necessary.


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: LATENT PROFILES OF ELITE MALAYSIAN ATHLETES’ USE OF PSYCHOLOGICAL SKILLS AND TECHNIQUES AND RELATIONS WITH MENTAL TOUGHNESS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The revised version has improved much comparing to the first draft. Especially, I concerned with the rationale about the skills and techniques, as well as the association between psychological skills and mental toughness. The revised version addressed these very well.
Experimental design: Research questions are clearly stated, and the methods are reasonable and acceptable.
Validity of the findings: The specific contribution of this study was to adopt a person-centered approach to analyze the different profiles of PST used in training and competition, and their differences in metal toughness. Results not only found different profiles of PST used in training and competition but also found high level of PST use was associated with higher level of mental toughness. Results bring significant contributions to the field of sport psychology. Also, the research approach is inspiring for future researchers.
Additional comments: Congratulations to successfully accomplished a significant paper that address the relationship between PST and mental toughness. Results were inspiring.


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: LATENT PROFILES OF ELITE MALAYSIAN ATHLETES’ USE OF PSYCHOLOGICAL SKILLS AND TECHNIQUES AND RELATIONS WITH MENTAL TOUGHNESS
Review round: 2
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: Overall, I thought the authors did an excellent job with the revisions. It was great to read the revised version. That said, I still have a few reservations that I think need to be addressed before publication. My comments are below.

Introduction

Lines 116-120: It’s noted the authors have attempted to address the issue surrounding mental toughness and performance. The references provided, however, could better reflect the breadth of literature available rather than focusing on two major groups of researchers alone. There are at least two reviews on mental toughness and achievement/performance, one in sport and one in more general contexts. A review along these lines is certainly preferred over single papers that address the same issue. The two articles I refer to are below; at least one should be mentioned in light of the statement on mental toughness and performance.

Cowden, R. (2017). Mental toughness and success in sport: A review and prospect. The
Open Sports Science Journal, 10. doi:10.2174/1875399X01710010001

Lin, Y., Mutz, J., Clough, P., & Papageorgiou, K. (2017). Mental toughness and individual differences in learning, educational and work performance, psychological well-being, and personality: A systematic review. Frontiers in Psychology, 8, doi:10.3389/fpsyg.2017.01345

Results

Lines 213 to 215: A reference is required for statements about acceptable levels of normality and outlier detection cut-offs.

Lines 241 to 247: The additional analyses predicting class membership are interesting and as one might have expected. This doesn’t necessarily negate the issue of needing to control for at least some of these variables in the analysis involving mental toughness (the most important outcome variable). How do we know that the profiles show differences in mental toughness when you control for relevant variables such as playing experience? I didn’t pick up in the text where the authors had reported controlling for some of these demographics in this analysis. At a very minimum, the authors should check and report the relationships between mental toughness, age, sex, and playing experience. If none are associated with mental toughness, one could see an argument being made for omitting them from the analysis involving mental toughness. Without reporting these the reader is left wondering what effect the large age and playing experience ranges, in particular, have on the outcome of the analysis with mental toughness.


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: PROTECTION OF CULTURED BRAIN ENDOTHELIAL CELLS FROM CYTOKINE-INDUCED DAMAGE BY Α-MELANOCYTE STIMULATING HORMONE
Review round: 1
Reviewer: 1

Output:
Basic reporting: excellent study
Experimental design: excellent design
Validity of the findings: novel and inovative
Additional comments: Harazin and coauthors have investigated the protective effect of alpha-MSH on brain endothelial cells and in vitro BBB model system. They found that alpha-MSH attenuated the cytokines –induced- BBB permeability, reduced production of reactive oxygen species, and NF-kB translocation.

This is one excellent study.  In general, I found the manuscript well prepared and study is carefully designed and mapped out.  Data are convincing, well presented and discussed. I did not have any specific concerns regarding 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: PROTECTION OF CULTURED BRAIN ENDOTHELIAL CELLS FROM CYTOKINE-INDUCED DAMAGE BY Α-MELANOCYTE STIMULATING HORMONE
Review round: 1
Reviewer: 2

Output:
Basic reporting: In this study ‘Protection of rat brain endothelial cells from cytokine-induced damage by alpha-melanocyte stimulating hormone’ by Harazin et al., the authors established an in vitro culture model of BBB and studied the protective effects of alpha-MSH on the integrity of BBB from cytokines IL-1beta and TNF-alpha induced damage. The study’s background, experimental flow and data analysis were clearly reported, and appropriate publications have been cited to support the author’s work.
Experimental design: The idea of using alpha-MSH to protect BBB from inflammation-induced damage is in general well tested in vitro. However, there are some limitations regarding the experimental set up and design. First, the authors used an overly simplified model of just two cytokines at one dose to mimic neuroinflammation. It is unclear why a fixed dose of cytokines were chosen without titration, and why the authors only included two cytokines while in reality many cytokines are involved in neuronflammation (i.e IL-6, IL-18, IL-33). The authors should confirm their studies with a more comprehensive cytokine cocktail and also try a series of doses to demonstrate the true efficacy of alpha-MSH.

