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: LEGACY EFFECTS OF HISTORICAL GRAZING ALTER LEAF STOMATAL CHARACTERISTICS IN PROGENY PLANTS
Review round: 1
Reviewer: 2

Output:
Basic reporting: I think the manuscript is clearly written and relevant in the context of stomata on the legacy effect of historical overgrazing on the grassland plant. abs the literature references were sufficient.and the structure were professional.
Experimental design: The experiment design and techniques employed are appropriate. Relevance and novelty of the study are obviously.
Validity of the findings: This paper is valuable on exploring the role of stomata on the legacy effect of historical overgrazing on the grassland plant. It will be helpful for us to better understand the possible mechanism how plant adapting to long term grazing.
Additional comments: Stoma is one of the most important organs for plant photosynthesis and water transportation. I think the manuscript is clearly written and relevant in the context of stomata on the legacy effect of historical overgrazing on the grassland plant. The experiment design and techniques employed are appropriate. Relevance and novelty of the study are obviously. This paper is valuable on exploring the role of stomata on the legacy effect of historical overgrazing on the grassland plant. It will be helpful for us to better understand the possible mechanism how plant adapting to long term grazing. The paper can be considered for publication after addressing the following questions.

Abstract，
1. The abstract is too long, there are so much words for the background. It is best to cut back and leave the important part.
“Grazing, one of the primary modes in the utilization of grassland, is the main cause of grasslands degradation. Long-term overgrazing results in dwarf phenotype of perennial plants and decreases photosynthesis, which is not only a contemporary phenomenon, but also sustainable in future generations. However, it is unknown what is the mechanism of legacy effect of lower photosynthesis from historical grazing. Stomata, as an important regulatory organ of photosynthesis, is one of key elements to control the efficiency of photosynthesis. What role stomata play in this low- photosynthesis induced by long-term overgrazing still have no answer”.
2.  The Keywords not include the main key word. I suggest genes expression should be added in
Introduction
1. Line 39 (Lohmann et al., 2012; Wang, Wang & Wang, 2006; Sasaki et al., 2008)
Should be the order (Wang, Wang & Wang, 2006; Sasaki et al., 2008; Lohmann et al., 2012)
The reference are also mess and need to be revised.
Wang W Y, Wang Q J, Wang HC. 2006. The effect of land management on plant community composition, speciesdiversity, and productivity of alpine Kobersia steppe meadow. Ecological Research 21:181-187 DOI 10.1007/s11284-005-0108-z.
3. Line 80 Leymus chinensis, which is a perennial, rhizomatous clonal grass with high palatability and forage value (Liu, 2006), covers a large area of the eastern Eurasian temperate grassland.
    The author should mention the importance of Leymus chinensis in the local grassland ecosystem.
4. The important and research of gene expression in grassland should be supplemented.
Materials and Methods
1. As to grazing treatments, 6 sheep were continuously grazing in the plot with the area of 0.64 hm2 during the growing season from May to October each year.
Grazing treatments should be converted into grazing intensity
2. As to plant materials, why the buds were taken and planted in greenhouse instead of taking plant materials in the field directly on summer?
3. The temperature was about 25℃ during the day and 15℃ at night. The phenotypic traits, gas exchange parameters, stomatal traits and related gene expression studies were tested successively after plant transplantation.
    The exact test time should be stated.
4. As to real-time PCR analysis, the samples were taken from two treatments long-term enclosure and overgrazing? Are these two treatments the same with the Grazing and No grazing treatment? If not, please describe the difference?
5. As to real-time PCR analysis, how the 8 genes related to stoma development and regulation were selected?
Results
1. The authors mentioned the progeny developed from the grazed and un-grazed parents, How did the progeny obtained? Are they sexual or asexual progeny?
2. The authors should provide a microscopic stomatal structure picture, so it is very intuition for the readers
 Discussion,
1. How to explain the up-regulation of cycp3L -1? Doe s the stoma data support this findings?
Conclusion
Line 274 which resulted in the dwarf phenotype of offspring plants (Fig. 6).
    The (Fig.6) should be deleted


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: LEGACY EFFECTS OF HISTORICAL GRAZING ALTER LEAF STOMATAL CHARACTERISTICS IN PROGENY PLANTS
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript reports on the effects of grazing on a suite of leaf traits, with particular focus on stomatal density. The research is important and of interest.  Overall the manuscript is coherent and well put together. However, it requires extensive editing for grammar and word usage.
Experimental design: The experimental design is appropriate. There is ambiguity in how plants were sampled in the field that requires more detail (e.g. how many samples, number per paddock, did they come from different parental plants, etc.).
Validity of the findings: The discussion needs revision. Because the research was done in the greenhouse and because there was little ancilliary data provided to explain some of the patterns much of the discussion is speculative. For example, water supply can have a significant impact on stomatal density and there is much speculation around this in the discussion, but not accompanying data - this is not necessarrily a problem, but the amount of discussion to this end needs to be reduced.

The mass of the leaves and the mass of the plants should have been measured.  If this data is available it must be included with this manuscript. This would enable 1) better information on plant growth, as height is a poor indicator and 2) enable the authors to relate patterns to specific leaf area (SLA) a key metric for understanding plant growth in response to environmental change.

Wording throughout the manuscript around the idea of offspring and legacy effects needs to be written with clarity. This study uses clones from the parental plant and this must be made absolutely clear. The findings essentially represent phenotypic plasticity, an idea which is not dealt with to sufficient extent in this manuscript.
Additional comments: I have provided additional minor comments in the pdf.


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

Input:
Title: LEGACY EFFECTS OF HISTORICAL GRAZING ALTER LEAF STOMATAL CHARACTERISTICS IN PROGENY PLANTS
Review round: 2
Reviewer: 1

Output:
Basic reporting: I think the manuscript is clearly written and relevant in the context of stomata on the legacy effect of historical overgrazing on the grassland plant. abs the literature references were sufficient.and the structure were professional.
Experimental design: The experiment design and techniques employed are appropriate. Relevance and novelty of the study are obviously.
Validity of the findings: This paper is valuable on exploring the role of stomata on the legacy effect of historical overgrazing on the grassland plant. It will be helpful for us to better understand the possible mechanism how plant adapting to long term grazing.
Additional comments: This paper is valuable on exploring the role of stomata on the legacy effect of historical overgrazing on the grassland plant. It will be helpful for us to better understand the possible mechanism how plant adapting to long term grazing.


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: LEGACY EFFECTS OF HISTORICAL GRAZING ALTER LEAF STOMATAL CHARACTERISTICS IN PROGENY PLANTS
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors have made appropriate changes to the manuscript and I have no concerns with their revisions.