The in vitro protective effect of alpha-MSH was modest in most experiments, therefore one would question its efficacy in vivo, when delivery route and other barriers may further limit the final amount of alpha-MSH that could actually exert effect on brain endothelial cells. The authors should set up at least some in vivo assays to show the protective effects of alpha-MSH in vivo. In addition, as the authors mentioned alpha-MSH has receptors on different tissues, the authors should carefully address the potential issue of toxicity/off-target effect of using alpha-MSH in vivo.
Validity of the findings: As mentioned above, the neuronal protective effect of alpha-MSH was modest in most experiments. Especially in the first in some assays, the effects at higher 10uM concentration were actually more modest than those at a lower dose (1uM) (i.e. data in Figure 3). The authors should at least discuss why alpha-MSH effect did not correspond well with the applied dose.

Figure 1: the authors only showed the expression of MC1R on cells cultured in vitro, which is somewhat artificially and could be modulated by culture conditions. It is better to actually do IHC or IF on tissue sections to demonstrate MC1R expression in vivo.

Figure 5: The difference of barrier integrity as shown by claudin-5 and beta-catenin expression as shown was at best modest. It is hard to tell the difference by looking at the picture and find where the junction was breached. It would be much better to quantify the data and point out where the damages are in the picture. Also the authors need to clarify how many pictures they actually quantify, more than one experiments should be done.
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: PROTECTION OF CULTURED BRAIN ENDOTHELIAL CELLS FROM CYTOKINE-INDUCED DAMAGE BY Α-MELANOCYTE STIMULATING HORMONE
Review round: 1
Reviewer: 3

Output:
Basic reporting: The article 'Protection of rat brain endothelial cells from 1 cytokine-induced damage by α-melanocyte
stimulating hormone' by Harazin et al, have explored the inhibitory role of MSH in neuroinflammation specifically in the context of blood brain barrier. In my opinion, the authors report important and translationally relevant implications of this biology that will be of interest to the field in general. The observations appear to be robust and have been effectively described. Overall, the link between MSH and blood brain barrier protection has been established through well-designed experiments in multiple settings, suggesting a general role for MSH in regulating blood brain barrier function. I have no major concerns. I strongly suggest the manuscript for publication.
Experimental design: No comment
Validity of the findings: No comment
Additional comments: No additional comments


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

Input:
Title: UNFOLDING OF Α-HELICAL 20-RESIDUE POLY-GLUTAMIC ACID ANALYZED BY MULTIPLE RUNS OF CANONICAL MOLECULAR DYNAMICS SIMULATIONS
Review round: 1
Reviewer: 1

Output:
Basic reporting: This is a tedious but useful study of the 20-residue poly-glutamic acid.
The poly-glutamic acid may come with various lengths and its properties depend on the length. There is no mention of this in the manuscript. One paper that should be referred to in this context is Gomez-Sicilia et al. PLoS Comp. Biol. 11: e1004541 (2015). The authors comment about the necessity of having force fields that would be more adequate for the IDP systems. There is already at least one such a force field available: Huang et al. Nature Methods 14: 71 (2017). At length 20, the system is also an IDP but these aspects are not characterized adequately. One way to do it is to provide values of the average end-to-end distance, its dispersion, and the average radius of gyration. The time-dependent structure has other interesting features than merely the secondary-structure content.
Experimental design: The theoretical set-up is appropriate.
Validity of the findings: The findings appear to be valid but limited.
Additional comments: Same as in point 1.


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: UNFOLDING OF Α-HELICAL 20-RESIDUE POLY-GLUTAMIC ACID ANALYZED BY MULTIPLE RUNS OF CANONICAL MOLECULAR DYNAMICS SIMULATIONS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Clear and unambiguous, professional English used throughout.

Literature references, sufficient field background/context provided.

Professional article structure, figs, tables. Raw data shared.

Self-contained with relevant results to hypotheses.
Experimental design: Original primary research within Aims and Scope of the journal.

Research question well defined, relevant & meaningful. It is stated how research fills an identified knowledge gap.

Rigorous investigation performed to a high technical & ethical standard.

Methods described with sufficient detail & information to replicate.
Validity of the findings: Data is robust, statistically sound, & controlled.