The authors may find this paper relevant to their work:Oesterheld, M., McNaughton, S.J. Intraspecific variation in the response of Themeda triandra to defoliation: the effect of time of recovery and growth rates on compensatory growth. Oecologia 77, 181–186 (1988). https://doi.org/10.1007/BF00379184
Experimental design: No concerns
Validity of the findings: No concerns
Additional comments: Thank you for making revisions to the 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: NOVEL RAPID IDENTIFICATION OF SEVERE ACUTE RESPIRATORY SYNDROME CORONAVIRUS 2 (SARS-COV-2) BY REAL-TIME RT-PCR USING BD MAX OPEN SYSTEM IN TAIWAN
Review round: 1
Reviewer: 1

Output:
Basic reporting: The report describes the use of the automated BD MAX PCR system for detection of SARS-CoV2 using a lab-developed protocol. Advantages over manual RT-PCR are the decrease in hands-on-times, decrease in turnaround-times and potentially, less contamination risk due to fully automated handling. The authors describe the method and evaluate clinical performance in comparison to the standard lab method employing a validated protocol.
Experimental design: The report is clear and concise, the method and the results are well described. The data set for validation is very limited as only 10 samples among the 300 samples were positive.
Validity of the findings: see below
Additional comments: The following issues should be solved:
•	l. 70, what kind of swab have been used to sample the throat?
•	l. 74: When using sputa, how many samples were inhibited despite of liquefaction? Did the authors use quality criteria for the internal control (a range of Cts for acceptance)?
•	For the turnaround times: Did the authors include the time to prepare the mastermix in the BD MAX hands-on times?
•	Remove Figure 3, this information is sufficient as text
•	Figure 4: There is a clear reduction in the sensitivity to detect the 1e-4 diluted sample by BD MAX. Also, replicates for this assay comparison are missing. Moreover, for none of both assays there seems to be a good PCR efficiency. The latter can and should be calculated from the dilution series.
•	10 positive samples are not many samples to state 100% agreement as a general statement. The limitation of only 10 pos. samples should be stated in the abstract.
•	Discussion: the authors should discuss how to optimize reagent handling to facilitate handling on the BD MAX: Is it possible to prepare read-to-use tubes? Can the mastermix be prepared in advance and stored?
•	Figure 5: what are the orange dots? must be removed!

Minor
•	l. 36 “and” instead of “band”
•	l. 107-112; incomplete sentences, english to be reworked
•	BD MAX is not a truly high-throughput machine (change wording)


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 RAPID IDENTIFICATION OF SEVERE ACUTE RESPIRATORY SYNDROME CORONAVIRUS 2 (SARS-COV-2) BY REAL-TIME RT-PCR USING BD MAX OPEN SYSTEM IN TAIWAN
Review round: 1
Reviewer: 2

Output:
Basic reporting: This manuscript by Perng et al. describes a laboratory-developed test for SARS-CoV-2 on the BD Max system. The manuscript has clear and concise details concerning usage of this laboratory developed test for RNA extraction and RT-PCR analysis, however, key test performance characteristics were not determined, positive sample size (n=10) is too low and data requires reanalysis and reconfiguration prior to resubmission.
Major critiques
-	Positive sample size needs to be increased. 10 positive out of 400 samples is insufficient for accurate analysis of clinical sensitivity
o	Concordance levels would shift and show variability with lower-level positives from their LDT comparative to the BD Max. The authors (Fig 4) show a clear difference between detection between their assays (BD Max appears less sensitive). The reviewer thinks that the authors should expand this to a larger range of positive samples (10-20 more positives minimum), or use spiked in positive control (plasmid or synthetic RNA) in primary samples to show that at low levels of detection that the concordance remains the same.
-	Although precision is presented linearity / limit of detection studies are required to complete analytical test performance characteristics.
-	Present in silico or in vitro specificity data with known coronaviruses and other respiratory pathogens to determine analytical specificity
-	Internal controls innate to the sample (not spike in external controls) are essential to determine integrity of the RNA in original samples during transport. Consider incorporation of targets such as human Rp
-	Define the ORFs themselves used as targets for the PCR – the RNA-dependent RNA-polymerase is a multimeric subunit protein in SARS-CoV2 (nsp7/8 and nsp12) and there have been implications of potentially two subunits with RdRp activity.
Experimental design: Minor critiques
-	Clearly define the nature of the positive control used for each assay
-	Spell out abbreviations (Ex. LDT (laboratory developed test), TAT (turn-around time)
-	Line 136-137 suggest inter and intra-well replicates with data for cross-reactivity analysis. Please show this data.
-	Line 26, Severe Acute Respiratory Distress Syndrome Virus 2 should be capitalized as it is the name of the virus.
-	Review and correct punctuation and grammar throughout the manuscript
Validity of the findings: see above
Additional comments: see above


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

Input:
Title: NOVEL RAPID IDENTIFICATION OF SEVERE ACUTE RESPIRATORY SYNDROME CORONAVIRUS 2 (SARS-COV-2) BY REAL-TIME RT-PCR USING BD MAX OPEN SYSTEM IN TAIWAN
Review round: 2
Reviewer: 1

Output:
Basic reporting: My minor initial concerns have been adressed sufficiently. Number of pos. samples has been increased. Although the number is still low, Í think that a well justified first evaluation is now possible. There is a need for SARS-CoV2 assays on automated PCR devices like the BD MAX and thus protocols should be published rapidly.
Would have been fine to also calculate the PCR efficiency, but at least limit of detection is now given.
No further substantial criticism.
Experimental design: ok
Validity of the findings: ok
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: NOVEL RAPID IDENTIFICATION OF SEVERE ACUTE RESPIRATORY SYNDROME CORONAVIRUS 2 (SARS-COV-2) BY REAL-TIME RT-PCR USING BD MAX OPEN SYSTEM IN TAIWAN
Review round: 2
Reviewer: 2

Output:
Basic reporting: Addressed all concerns
Experimental design: Addressed all concerns
Validity of the findings: Addressed all concerns
Additional comments: Addressed all concerns


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: ISOLATION, DIVERSITY, AND ANTIMICROBIAL ACTIVITY OF FUNGAL ENDOPHYTES FROM ROHDEA CHINENSIS (BAKER) N.TANAKA (SYNONYM TUPISTRA CHINENSIS BAKER) OF QINLING MOUNTAINS, CHINA
Review round: 1
Reviewer: 1

Output:
Basic reporting: Major points:
1. In general, there is a lack of description or reference of known species identified in this study. Only observation was provided in some cases with no explanation or further discussion (e.g. on line 181, it’s not clear why Sordariomycetes and Dothideomycetes were most abundant groups). How are the relative abundance of these microorganisms compared to the previously reported data in the same region if any? On line 164, what’s the implication of having these six species in both tissues? Is it surprising to see this result? It’s not clear what these discoveries mean. Although the major focus of the paper is to look for new bioactive substance, it’s important to discuss the biodiversity of endophytic fungi of this plant since authors claimed that this was the first time it had been done.
2. Authors only include the positive data in this manuscript. The negative results should also be reported. On line 199, the data from the other 85 extracts that did not show inhibitory activity are not included.

Minor points:
1. There are some small grammar mistakes like on line 79 “As far as we known” should read “As far as we know”.
2. Appropriate citations should be included for methodology used. For instance, on line 189, “Neighbourhood-joining method” was originally described in Saitou N, 1987, Mol Biol Evol.
Experimental design: Major points:
1. There is no control in the antimicrobial activity screening done by authors. It’s not clear whether the inhibitory activity is truly due to the extracts or due to other factors like contamination. The results from the three replicates are not clearly presented. It’s not clear whether the higher antibacterial activity from F8047 and F8075 is significant or not. How does the inhibitory activity of these extracts compare to the extracts of fungi that are known to produce antibiotics? This comparison is crucial to determine whether there are truly bioactive substances.
2.  Authors should specify “The other statistical analyses” on line 147. The statistical analysis is not clearly presented with no p-value or statistics given (e.g. line 163, 167).

Minor points:
1. It’s not clear how antimicrobial activities were evaluated. How were the proportions on line 201 obtained? Is there a cutoff for the change of diameter?
Validity of the findings: Major points:
1. Most data were presented in tables rather than figures. The content in all three tables can be better visualized with bar plots and/or pie plots. (e.g. https://www.nature.com/articles/s41598-019-50602-5.pdf)

Minor points:
1. On line 257, “This result suggested that the T.chinensis Baker plant harbor a great diversity of endophytic fungi resources.” This is not an accurate statement without comparing with other plants. Having four new species doesn’t mean “a great diversity”.
2. On line 152, what’s the percentage of tissue segments with fungal colonies? What’s the distribution of 371 colonies among the collected samples?
Additional comments: Chao el at reported 371 endophytic fungal colonies extracted from T. chinensis Baker at Qinling mountain in China. Using previously published fungal isolation and cultivation techniques, 65 species of endophytic fungi were identified as endosymbiont with the plant. Among these strains, three new species were identified as well as new antibacterial compounds. Authors claimed that these findings would make fungi from T. chinensis Baker a potential novel source for new bioactive compounds.
The question of this study is well-formed but it lacks proper controls, raw data including negative results, sufficient discussion of the findings and detailed statistics. Therefore, I suggest a major revision.


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: ISOLATION, DIVERSITY, AND ANTIMICROBIAL ACTIVITY OF FUNGAL ENDOPHYTES FROM ROHDEA CHINENSIS (BAKER) N.TANAKA (SYNONYM TUPISTRA CHINENSIS BAKER) OF QINLING MOUNTAINS, CHINA
Review round: 1
Reviewer: 2

Output:
Basic reporting: Please see below. My complete review is included in the Validity of findings section
Experimental design: My complete review is included in the Validity of findings section
Validity of the findings: Manuscript. No.: #40649

Title: Isolation, diversity, and antimicrobial activity of culturable fungal endophytes of Tupistra chinensis Baker from Qinling Mountains, China

Authors: Chao An, Saijian Ma, Xinwei Shi, Wenjiao Xue, Chen Liu, Hao Ding

This study’s background information is good but I am not convinced the results/conclusions provided meets the study goals based on the data provided in this manuscript. Methods section also need more detail for better understanding of their experimental design.

Overall, the authors need to clarify the following points in order to justify their data, results and conclusions:

1) The phylogenetic analysis as shown in Figure 1 and 2 and all subsequent conclusions based on this as mentioned in the results section needs to be supported with better bootstrap values. At this point, the bootstrap values are unacceptable to draw such conclusions.

2) The authors need to provide more details on how the antimicrobial activity was measured – on what criteria the zone diameters were measured, how the proportions of inhibitory activity were measured.
Please see additional comments/ suggestions below.

Introduction:
Lines 63-62 – Please elaborate what you mean by this and provide a reference.
Lines 66-68 – This sentence needs to be rephrased for better understanding of the readers. What do you mean by ‘excavation’?
Lines 67-69 – Sentence ‘For this reason…” needs references.

Materials and methods:
Lines 84-88 – Plant material – Authors need to provide the following information- a) how many plants were collected? b) How many of these were used for fungal isolation? c) How were the collected plants transported to the laboratory- under what conditions?
You mention some of these between lines 234-235 in discussions, this needs to be clearly included in methods.

Line 94 – please elaborate what ‘modifications’ were used.
Lines 95 – Expand what you mean by PDA, MEA and SDA medium, abbreviations need to be expanded and explained on first use.
Line 101 – At what conditions were the pure isolates stored? Please explain.
Line 119 – What is ‘levers’?
Line 128 – what is r? Is this rpm or rcf?
Line 131 – what do you mean by a ‘cupboard’?
Line 133 - please elaborate what ‘modifications’ were used.
Line 136- 141 – Antimicrobial activity – this section needs to much more detailed. What were the standards used for diameter measurements to determine antimicrobial activity? How were the proportion of inhibitory activity calculated?
Line 147 – What do you mean by ‘other statistical analyses’? Please elaborate.

RESULTS:
Line 165 – it is unclear what ‘observed simultaneously in two plant tissues’ means here.

Figure 1 – The bootstrap values displayed in this phylogenetic tree is unacceptable at this point. Your bootstrap values are too low for most of the phylogenetic clades to confirm statistical significance of this tree, and the subsequent conclusions displayed under the results section.
Although this is not the only one, for your benefit, I am sharing one reference paper here: https://academic.oup.com/sysbio/article-abstract/42/2/182/1730933/.

Figure 2 – (Lines 189 – 192) – In figure 2, the bootstrap values supporting this specific clade is unacceptable to conclude this.

Lines 197 – 211 – Authors need to further elaborate on how exactly these inhibition zones were traced, and using what standards zones of inhibition were measured. Authors also need to elaborate on how the proportion of inhibitory activity were calculated.
Additional comments: My complete review is included in the Validity of findings section


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

Input:
Title: ISOLATION, DIVERSITY, AND ANTIMICROBIAL ACTIVITY OF FUNGAL ENDOPHYTES FROM ROHDEA CHINENSIS (BAKER) N.TANAKA (SYNONYM TUPISTRA CHINENSIS BAKER) OF QINLING MOUNTAINS, CHINA
Review round: 1
Reviewer: 3