Conclusion are well stated, linked to original research question & limited to supporting results.
Additional comments: The manuscript studies helical unfolding or helix/coil transition of a 20-residue Polyglutamic acid (PGA) in an acidic environment through explicit canonical (NVT) MD simulations to verify with two previously reported helical structures of PGA: single alpha-helix or short alpha helices separated by coil. The authors ran simulations with two different alpha helical conformations as starting structure – one with a helical conformation obtained from their previous MD study and the other, which they refer to as ideal alpha helix, was curated with all backbone phi and psi angles in the helical range. A total of 8 and 20 simulations with different starting velocities were run with the helical structure obtained from their MD study and with the ideal helical structure respectively. Among these, 3 simulations with the MD-obtained helix was run for 3 microseconds to account for long-term dynamics, the rest being all 400 ns each. The authors observed different unfolding times for the first three simulations, with transient helix formation after the first unfolding event, except for one of the simulations. The majority of simulations showed helix-coil transitions starting from both the termini and extending to the middle segment, and that the turn conformations were highly coupled with such transitions as obtained from their ensemble averaged transition probabilities starting from the ideal helix. They also obtained coupling of the 3-10 helices with alpha helices. They claimed that both the turns and 3-10 helices as kinetic intermediates in the helical unfolding pathway.
Overall, the manuscript reports well on conformational changes on a set of long simulation results from two different structures of a single protein, which is extensive. The secondary structure transition probability analyses, although ensemble averaged is satisfying. However, there are few things that may need to be clarified. Firstly, in the simulation method, they have used ensembles generated by NVT. There must be a good reason for this. In order to model conformational changes, that is helix-coil transitions in this case, it may be a good idea to use NPT as most experimental measures are obtained under constant pressure. This makes more sense in the light of their introduction to the topic, where the authors indicate towards verifying their findings with previously reported FTIR and CD spectroscopy. Secondly, in Figure 2, which shows the cumulative helix averages over the 3.0 microseconds runs (Sim1, Sim2 and Sim3) with same initial structure, but different starting velocities record quite different helical probabilities: especially with Sim 1 there is a sudden increase in helices between 0.5 and 1.5 microseconds. The authors need to give better explanation (rather than sampling time) why such a trend is seen with one simulation (may be some close side chain contacts stabilizing the helices?), but not the other two, although they have noted this in the text (lines 156-159). Finally, the claim in the conclusions and the abstract that turn and 3-10 helices are kinetic intermediates in the helix-coil transitions are based only on 3 (N-term, C-term and middle) out of a total of 20 residues in their analyses. In order to gain statistically significant numbers (and the kinetic intermediate claim), one has to consider the evolution/probabilities of at least 11 out 20 residues (>50%).


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: UNFOLDING OF Α-HELICAL 20-RESIDUE POLY-GLUTAMIC ACID ANALYZED BY MULTIPLE RUNS OF CANONICAL MOLECULAR DYNAMICS SIMULATIONS
Review round: 2
Reviewer: 1

Output:
Basic reporting: I am satisfied with the revised version of the manuscript.
Experimental design: Appropriate.
Validity of the findings: Appear to be valid.
Additional comments: The revised version is ready for publication.


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

Input:
Title: AN EXAMINATION OF THE IMPACT OF OLSON’S EXTINCTION ON TETRAPODS FROM TEXAS
Review round: 1
Reviewer: 1

Output:
Basic reporting: General comments: This manuscript investigates evidence for “Olson's extinction” and apparent decline in tetrapod diversity near the end of the early Permian. The reality of this extinction has been debated for some time based on potential problems associated with differences in geographic sampling, binning of samples, and correlations among different rock units (and with the SGCS). This paper attempts to deal with these issues by focusing on diversity patterns from just one geographic area (the classic Permian redbeds of Texas), utilizing a specimen-level dataset. In many ways it is a follow-on and response to Lucas' (2017) paper in Earth-Science Reviews, which looked at a similar question but using a less detailed data set and without attempting to control for sampling effects. The result here suggest that there is indeed a decline in tetrapod diversity, and an increase in extinction rates, at this time, regardless of how samples are binned temporally and whether sampling is controlled for. I found the paper to be very clear and well-written, and I think the analyses are convincing. I have a few comments and questions below, but I think most of them should be fairly easy to address. Of these, I think the most important are 1) why the older Plummer and Moor lithostratigraphy is used instead of the more recent (and arguably more directly applicable to the specific rocks in question) revision of Hentz (1988); and 2) how the seemingly very precise numerical ages were assigned to the beginnings and ends of the LVFs. The author says that the dates were taken from Lucas (2017), but as far as I can tell he doesn't assign numerical ages to the the boundaries in either his Earth-Sciences Reviews paper or his Geological Society of London book chapter. Overall, I think the paper should be acceptable with minor revisions.
Experimental design: see comments to author
Validity of the findings: see comments to author
Additional comments: Abstract first sentence: ...is not actually a sentence.

Line 31: whereas instead of while

Line 35: I don't know if I would characterize the transition Olson described as resulting in more trophic levels. It's more of a transition in the main path of energy flow in the terrestrial component of the ecosystems, with terrestrial productivity being passed more directly to terrestrial secondary consumers via tetrapod (and/or insect) herbivores.

Line 57: whereas instead of while

Line 59: The shift in geographic areas of fossil preservation going from the early to middle Permian doesn't seem like the key issue in figuring out the timescale of the event. Instead, the lack of radiometric dates for key formations on either side of the event is the most significant hindrance.

Line 74: remove the before extinctions

line 75: change they to he

Line 77: I recommend starting a new paragraph here. The discussion of the debate over the age of the formations seems distinct enough to warrant it.

Line 112: previous instead of previously

Line 122: General nitpicky semantic comment: data are plural, so I recommend chaning your verbs to reflect this throughout the manuscript

line 124: change on to in

Line 133: Because your dataset includes numbers of specimens, it can address how much of a problem Lucas' concern with singletons might be. Therefore, I think it would be useful to provide a couple of summary statistics about this (e.g., percentage of taxa in your dataset that are known from just one specimen, percentage that just occur in one stratigraphic or biochronological unit), probably at the start of the results.

Line 140: It might be worth noting that they are biostratigraphic bins that are essentially based on the section in which you're working. As such, they likely capture details that might be obscured in the global timescale, which is patched together with information from various geographic areas and environments.

Line 152: I'm not sure if you provide code for the analysis in your in press Evolution paper. If you don't, you should consider doing so here (and you may want to even if you do include code in the Evolution paper).

Line 158: These lithostratigraphic units certainly provide a good connection with historical research in the area. However, Hentz (1988) proposed a revised lithostratigraphy that applied more directly to the terrestrial strata. Is there a reason that you didn't use that? Even if it is something as simple as Hentz's scheme not providing many more bins than the LVF binning scheme, you should note this.

Line 168: A few comments here. First, the paper you cite for the ages is Lucas' Earth-Science Review paper from 2017, but that paper doesn't delve into the details of how the correlations between his LVFs and the SGCS were determined. Instead he refers readers to his 2017 Geological Society of London book chapter, which does provide the details. Since the source of the ages is critical to your inferences about rates, I think it would be good to cite this paper as well. Second, the it is perhaps worth noting that Lucas' ages for the Texas LVFs are mostly based on (marine) biostratigraphy and radiometric ages from mostly geographically distant areas. Third, Lucas doesn't specify exact numerical dates for the start and end of his different LVFs, so it would be useful to describe how you determined the seemingly high-precision dates that you present in the supplement, and which are presumably bound up in the rates that you calculated.

Line 187: estimated instead of estimates

Line 196: again, consider presenting code in the supplement

Fig. 1: The lettered labeling for the parts of this figure seem awkward. I don't think the dendrogram needs a label, and I would change the labels on the time scale to SGCS, this study, and Lucas (2017), respectively.

Line 208: Do you think the change in the placement of the boundary of the Coyotean here stems at all from the fact that the LVF is defined based on a fauna from New Mexico, whereas the others in this sequence are defined based on faunas from Texas?

Line 212: I think you mis-characterize the results in Fig. 2 a bit here. Yes, there is a substantial drop between the Redtankain and Littlecrotonian when data are binned at the LVF scale, but your finer binning schemes seem to indicate that most of that drop was in the Redtankian, and potentially quite early in the Redtankian, such that the observed low diversity of the Redtankian is more a continuation of an established parttern than something new.

Line 232: there seems to be something missing from this sentence. I assume you mean something like: “Having argued against an extinction of tetrapods at the Kungurian/Roadian boundary (due to...”

General comment on the discussion: Is it worth considering how origination rates changed across this time as well? Lucas (2017) talks a little bit about this, and suggests that origination rates were somewhat elevated in the Redtankian and Littlecrotonian. It would be interesting to see what the patterns look like given your revised/finer scale binning. Your argument of a mass extinction would also be further reinforced if you could show a decrease in origination rates corresponding with an increase in extinction rate.

Line 331: you should note here that this is observed in the Texas section

Ken Angielczyk


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: AN EXAMINATION OF THE IMPACT OF OLSON’S EXTINCTION ON TETRAPODS FROM TEXAS
Review round: 1
Reviewer: 2

Output:
Basic reporting: REVIEW OF :
An examination of the Impact of Olson’s extinction on Tetrapods from Texas – by N Brocklehurst

The matter of the validity and impact of Olson’s extinction on early- middle Permian tetrapod faunas has been the subject of much debate in the scientific literature but there has not been clear resolution largely because of the geographically disparate nature of the sedimentary successions hosting relevant fossil faunas. The subject is important as this is the first large extinction event affecting tetrapod faunas in the continental realm.

This paper examines the tetrapod fossil record from different formations of the Texas sedimentary succession, arguably the temporally most complete Early Permian succession, at a finer stratigraphic level than that used in previous analyses. Species richness in the different formations has been determined using four different binning schemes. The author convincingly makes a case that extinction rates are greater than background rates, but the peak occurs in the Choza Formation. He concludes that Olson Extinction is a real event and is not an artefact of binning methodology. In my opinion this is a useful paper and should be published.