Output:
Basic reporting: 1.	In this manuscript, the authors have isolated and characterized the endophytic fungi of Tupistra chinensis Baker from Qinling Mountains, China. Though the work has its scientific value, however the manuscript in the current form suffers from various flaws. So following are my comments/suggestions.
The manuscript suffers from a significant linguistic problem.
For example:
i.	The genetic and metabolite levels of the endophytes have changed owing to resisting the stress from their habitats.
ii.	The result showed that the PDA medium isolated more endophytic fungal (153, IR 41.24%), followed by SDA medium (142, IR 38.27%) and MEA medium (76, IR 20.49%).
iii.	Five genera with higher isolation frequency (IF) were Phyllosticta spp (IF, 21.65%), Colletotrichum spp (IF, 13.40%), Arthrinium spp (IF, 8.76%), Aspergillus spp (IF, 7.22%), and Nemania spp (IF, 6.19%) in the root tissues, respectively. Nevertheless, those higher isolation frequency genera were Colletotrichum spp (IF, 22.03%), Dactylonectria spp (IF, 11.30%), Fusarium spp (IF, 11.29%), Aspergillus spp (IF, 9.03%),Cladosporium spp (IF, 3.95%), in the stem tissues, respectively.
iv.	The composition and distribution of endophytic fungi exhibited significantly different in different host plants (Yuan et al., 2011; Jia et al., 2016).
v.	Though most genera of the 15 endophyte funguses, which were isolated from of other ecological environment, have been reported to produce antibacterial compounds.
Besides these examples, there are such linguistic errors at several places. Thus, the English language must be significantly improved to ensure the clear delivery of your message to the readers. Besides, authors must ensure consistency throughout (eg. Use either fungi or funguses).
2.	The introduction section lacks the motivation for the work carried out by the authors. The authors must provide sufficient literature in the Introduction section to highlight the gaps in this field of research in the current scenario. Besides, there is redundancy in their Introduction and Results section. For example, authors have provided the literature about the bioactive metabolites, steroids etc., however, in the current work, they are “just” checking the antimicrobial activity of the endophytic fungi. In this direction, the authors must add more literature.
Importantly, authors must discuss their results in parallel to in the Introduction section.
Experimental design: 1.	Here authors have explored the fungal diversity of a Tupistra chinensis Baker from Qinling Mountains, China. However, there is no novelty as authors have identified neither some novel fungal strains nor some novel bioactive compounds. Authors have made very superficial conclusions/speculations only and no validation was carried out for these speculations, which make this a shallow study. Such shallow investigations do not qualify the standards and the scope of PeerJ.
2.	Research questions are not well defined as there is a lack of literature support to display the gap which authors have attempted to fulfill.
3.	The molecular identification and phylogenetic analyses carried out by authors seem to be very shallow and lack rigorous investigation. Below are some points which authors must address:
4.	There are no details of the taxonomic assignment and phylogenetic analysis in the manuscript. What were the cutoff values of ITS similarity with reference sequences for species, genus, order, family, and phyla delineation and reference(s) for these cutoff values? How have authors selected members for phylogenetic analysis? How have the aligned the sequences? Have they performed bootstrapping or not?
5.	Authors have not provided the method details and reference of filtering paper method for antimicrobial assays.
Validity of the findings: 1.	Here authors have explored the fungal diversity of a Tupistra chinensis Baker from Qinling Mountains, China. However, there is no novelty as authors have identified neither some novel fungal strains nor some novel bioactive compounds. Authors have made very superficial conclusions/speculations only and no validation was carried out for these speculations, which make this a shallow study.
2.	As mentioned above, the molecular identification and phylogenetic analyses carried out by authors seem to be very shallow and lack rigorous investigation.
3.	Conclusions do not match the objectives mentioned by authors.
Additional comments: 1.	What are the isolation rate and isolation frequencies?
2.	Authors have provided scientific names of few plants and common English names of few others. I recommend that authors should provide the scientific name and well as the common English name of the plants mentioned in the manuscript.
3.	There are lots of errors in scientific names. Authors must correct these names. (For example, P.earuginosa to P. aeruginosa.; C.albicans to C. albicans)
4.	The scientific names mentioned for the first time in the manuscript should be expanded.
5.	The authors must adhere to the nomenclature, for example, Penicillium sp to Penicillium sp.
6.	You have mentioned in the paragraph “As presented in Table 2, the strains isolated from T.chinensis Baker were grouped into two 174 phylum  (Ascomycota (IF, 97.57%) and Zygomycota (IF, 2.43%))”. However, in Table 2, this are only two phyla viz., Ascomycota and Basidiomycota.
7.	The paragraph “As presented in Table 2, the strains isolated from T.chinensis Baker were grouped into two phylum” is redundant. The results are already appended in Table 2. So, remove this section.
8.	Authors have claimed that “As shown in Figure 2, the 190 results showed that the isolates F8001 were identified only at the genus level belonged to Leptospora spp and showed distinct clades with the its closest relatives species, which suggested that F8001 may be a potential new species of Leptospora spp.” Here, F8001 is clustering with Pleosporales sp. 38. So, how do you suggest it as a novel species of the genus Leptospora but not as a novel genus itself in the order Pleosporales.
9.	Authors speculated that F8067 and F8079 are new species of the genus Nemania. What do you reason behind this hypothesis as the genus Nemania is itself scattered in the tree?
10.	Authors have mentioned that the relatively low proportion of activity against the Gram-negative bacterium such as E.coli (2%) and P.earuginosa (2%) were observed. R. solanacearum is another Gram-negative bacterium yet showing 5% activity.
11.	Authors have mentioned that “Based on the Ouyang et al. (2018) research, a new antibacterial compound named molicellins O, which was isolated from the endophytic fungus Chaetomium sp. Edf-10, exhibited antibacterial antibacterial activities against Staphylococcus aureus ATCC 29210”. But original paper cited here has not bacterial strain Staphylococcus aureus ATCC 29210. Clarify it.
12.	The discussion part includes majorly the literature without any relevant relation to the authors’ work. It seems that authors are supporting the literature with their work, whereas they are supposed to support their work with literature. If there is not enough discussion then authors must merge the Results and Discussion section.
13.	The conclusion is very shallow.
14.	References are not as per the required format
For example:
i.	Bailey AM, Alberti F, Kilaru S, Collins CM, de Mattos-Shipley K, Hartley AJ, Hayes P, Griffin A, Lazarus CM, Cox RJ, Willis CL, O'Dwyer K, Spence DW, Foster GD. 2016.  Identification and manipulation of the pleuromutilin gene cluster from Clitopilus passeckerianus for increased rapid antibiotic production. Scientific reports 6: 25202-25212 DOI 10.1038/srep25202. (Scientific report to Scientific Reports).
ii.	Cheng Z, Xu W, Liu L, Li S, Yuan W, Luo Z, Zhang J, Cheng Y, Li Q. 2018. Peniginsengins B⁻E, New Farnesylcyclohexenones from the Deep Sea-Derived Fungus Penicillium sp. YPGA11.Marine drugs 16(10):358-368 DOI 10.3390/md16100358. (Marine drugs to Marine Drugs).


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: ISOLATION, DIVERSITY, AND ANTIMICROBIAL ACTIVITY OF FUNGAL ENDOPHYTES FROM ROHDEA CHINENSIS (BAKER) N.TANAKA (SYNONYM TUPISTRA CHINENSIS BAKER) OF QINLING MOUNTAINS, CHINA
Review round: 2
Reviewer: 1

Output:
Basic reporting: The revised manuscript is better structured.
In the revised manuscript, authors provide a better description of the scientific question and a deeper discussion about the impact of this study.
Experimental design: Proper controls are clearly explained in the revised manuscript.
The methods include more detail to replicate the experiments.
Validity of the findings: The detailed analysis of data is provided in the revised manuscript.
Supplementary data are provided.
Additional comments: Higher resolution images should be used for Figures.
The statistical analyses section in the results (line 162) does not include any hypothesis testing. It's more like taxonomic analysis.
Acknowledgements should not include funding statement.


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: ISOLATION, DIVERSITY, AND ANTIMICROBIAL ACTIVITY OF FUNGAL ENDOPHYTES FROM ROHDEA CHINENSIS (BAKER) N.TANAKA (SYNONYM TUPISTRA CHINENSIS BAKER) OF QINLING MOUNTAINS, CHINA
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors have made all changes to this manuscript based on my previous suggestions, and the manuscript is more articulate now. The English is a lot clearer, and overall the paper looks great now after all the revisions.
Experimental design: see above for my comments.
Validity of the findings: see above for my comments.
Additional comments: Thank you for making the changes to the manuscript based on my previous suggestions.


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: ISOLATION, DIVERSITY, AND ANTIMICROBIAL ACTIVITY OF FUNGAL ENDOPHYTES FROM ROHDEA CHINENSIS (BAKER) N.TANAKA (SYNONYM TUPISTRA CHINENSIS BAKER) OF QINLING MOUNTAINS, CHINA
Review round: 2
Reviewer: 3

Output:
Basic reporting: Line numbers are those in the review PDF

There are many grammatical / language errors throughout. I suggest to have a native English speaker edit. References for methods are often to the most recent publication rather than the original. I suggest to cite the originals for clarity. The raw data for constructing the phylogeny and the phylogeny itself are not made public - please make public. The sequence data in GenBank has the wrong host species listed - please fix.

Abstract
- There are a few grammatical / language mistakes:
  - line 27: ... segments of T. chinensis Baker plant... (i.e., plant is awkward)
  - line 30: numbers < 10 should be spelled out
  - lines 33-34: ... were most frequently from the tissues...
  - line 38: ... one of the pathogens microorganism.