Basic reporting

The paper is well written with good language usage, and the structure conforms to PeerJ standards.  The abstract is concise, and the introduction provides a comprehensive literature review setting out the problem which is being addressed. The Results and Discussion are appropriately presented. I suggest that the Conclusion needs to be expanded to set out what has been achieved by this paper.
Literature is well referenced, and the figures are all relevant. The raw data is adequately presented in two tables providing number of specimens of particular genera and species from individual formations.
Experimental design: The project is not dependant on experimentation. The methodology is well set out and explained and the research question well defined and relevant. The paper sets out the intention to approach the question of the “existence” of Olson’s extinction from a different angel to that previously used by employing a binning strategy of the tetrapod  genera and species from different formations in the fossil record from the Texas stratigraphic sequence. Four methods are used to define time bins which are well explained and carried out. The methodology is well set up and the raw data adequately presented as two tables in subsidiary information. In my opinion the methods are described with sufficient detail to replicate.
Validity of the findings: CONISS clustering of the formations suggest that the Mitchellcreekian be shifted downwards to include the Belle Plains Formation.

By employing the methodology explained using the four time-binning schemes, in the results section the paper convincingly indicates a substantial drop in diversity between the Redtankian and Little Crotonian and shows the peak of taxonomic richness to be in the in the Arroyo Formation.

The paper further points out three peaks in extinction fate (both at genus and species level) at the top of the Belle Plains, Arroyo and Choza formations.
The paper convincingly points out that a substantial tetrapod mass extinction event occurred in the Redtankian and as such this information deserves publication in PeerJ.

The conclusion does not adequately summarise the findings of the paper and should be fleshed out.
Additional comments: I have pointed out various relatively minor editorial modifications with annotations in the attached pdf. The following are specific points which need attention.
L26 – add Permian as a key word.
L 79 – include comas
L 82 – and other places pointed out in the text (Lines 117, 205, 213, 228 ). In formal stratigraphic nomenclature Formation (in this case Blaine Formation) requires an upper case “ F”. if it is used in the plural eg. San Angelo and Chickasha formations then it is lower case ‘f”
L84 – Riesz should be Reisz
L 112 previously studies – shodul be previous studies


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: IMPACT OF FRONT RANGE SOURCES ON REACTIVE NITROGEN CONCENTRATIONS AND DEPOSITION IN ROCKY MOUNTAIN NATIONAL PARK
Review round: 1
Reviewer: 1

Output:
Basic reporting: The paper presents measurements of PAN, alkyl nitrates, NOx, NOy and NH3 from a site in Rocky Mountain National Park, Colorado, during the Front Range Air Pollution and Photochemistry Experiment (FRAPPE) in July-August 2014.  The paper is generally well written and professionally presented.  Sufficient background is provided for the reader to understand the importance and context of the measurements.

The link to the raw data directs you to the general FRAPPE data repository.  Finding the specific data covered in this article is not easy (I managed to find it after some puzzled searching), so I recommend that a more specific link be provided that sends the reader to the specific dataset(s) that are reported here.
Experimental design: The work presented here is original primary research and adds to the general understanding of atmospheric reactive nitrogen concentrations and deposition.  The methods used and data obtained are described in sufficient detail and demonstrate the authors' ability to perform this kind of research at a high technical and ethical level.
Validity of the findings: The findings of this research are well supported although they could be strengthened as described below.  The analyses are appropriate and statistically sound.  Although not earthshaking results, the data and analysis add to the general body of research of atmospheric reactive nitrogen.

The data in Figure 2 could be presented in a more powerful way by creating windrose plots for each of the species shown.  The time dimension of the data would be lost but there doesn't seem to be a substantial temporal variation to the data anyway.  Using windrose plots would effectively highlight how the species concentrations vary as a function of wind direction.  As it is presented now, the reader has to do quite a bit of work to determine what each species concentrations are for a given wind direction (e.g., upslope flow from the southeast).
Additional comments: Editing Suggestions

Line 137:  Be consistent with the format of dates … “July 23 – August 9” rather than 7/23 – 8/9.

Lines 194-197:  It’s not at all clear what days/times on which upslope (from the southeast) flow occurs and so it’s very difficult to correlate the wind direction with species concentrations.  The suggestion above was to create windrose plots of each species.  However, if this is not feasible, at least modify the wind plot of Figure 2 to allow the reader to easily see which wind directions are “upslope” flow.

Line 207: “July 22” rather than “7/22”.

Line 289:  Should be “raise” not ‘raising’.

Line 334:  “We have” not “We’ve”, or better yet “The contribution of dry NO deposition to total N deposition has been neglected, as …”.

Line 366-368:  Suggest the following … “No significant increase in the correlation with any of the RN compounds is observed when moving from a single regression to a multi-linear regression using ethane and C2Cl4.”

Line 439:  Should be “… estimates of deposition of PAN …”.

Figure 7:  The caption panel letterings do not correctly correspond to the figures as shown for (c) and (d), and (e) and (f).

Figure 8:  A different color scale for the MeONO2/EtONO2 ratio might help to make this plot more easily interpreted.  As it is presented now, the very dark color overwhelms the other colors.


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: IMPACT OF FRONT RANGE SOURCES ON REACTIVE NITROGEN CONCENTRATIONS AND DEPOSITION IN ROCKY MOUNTAIN NATIONAL PARK
Review round: 1
Reviewer: 2

Output:
Basic reporting: As I am not a native English speaker and I do not comment the language.
Experimental design: See General comments.
Validity of the findings: no comment
Additional comments: This is a compact MS about RN measurement and I have just minor comments:

Abstract: to A limited set of measurements was extended through October. ( this is a detail not for the abstract)

Indroduction: Information like the instruments and their time resolutions and details about the compounds measured should be written in the methods part, not in the introduction:  ….high time resolution (1 min to 1 hour)… 24 h.. two gas chromatograps…..

Methods
Write more logically and think what is relevant: some methods have more details about where there were situated (like mobile lab?) or about the calibration (pH buffers 4 and 7). Is it possible to have a table of the methods?

Results
Check the %-values in the text, they are not in agreement with the figures (or are the figures correct):

r. 253-  (Alkyl nitrate?) Measurements during FRAPPÉ were consistent with this, contributing 4% to NOy and 2% (I got 2.7% ?) to the total atmospheric RN budget during FRAPPÉ (Figure  4). The measured alkyl nitrates were primarily composed of i-propyl nitrate (26%), methyl nitrate (25%), and 2-butyl nitrate (23%). The remaining 26% were ethyl nitrate (9%), n-propyl nitrate (6%), 2-pentyl nitrate (6%) – in the figure 5%, and 3-pentyl nitrate (5%) – in the figure 6%.

r. 286 …than 2% (ΣR-ONO2 + PAN = 22%) – in the figure 6: 12.8% + 0.4 % ≠ 22%

It would be nice also to see clearly the deposition amounts
not only

Conclusions
r. 406 add FRAPPE 2014 (just easier to remember for the reader.)
r. 425-431	This part there is unclear speculation. Write this part more compact and clear. (other NH3 and RN measurements – which measurements – are beyond this study? Further analysis beyond of this study…)

Details:
Check the heading: sometimes like 2. Methods and sometimes 3.0 Results etc…
Figure 1 (map): add the scale.
r. 53	Elevated nitrogen deposition can alter ecosystems and the services they provide... services?
r. 99	NOy – not explaned
r. 117	NPS/CSU -not explaned?
r. 156-157 non-methane hydrocarbons (NHMCs) – Use NMHC or you do not need to mention this at all because there is already C2-C7 and C6-C10
r. 167	You do not need the abbreviation UHP, because you do not use it in the rest of manuscript.
r. 178	What is NCON? A company?
r. 183	Sample pH was measured after returning to the lab using a combination pH electrode calibrated with pH 7 and pH 4 buffers – too detailed or you should not write how you calibrated all the other instruments.
r. 206	Benedict et al., 2017  - Not mentioned in the reference list (there it is 2018?).
r. 289 fractions observed here raising – are raising?
r. 299	At the measurement site in ROMO average precipitation from July to September for the period of 1995-2016 is 179 mm – so this means the average precipitation for the 3 months period (Jul-Sep)
r. 318	hourly monthly averages?
r. 357	it is used a dry…
r. 439	This is the first study in ROMO that includes estimates deposition of PAN – estimated or estimates of PAN deposition..
r. 401 (Gschwend, MacFarlane & Newman, 1985; - should be
Gschwend et al. 1985

Figure 2. Precipitation: I have difficulties to see the green line close to the black one (WD).


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: IMPACT OF FRONT RANGE SOURCES ON REACTIVE NITROGEN CONCENTRATIONS AND DEPOSITION IN ROCKY MOUNTAIN NATIONAL PARK
Review round: 1
Reviewer: 3

Output:
Basic reporting: In their manuscript, Benedict et al. describe measurements on reactive nitrogen (RN) species during a field study (i.e., FRAPPÉ) in summer 2014 in the Rocky Mountain National Park. In particular, they measured PANs, C1-C5 alkyl nitrates, NOx, NOy, and ammonia. Additionally, VOC measurements were conducted, using in situ GC-ECD and GC-FID. From these measurements, the authors conclude that concentrations of RN mostly correlate with emissions from nearby oil and gas operations as well as emissions from agriculture and urban areas. For some periods, long-range transport is also suggested as source of RN in the study area. Moreover, the authors calculate an atmospheric RN budget, including a nitrogen deposition budget for dry and wet removal processes.