Introduction
- There are several grammatical / language mistakes:
  - lines 48-49: ... discovery of new structure active ingredient...
  - line 64: ... endophytic fungi... is scarcely explored...
- line 56: Citations here are not ordered by year within author.
- line 66: It would be nice to have some taxonomy beyond genus described for the plant host - such as family.
- line 83: I recommend to avoid beginning sentences with numbers, and to write them out instead.
Experimental design: Many details of the culturing, extractions, and assaying could be explained further, for reproducibility. No real research question was defined. While the work is novel in that it is the first study of endophytes from this particular host, no new fungi nor metabolites were described. I suggest to describe the unknown fungi and confirm the identity of the metabolites described.

Materials and methods
Plant material
- lines 84-86: The citation Tan et al. 2014 describes collection somewhat differently than reported here, in that they first transported plants to the lab and then placed them into a refrigerator. I suspect the order of '... packed into a refrigerator (4 degrees C) and immediately transported back to the lab...' is reversed. If not, please describe in further detail the instrument / refrigerator used to transport the plants to the lab.
Fungal isolation and cultivation
- lines 89, 95: Which parts of the plant were fungi isolated from? Where did the 510 segments come from with respect to the 20 plants collected?
- lines 98-99: What size Petri plates were used, and how many tissue segments were plated on each plate? Were plates sealed with Parafilm, or otherwise contained?
Molecular ID and phylogenetic analysis
- There is a discrepancy between reporting only the forward primer used in White et al. (1990) and the PCR recipe reported that contains 2 µL of each primer.
- line 112: The word preceding 'Standard Gradient' appears misspelled.
- line 120: GenBank is misspelled, and the accessions have the wrong plant host species listed.
Antimicrobial activity
- lines 134-135: Italics are used incorrectly in some cases: 'pathogenic bacterial' should not be italicized, whereas oryzae (following pv.) should be. I see that you refer to 'pathogenic bacterial' again on line 145, but urge you to highlight this group otherwise, if even necessary, to avoid confusion with the latin nomenclature here.
- What size Petri dishes were used throughout?
- line 147: Is the use of methanol as a negative control appropriate? Wouldn't using ethyl acetate, or water, be more appropriate given the compounds being assayed were extracted using ethyl acetate?
- I think more needs to be described regarding how the ethyl acetate extracts were produced, beyond citing Wang et al. 2019 that has little additional details in that regard. For example, how were cultures grown prior to extraction? How much volume or biomass was extracted and using how much solvent? Please elaborate for clarity and transparency.
ID of compounds
- Much of the text in this section appears nearly identical to that in the citation Tan et al. (2018). Also, that citation cites another paper, "Fu, Y. J. et al. An analytical pipeline to compare and characterise the anthocyanin antioxidant activities of purple sweet potato cultivars. Food Chemistry 194, 46–54 (2016)", for some of the analysis in this section.
Statistical analyses
- line 171: 'The procedure' is the Shannon-Weiner identity and Tan et al. (2018) is not the original citation - I recommend to define it here, and/or use the original citation.
Results
- line 191: The citation here should also be reported during the method's first mention in the methods section
- Figure 1: The inclusion of only one taxon per genus compromises your ability to classify things beyond genus - you need to include sequences from other species in each genus in order to see where your sequences are placed in that context. However, I see that your longest sequence is around 650 bp, which is quite short. It is not acceptable to infer a phylogeny across this diversity using only the ITS region.
- Figure 2: I recommend to use colors and/or patterns that are more clearly distinguishable. Also, consider using stacked bar plots, rather than pie charts, for clarity.
- line 198: By different plants, do you mean among the 20 individuals you sampled? Please clarify.
- line 221: Leptosporium is misspelled
Validity of the findings: While classification to genus of the fungi sequenced, I suspect that they are not an accurate representation of all isolated. The estimation of isolation frequencies among the taxa reported are skewed due to certain isolates being assigned incorrectly to species. Phylogenetic placement of sequences using non-model-based inference did not include an inclusive sample of taxa within each genus such to provide additional insight beyond BLAST.

Sequence data at NCBI: Upon checking several accessions from this study, I notice that all have Disporopsis fuscopicta listed as the host plant species, as well as an unpublished study focused on that species listed as the publication source.

The multiple sequence alignment and resultant phylogeny are not accessioned or made public.

Molecular ID and phylogenetic analysis
- line 121: How many sequences were included in your phylogenetic analysis? What was the length of your multiple sequence alignment? If the number of sequences is close to the number of positions, constructing a de novo phylogeny may not be appropriate. Also, I recommend to use an inference method that uses an explicit model of sequence evolution, such as maximum likelihood or Bayesian methods. RAxML or MrBayes are good tools to use. Finally, please accession your alignment and phylogeny someplace where we can access them.

- Table 1: The taxon names here include species and often many within a single genus. I recommend to only classify taxonomy based on ITS sequences to the genus level. The phylogenetic analysis does not support classifying to species level because you did not include multiple species within each genus. The isolation frequencies are not accurate because of this. More importantly, many fungi that appear the same are often not, and some that look distinct can be the same (i.e., at the sequence level). I suspect that by assigning morphotypes and sequencing a single representative of each, that your conclusions based on that subsample do not accurately represent the fungal diversity in your entire culture collection.
- Table 3: Nice table! However I do not think you should classify your sequences past genus based on these data.
- Table 4: Also a cool table. However, work in foliar endophytes has shown species to diverge at ca. 95% ITS sequence similarity, so you are right on the line here, especially for some of the groups reported here (U'Ren et al. 2009 - Diversity and evolutionary origins of fungi associated with seeds of a neotropical pioneer tree - case study for analysing fungal environmental samples). I would hesitate to conclude that these are novel taxa without also stating that performing a multi-locus phylogenetic, or phylogenomic analysis with other members for each group would provide better insight, given the variability of ITS sequences even within a single species (and in some cases even a single spore).
Additional comments: Line numbers are those in the review PDF

In the abstract and introduction sections below, I highlight few instances of grammatical / language mistakes, but stop after that due to their consistent presence in the manuscript. I suggest the authors have a native english speaker go through the draft for clarity.

Abstract
- There are a few grammatical / language mistakes:
  - line 27: ... segments of T. chinensis Baker plant... (i.e., plant is awkward)
  - line 30: numbers < 10 should be spelled out
  - lines 33-34: ... were most frequently from the tissues...
  - line 38: ... one of the pathogens microorganism.
- lines 34-36: What are the values of species richness and diversity being compared to, such that they are 'high'?

Introduction
- There are several grammatical / language mistakes:
  - lines 48-49: ... discovery of new structure active ingredient...
  - line 64: ... endophytic fungi... is scarcely explored...
- line 56: Citations here are not ordered by year within author.
- line 66: It would be nice to have some taxonomy beyond genus described for the plant host - such as family.
- line 83: I recommend to avoid beginning sentences with numbers, and to write them out instead.