The language quality is high and the author discuss their findings mostly in a clear way. Nonetheless, I would suggest shortening some rather long sentences and rephrasing certain sections, to improve the clarity of the manuscript (see general comments).
The article includes sufficient introduction and background and generally references relevant literature (see the general comments for some further suggestions).

All figures presented are relevant, high quality, and well described.

Raw data is not supplied with the manuscript and can ,therefore, not be assessed.

I would suggest publication of the manuscript after major revisions of the findings, as stated in the comments below.
Experimental design: The research question is well defined and the experimental setup and design appropriate to answer it. All methods applied are well described with sufficient detail to replicate such measurements.
Validity of the findings: Figure 1: The source of the data shown in this map remains unclear.
L207-211 / Figure 2: Please give more evidence to support your suggested correlation between PAN, RONO2, ethane, and C2Cl4. I would suggest depicting the relationships in a correlation plot rather than in a timeline plot. For the reader, it is hard to tell from a timeline if the signals really correlate or not.
Figure 3: Instead of showing every data point, I would suggest to show the standard deviation of the hourly averages in each panel. This would substantially improve the readability of the figure and make it easier to the reader to assess the total variability.
L216 / Figure 3: I cannot see a peak in ammonia for the early morning hours in Figure 3! From the depicted data, I would actually say that there is a peak between 2-4 PM. Thus, your suggested explanation that this peak reflects volatilization from evaporating dew cannot be correct.
L221: I do not understand why you remove all data for ammonia from 6-10 AM. Even if there was an early morning effect, it would not be a measurement artifact, but reflect actual RN concentrations in the air. This point is crucial, since it affects the entire data set and calculations of the study.
L245-249: If you state that “overall partitioning […] is consistent with previous years”, you have to show it either by giving references or by showing the data. Moreover, it remains unclear, which studies you are discussing when it is stated “All four studies […]” (L247) and “the other three studies” (L249).
L263: To me it seems very interesting that you can measure such long-range transport of marine tracers. However, marine sources are not the only possible explanation for the abundance of CHBr, as you also admit later in section 3.4 (see L399). Thus, I would strongly recommend adding backward trajectories (e.g., from HYSPLIT) to demonstrate the origin of the air masses arriving at the site.
L283: It would be interesting to know a bit more about the uncertainty range in these calculations.
L295-298: This (very long) sentence remains completely unclear to me. What message do you want to convey here? Which other studies do you mean?
L299-308: The discussion of the precipitation results seems incomplete to me. Do you see any other correlations? Why was 2015 different?
L318: What do you mean by “historical data”? Why do use a scaling factor of 0.7?
L328: If there are more appropriate values published for PAN deposition velocities (Wolfe, 2009), why do you use others?
L340: As already in L318, why do use this scaling factor?
L362-364: I am not convinced of this suggested correlation. For NOy, I think it is reasonable to state that there is some significant correlation with CCl4. However, for PAN it seems rather ambiguous whether the correlation is significantly stronger with ethane (R2=0.27) or with CCl4 (R2=0.35). Actually, I would say there is neither for ethane nor for CCl4 a good correlation to PAN.
L367: If there is no benefit from a multilinear regression, it is not necessary to discuss this. Please remove it also from table 2, since this only creating confusion.
L379: To me it does not look solely like scatter for the data below 2 ppb. In general, the ratio of i/n butane seems to be significantly larger for lower concentrations for any of the measured species. Is there any explanation for this?
L382-384: Where is the difference between PAN/NOy and the alkylnitrates? If I understand it correctly, all species exhibit i/n butane ratios similar to the DJ Basin.
Fig 7: In general, I would guess the strongest correlation here is observed for the i/n butane ratios and the wind direction. This should be pointed out more clearly in the discussion of the figure.
Fig 8: Units are missing in all panels!
L394: Units for CHBr are missing!
L390-394: I think you have to be careful here with the interpretation. As suggested above, it would be really beneficial to see some backward trajectories, since CHBr might also have other sources.
Additional comments: L195: I would suggest giving a wind direction instead of using the word “upslope”, since this seems a bit ambiguous. I would suggest changing this throughout the entire manuscript.
L200: What previous work? Please give a reference or show the data.
L282: I would recommend using SI units, i.e., kg N / m2
L288f: This is to general. Either you discuss possible future research directions or you better remove it.
L312: What is Vd?
L333: What other studies were publsihed?
L373 & Fig. 7: Is it Piceance or Pieance? The name in the figure differs from the one in the text.
L382 & Fig 7: I would suggest adding the letters of the panels to the text, where they are discussed.
L426-433: I would recommend removing the discussion on preliminary results from the conclusion, since you do not show any of these data.
L435: What previous work?