Materials and methods
Plant material
- lines 84-86: The citation Tan et al. 2014 describes collection somewhat differently than reported here, in that they first transported plants to the lab and then placed them into a refrigerator. I suspect the order of '... packed into a refrigerator (4 degrees C) and immediately transported back to the lab...' is reversed. If not, please describe in further detail the instrument / refrigerator used to transport the plants to the lab.
Fungal isolation and cultivation
- lines 89, 95: Which parts of the plant were fungi isolated from? Where did the 510 segments come from with respect to the 20 plants collected?
- lines 98-99: What size Petri plates were used, and how many tissue segments were plated on each plate? Were plates sealed with Parafilm, or otherwise contained?
Molecular ID and phylogenetic analysis
- There is a discrepancy between reporting only the forward primer used in White et al. (1990) and the PCR recipe reported that contains 2 µL of each primer.
- line 112: The word preceding 'Standard Gradient' appears misspelled.
- line 120: GenBank is misspelled, and the accessions have the wrong plant host species listed.
- line 121: How many sequences were included in your phylogenetic analysis? What was the length of your multiple sequence alignment? If the number of sequences is close to the number of positions, constructing a de novo phylogeny may not be appropriate. Also, I recommend to use an inference method that uses an explicit model of sequence evolution, such as maximum likelihood or Bayesian methods. RAxML or MrBayes are good tools to use. Finally, please accession your alignment and phylogeny someplace where we can access them.
Antimicrobial activity
- lines 134-135: Italics are used incorrectly in some cases: 'pathogenic bacterial' should not be italicized, whereas oryzae (following pv.) should be. I see that you refer to 'pathogenic bacterial' again on line 145, but urge you to highlight this group otherwise, if even necessary, to avoid confusion with the latin nomenclature here.
- What size Petri dishes were used throughout?
- line 147: Is the use of methanol as a negative control appropriate? Wouldn't using ethyl acetate, or water, be more appropriate given the compounds being assayed were extracted using ethyl acetate?
- I think more needs to be described regarding how the ethyl acetate extracts were produced, beyond citing Wang et al. 2019 that has little additional details in that regard. For example, how were cultures grown prior to extraction? How much volume or biomass was extracted and using how much solvent? Please elaborate for clarity and transparency.
ID of compounds
- Much of the text in this section appears nearly identical to that in the citation Tan et al. (2018). Also, that citation cites another paper, "Fu, Y. J. et al. An analytical pipeline to compare and characterise the anthocyanin antioxidant activities of purple sweet potato cultivars. Food Chemistry 194, 46–54 (2016)", for some of the analysis in this section.
Statistical analyses
- line 171: 'The procedure' is the Shannon-Weiner identity and Tan et al. (2018) is not the original citation - I recommend to define it here, and/or use the original citation.
Results
- line 191: The citation here should also be reported during the method's first mention in the methods section
- Figure 1: The inclusion of only one taxon per genus compromises your ability to classify things beyond genus - you need to include sequences from other species in each genus in order to see where your sequences are placed in that context. However, I see that your longest sequence is around 650 bp, which is quite short. It is not acceptable to infer a phylogeny across this diversity using only the ITS region.
- Figure 2: I recommend to use colors and/or patterns that are more clearly distinguishable. Also, consider using stacked bar plots, rather than pie charts, for clarity.
- line 198: By different plants, do you mean among the 20 individuals you sampled? Please clarify.
- line 221: Leptosporium is misspelled
- Table 1: The taxon names here include species and often many within a single genus. I recommend to only classify taxonomy based on ITS sequences to the genus level. The phylogenetic analysis does not support classifying to species level because you did not include multiple species within each genus. The isolation frequencies are not accurate because of this. More importantly, many fungi that appear the same are often not, and some that look distinct can be the same (i.e., at the sequence level). I suspect that by assigning morphotypes and sequencing a single representative of each, that your conclusions based on that subsample do not accurately represent the fungal diversity in your entire culture collection.
- Table 3: Nice table! However I do not think you should classify your sequences past genus based on these data.
- Table 4: Also a cool table. However, work in foliar endophytes has shown species to diverge at ca. 95% ITS sequence similarity, so you are right on the line here, especially for some of the groups reported here (U'Ren et al. 2009 - Diversity and evolutionary origins of fungi associated with seeds of a neotropical pioneer tree - case study for analysing fungal environmental samples). I would hesitate to conclude that these are novel taxa without also stating that performing a multi-locus phylogenetic, or phylogenomic analysis with other members for each group would provide better insight, given the variability of ITS sequences even within a single species (and in some cases even a single spore).


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: IDENTIFICATION OF CANDIDATE GENES AND PROGNOSTIC VALUE ANALYSIS IN PATIENTS WITH PDL1-POSITIVE AND PDL1-NEGATIVE LUNG ADENOCARCINOMA
Review round: 1
Reviewer: 1

Output:
Basic reporting: In this manuscript, the authors identify the DEG and different biological pathways that are involved in the patients of PDL-1 positive and negative lung adenocarcinoma. Furthermore, the authors report the key genes that can be used as a biomarker for lung cancers.

There is no sufficient background provided about the role of PDL-1 in lung cancer, hard to understand the purpose of the study. I think the authors should include more background and details from the previous reports in the "Introduction" section for better understanding to broad interest readers. Re-write the abstract and introduction section.

The authors follow the journal's guidelines for article structure and figures and table preparation.
Experimental design: The data presented in the manuscript are clear and corroborate the conclusion made by the authors.

The major issue with the experimental design is related to a limited (3 patients) number of the dataset in each group which I think is very low and seems insufficient to make any conclusive interpretation. Please comment.

However, I would like to provide a few suggestions that will improve the quality of the manuscript:

1. Re-write the materials and methods section (provide more details); seems incomplete
2. In the figure section, there are a few minor errors:
a) Figure 1 legend, Typo-error: change "GEGs" to "DEGs"
b) Figure 1B legend: There are no black and green color dots in Figure 1B. Change "green" to "blue" and "black" to "grey"
3) In Figures 5, 6, and 7: Write the name of the gene in the inset of the boxes will make it very easy for readers to follow.
Validity of the findings: No comments
Additional comments: There is already existing literature that highlights the role of BRCA1 and NDC80 in lung cancers and the authors fail to cite any article which supports their findings. I think the authors should re-write the "Introduction and Discussion" section with proper literature citation.
Cite this article:
1. Potential therapeutic targets of the nuclear division cycle 80 (NDC80) complexes genes in lung adenocarcinoma, Cancer 2020; 11(10):2921-2934. doi:10.7150/jca.41834.
2. Are we ready to use biomarkers for staging, prognosis and treatment selection in early-stage non-small-cell lung cancer? doi: 10.3978/j.issn.2218-6751.2013.03.06


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: IDENTIFICATION OF CANDIDATE GENES AND PROGNOSTIC VALUE ANALYSIS IN PATIENTS WITH PDL1-POSITIVE AND PDL1-NEGATIVE LUNG ADENOCARCINOMA
Review round: 1
Reviewer: 2

Output:
Basic reporting: In this work, the authors used bioinformatics tools to analyze gene expression dataset between PDL1-positive and PDL1-negative patients with lung adenocarcinoma and suggest BRAC1 and NDC80 as new prognostic indicators for patients with lung adenocarcinoma.

Sufficient background information not provided as further elaborated in general comments for the authors.
Experimental design: The aim of the study is well-defined and the methods used are appropriate.
Validity of the findings: Overall conclusion are supported by the results shown. However, the novelty of the findings is not assessed as further elaborated in general comments for the authors.
Additional comments: 1)	The introduction needs improvement as sufficient background covering previous bioinformatics work identifying hub genes and potential biomarkers for lung cancer (to mention a few, DOIs: 10.3892/ol.2018.8882, 10.3892/ol.2019.10796 etc.) is not provided to understand the significance and novelty of the work.
2)	In the discussion authors should elaborate more on the novelty of the hub genes identified in this work as previous literature have already associated the hub genes identified here with lung adenocarcinoma. For example, on lines 245-246, “…Our study revealed that BUB1B, BUB1, MCM2, and DTL might be new potential oncogenes.…”. However previous works have associated these genes with lung adenocarcinoma (DOIs: 10.18632/genesandcancer.53, 10.3892/ol.2019.10796, 10.1038/s41598-017-13440-x, 10.1038/s41598-017-00512-1). Additionally, on lines 263-264, “…Moreover, we identified some new hub genes, such as BRCA1 and NDC80, which were associated with prognosis in patients with lung adenocarcinoma.…”. Again, both BRAC1 and NDC80 have been associated with lung adenocarcinoma in previous studies (DOIs: 10.3816/CLC.2008.n.048, 10.20892/j.issn.2095-3941.2018.0506, 10.1158/0008-5472.CAN-06-2137).
3)	One limitation of the analysis is in the dataset used which seems to consists of data from only 3 patients in each of the two groups (PDL1-postive and PDL1-negative). On line 77, “…GSE99995 was retrieved.…”. Was this the only dataset retrieved? If not, why was this dataset selected in particular. Also, in the methods section authors should add the criteria on which the patients were divided into two groups.
4)	Line 130, “…Our results showed that the two groups comprised 34729 genes…”. Authors should write the names of the two groups.
5)	Figure 2 seems to present data only for the PDL1-negative group, this should be identified in the text and figure legend.
6)	There are typos at some places in the manuscript. For example, “GEGs” should be “DEGs” and “green” should be “blue” in Figure 1 legend, “he” should be changed to “the” on line 281, etc.


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: IDENTIFICATION OF CANDIDATE GENES AND PROGNOSTIC VALUE ANALYSIS IN PATIENTS WITH PDL1-POSITIVE AND PDL1-NEGATIVE LUNG ADENOCARCINOMA
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The revised manuscript is now very clear with a detailed description of the methods. The authors have now addressed all the critical issues raised by both reviewers.
However, the manuscript needs English language editing in the Introduction and Discussion sections. I think the authors can use language editing services provided by 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: IDENTIFICATION OF CANDIDATE GENES AND PROGNOSTIC VALUE ANALYSIS IN PATIENTS WITH PDL1-POSITIVE AND PDL1-NEGATIVE LUNG ADENOCARCINOMA
Review round: 2
Reviewer: 2

Output:
Basic reporting: NA
Experimental design: NA
Validity of the findings: NA
Additional comments: The authors have addressed all of my concerns. However, there are spelling and grammatical mistakes at several places in the manuscript that needs correction. To give a few examples: "differences expression genes" on line 26, "studies have shown indicated" on line 82, etc. Also, fix lines 97-99.
I would recommend publishing the manuscript provided authors proofread and correct for mistakes in English.


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: IDENTIFICATION OF CANDIDATE GENES AND PROGNOSTIC VALUE ANALYSIS IN PATIENTS WITH PDL1-POSITIVE AND PDL1-NEGATIVE LUNG ADENOCARCINOMA
Review round: 3
Reviewer: 1

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


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

Input:
Title: IDENTIFICATION OF CANDIDATE GENES AND PROGNOSTIC VALUE ANALYSIS IN PATIENTS WITH PDL1-POSITIVE AND PDL1-NEGATIVE LUNG ADENOCARCINOMA
Review round: 3
Reviewer: 2

Output:
Basic reporting: NA
Experimental design: NA
Validity of the findings: NA
Additional comments: Authors have performed suggested changes and the manuscript is now suitable for publication.


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

Input:
Title: IDENTIFICATION OF SPRR3 AS A NOVEL DIAGNOSTIC/PROGNOSTIC BIOMARKER FOR ORAL SQUAMOUS CELL CARCINOMA VIA RNA SEQUENCING AND BIOINFORMATIC ANALYSES
Review round: 1
Reviewer: 1

Output:
Basic reporting: This manuscript provides a proof-of-concept investigation on the role of SPRR3 in OSCC by sophisticated bioinformatic analysis of three OSCC patient samples. Although a few language errors do not hinder the reader’s understanding, I do recommend the authors re-polish their manuscript or have a native speaker proof-read this manuscript before resubmission.
Experimental design: The experimental design need to be addressed more precisely.
1.	Authors collected three OSCC patients sample for sequencing but why author choose mouse genome as the mapping reference (line 112)??
2.	In figure 2, author should clearly label the row and column in the heatmap and point out which one is SPRR3 clearly in both volcano plot and heatmap.
3.	Why author chose p value (rather than FDR) as one of the statistical cut-off?
4.	Author should merge table S3 and S4 together and also list clearly logFC, FDR and p value of each DEGs.
5.	GSEA should also be considered to cross-validate that findings from GO and KEGG analysis from their own 6 matched samples.
6.	In line 274, how do author determine the “low expression of SPRR3”? median cut-off?
7.	Authors performed Cox and LASSO regression in GSE42743 and they got SPRR3 was the most significant genes. Afterwards, they validated SPRR3 in TCGA by univariate and multivariate Cox regression analysis. This design might be confusing and mess. Why not authors perform univariate and multivariate Cox regression analysis and LASSO in one dataset, then validate what they found in anther?
8.	What kind of TCGA data was downloaded ? level 3 RNA seq or microarray? What is the meaning of “the relative expression” in the y axis label of figure 6?
9.	In panel I of figure 6, the label is missing.
10.	In GSEA, how did author divide the patients into groups for analysis. Authors mentioned “according to the expression of SPRR3”, but more details are required.
Validity of the findings: Authors performed IHC to validate what they found in bioinformatic analysis.
1.	In figure 4, each p value should be labelled in panel A. What does the star represent for in panel B and C? What kind of statistical analysis used in these heatmap?
2.	In figure 6 and according text, author should be cautious about “correlated”. Authors found the expression of SPRR3 is different between the subgroup of patients, but author could not draw that was a correlation. If authors would like to test the correlation, regression (or correlation ) analysis should be considered.
Additional comments: The identification of potential molecular biomarkers in cancer is relevant to improve its diagnosis and treatment. In this line, the bioinformatic approach described in this work is useful to detect target genes with an interesting functional profiling in the frame of OSCC.

However this manuscript needs a better explanation related to three key points: 1) an adequate details about the design used that ensures the validity of findings and 2) improve the description of methods that allows the reproducibility of the work, 3) more validation works (whether the key genes from the results of GO/KEGG or GSEA are positively correlated with SPRR3).