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: KNOWING YOUR NEIGHBOURHOOD—THE EFFECTS OF EPICHLOË ENDOPHYTES ON FOLIAR FUNGAL ASSEMBLAGES IN PERENNIAL RYEGRASS IN DEPENDENCE OF SEASON AND LAND-USE INTENSITY
Review round: 1
Reviewer: 1

Output:
Basic reporting: Some corrections of the English text are indicated in the revised pdf file.
Experimental design: The main objective of the study is to determine if the presence of Epichloë in plants of Lolium perenne affect the structure of other components of the fungal mycobiota of the plants. To answer this question, an indirect method of fungal detection and identification based on NGS has been used. To my knowledge, this an innovative approach not yet used to test the above hypothesis.
Comments to make more clear the description of  some methods have been made in the annotated pdf file.
Validity of the findings: Some remarks to improve the findings of the study:

1. The listing of the fungal taxa in Table 1 is fine, but  another  list with the dominant genera will be very useful to assimilate the findings, and to compare them with those of other surveys. You might want to add the frequencies of different OTU belonging to the same genus for this purpose.

2. In the first paragraph of the discussion section consider that the method for fungal detection used here was NGS, and not direct isolation like in the cited surveys of grass mycobiota.  Similar results are obtained in terms of some dominant taxa, supporting this claim. In  contrast with surveys based in direct isolation, a large number of basiomycota (33% of OTU) have been detected using NGS.

3. Provide in the text or in table 2 the species richness values for E+ and E- plants.

4. What is the species richness value used (e.g. in figure 2) ? number of taxa? specify this on the text, figure axis, or figure caption.
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: KNOWING YOUR NEIGHBOURHOOD—THE EFFECTS OF EPICHLOË ENDOPHYTES ON FOLIAR FUNGAL ASSEMBLAGES IN PERENNIAL RYEGRASS IN DEPENDENCE OF SEASON AND LAND-USE INTENSITY
Review round: 1
Reviewer: 2

Output:
Basic reporting: This is a very interesting report on fungal endophyte community assemblages in grasses. it is well written although has some important typos and inconsistencies that should be revised. The structure meets journal standards.
Experimental design: The research question is well defined, relevant and meaningful, however some methods, although well described, do not meet with high technical standards to support the results, discussion and conclusions presented. It is highly recommended the authors rewrite several parts to clarify changes within the methods and materials section that are not consistent. Also, they should include references regarding criteria for sampling design, storage, processing and bioinformatic analysis within the results and discussion section. One suggestion is this article: Conducting a Microbiome Study Goodrich, Julia K. et al.
Cell , Volume 158 , Issue 2 , 250 - 262.
Validity of the findings: My main concern is that the authors do not present solid foundations for their  conclusions. The results section is very short and the authors do not discuss putative pitfalls that they might have had during the study, as I can infer based on the inconsistencies presented in the methods and materials section.
Additional comments: This is a very interesting manuscript. This research area needs more information regarding how the microbiome interacts under different conditions. I recommend you rewrite this interesting study focussing on the technical part. I think you presented several inconsistencies based on the high volumen of data you had. Many researchers in this area need to have more systematic ways to design the experiments and to perform meaningful statistical analyses and present them in a very easy way for readers.


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: KNOWING YOUR NEIGHBOURHOOD—THE EFFECTS OF EPICHLOË ENDOPHYTES ON FOLIAR FUNGAL ASSEMBLAGES IN PERENNIAL RYEGRASS IN DEPENDENCE OF SEASON AND LAND-USE INTENSITY
Review round: 2
Reviewer: 1

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


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: KNOWING YOUR NEIGHBOURHOOD—THE EFFECTS OF EPICHLOË ENDOPHYTES ON FOLIAR FUNGAL ASSEMBLAGES IN PERENNIAL RYEGRASS IN DEPENDENCE OF SEASON AND LAND-USE INTENSITY
Review round: 2
Reviewer: 2

Output:
Basic reporting: The current version is now clear and unambiguous. I thank the authors for making new figures. Also, the materials and methods section now is very well explained . I detected several typos, please double check before the manuscript is accepted as it is.
Experimental design: Yes, now the current version shows a well defined research question. The materials and methods are well described.
Validity of the findings: I consider their results are valid. However, it has to be stated that their findings were obtained under certain conditions that not necessary are easy to replicate. Please add one sentence commenting this. I do not necessary have to review it.
Additional comments: I consider your manuscript almost ready for publication. I appreciate your consideration to my comments and suggestions. You nicely answer my questions and concerns. Only one last recommendation, please double check for any typo you might still have.


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: KNOWING YOUR NEIGHBOURHOOD—THE EFFECTS OF EPICHLOË ENDOPHYTES ON FOLIAR FUNGAL ASSEMBLAGES IN PERENNIAL RYEGRASS IN DEPENDENCE OF SEASON AND LAND-USE INTENSITY
Review round: 3
Reviewer: 1

Output:
Basic reporting: Manuscript is ready for publication.
Experimental design: Manuscript is ready for publication.
Validity of the findings: Manuscript is ready for publication.
Additional comments: Manuscript is ready for publication.