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: IDENTIFICATION OF SPRR3 AS A NOVEL DIAGNOSTIC/PROGNOSTIC BIOMARKER FOR ORAL SQUAMOUS CELL CARCINOMA VIA RNA SEQUENCING AND BIOINFORMATIC ANALYSES
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1. The deposition of the generated data is better mentioned right after the description of the data in the materials and methods section.
2. The introduction states that SPRR3 was “determine as our gene of interest and the objective of this study was to verify its diagnostic and prognostic value ...” based on the known function of the gene in other tumors. However, the rest of the study presents the findings as being identified by screening for more important gene.
3. No documentation of related to the use of human samples is attached to the manuscript
4. The section on differential expression analysis details the steps of the analysis (first paragraph) and then describes the data collected from different sources and how it was processed. The authors may consider writing about data collection and data processing in separate subsections.
5. The criteria of significance of differentially expressed genes doesn’t include adjustment for multiple testing. The criteria of significance in enrichment analysis is not described at all.
6. Data (GSE42743 and TCGA), annotations(mm10 genome, GO and KEGG), and tools (HISAT2, HTSeq, R, Bioconductor, gplots,, affy, imput, edgeR, DAVID, Firebrowse and Cytoscape) were not cited properly in the text.
7. Figure 2A. The volcano plot has a peculiar shape. Namely, many genes with high log2 fold change have large p-values. This might be a result of inappropriately including more than one variables in the model for the differential expression analysis. Also, the significance criteria for the fold-change and p-values are different than what is declared in the text.
8. The axis labels in Figures 2A, 2B, 4A and 6I are missing.
9. The text and the references (in line citation) would benefit greatly from another round of revision and editing. On several occasions, the meaning of the sentences are not clear because of inappropriate word use or grammatical errors.
Experimental design: 1. Several datasets from GEO were used in the analysis without describing what they represent or why they are relevant to the current analysis.
2. The results of the differential expression and GO/KEGG enrichment cannot be trusted.
3. The meaning of MCODE score is not described and is not clear why it should be significant in this context.
4. Is the differential expression presented in Figure 4A independent of the one presented before? Figure 4B. presents uses two datasets from GEO without explaining what they represent or why the add value to the analysis. The same could be said about the analysis presented in Figure 5A&B.
5. The results of the survival analysis (Figure 5C&D) are different. Can the discrepancies be explained? Why isn’t the regression analysis (Table 3) applied to the GEO dataset?
Validity of the findings: The findings presented in this manuscript cannot be considered reliable enough to support the conclusions. Firstly, it is not clear whether the authors are using the workflow to arrive at the most significant gene or to justify their prior choice of SPRR1 as interesting based on previous literature. Secondly, there are several issues with the design and reporting of the study. Even when disregarding the first point, some of the analysis steps were not fully described, or missing.
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: IDENTIFICATION OF SPRR3 AS A NOVEL DIAGNOSTIC/PROGNOSTIC BIOMARKER FOR ORAL SQUAMOUS CELL CARCINOMA VIA RNA SEQUENCING AND BIOINFORMATIC ANALYSES
Review round: 1
Reviewer: 3

Output:
Basic reporting: This paper presents an equivalent method to identify the prognostic and diagnostic biomarker SPRR3 for OSCC, and builds up an experiment system for validation. Even though this paper contains interesting results which merit publication, for the benefit of the reader, however, should like to suggest that the author seek the advice of someone with a good knowledge of English, preferably native speaker to help proofread and reorganize sentences to improve the professional scientific expression.
Furthermore, a number of points need clarifying and certain statements require further justification.
Experimental design: This is a carefully done study, advanced bioinformatics tools were utilized to address specific questions, however, a number of points need clarifying and certain statements require further justification.
1.	In line 44, the author claimed SPRR3 as “a potential tumour suppressor gene”, but there is no following up reference or explanation about the role that SPRR3 played as a tumor suppressor, more details are supposed to be included in the background part.
2.	Line 260-261 mentioned that “SPRRs was reported to be functional”, what function??? Please provide more details.
3.	Some of the figures haven’t been clearly labeled, e.g. figure 2B heat map, figure 6I
4.	In 3.6 the author claimed that low expression of SPRR3 is associated with poor prognosis, however, low expression and high expression should have been defined in the first place
Validity of the findings: The work is meaningful in terms of OSCC diagnosis and potentially provides an indication for OSCC treatment. The conclusion "lower expression of SPRR3 associated with poor prognosis" has been well stated, expression in transduction level and protein level have been provided. However, the explanation in the functional aspect is still not very convincing and more evidence is supposed to be provided.
Additional comments: The work of identifying novel biomarkers for cancer diagnosis and prognosis is meaningful, this paper presents an equivalent method to identify the prognostic and diagnostic biomarker SPRR3 for OSCC, and advanced bioinformatics tools and methods have been applied. However, the main issues are as follows,

1. The scientific expression should be better polished by someone with a good knowledge of English, preferably a native speaker to help proofread.
2. More evidence demonstrates the role that SPRR3 played in tumorigenesis is supposed to provide to make the conclusion more convincing
3. Make sure all the figures are clearly labeled to help readers better understand


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: IDENTIFICATION OF SPRR3 AS A NOVEL DIAGNOSTIC/PROGNOSTIC BIOMARKER FOR ORAL SQUAMOUS CELL CARCINOMA VIA RNA SEQUENCING AND BIOINFORMATIC ANALYSES
Review round: 2
Reviewer: 1

Output:
Basic reporting: /
Experimental design: /
Validity of the findings: /
Additional comments: The revised manuscript is much more improved and the workflow is explicitly displayed.
If authors used trim galore to remove the adapter of the raw reads, this information should be added. The key parameters of trim galore and HISAT2 are also disclosed.


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: IDENTIFICATION OF SPRR3 AS A NOVEL DIAGNOSTIC/PROGNOSTIC BIOMARKER FOR ORAL SQUAMOUS CELL CARCINOMA VIA RNA SEQUENCING AND BIOINFORMATIC ANALYSES
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The revised manuscript addresses most of the issues raised in the review. In particular, the motivation of the study was further explained and many of the methodological details were added. Moreover, proper citations were added to the tools and datasets that were used in the study. Finally, references to the data repository where the raw data would be hosted was provided and documentation for the use of human samples were submitted to the journal.
One critical issue remains however. The initial step in the workflow, namely the differential expression analysis doesn’t inspire confidence. The shape of the volcano plot (the only provided diagnostic) is off. It’s unlikely that the small sample size is the reason. Given the importance of this step, especially because the downstream steps are all based on its results, a proper investigation should be conducted. For example, performing the differential expression with alternative models (design matrices) and showing more analysis prognostics.


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: CRISPR 2 PCR AND HIGH RESOLUTION MELTING PROFILING FOR IDENTIFICATION AND CHARACTERIZATION OF CLINICALLY-RELEVANT SALMONELLA ENTERICA SUBSP. ENTERICA
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: This study has been well done. However, Major revisions are needed. First, there are many errors of program and syntax in the manuscript which should be entirely revised. Second, the originality of the work is very limited. The authors have performed quite similar studies on the serotyping of Salmonella with similar methods in recently published articles. Third, it is not easy to understand the inconsistence of the number of Salmonella isolates (Line 27:26 isoaltes, Line 36: 68 isolates,Figure 7: 64 patients).


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: CRISPR 2 PCR AND HIGH RESOLUTION MELTING PROFILING FOR IDENTIFICATION AND CHARACTERIZATION OF CLINICALLY-RELEVANT SALMONELLA ENTERICA SUBSP. ENTERICA
Review round: 1
Reviewer: 2

Output:
Basic reporting: Wisitipanit and colleagues describe in their paper a technic to type and characterize Salmonella enterica subsp. enterica Enteritidis, Typhimurium and its monophasic variant. The text is well-written and the introduction brings the sufficient basis for the reader to understand the context.
Experimental design: The experiments are very well described and detailed.
Validity of the findings: no comment
Additional comments: The text should be amended to have the species and the gene name in italics.


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: CRISPR 2 PCR AND HIGH RESOLUTION MELTING PROFILING FOR IDENTIFICATION AND CHARACTERIZATION OF CLINICALLY-RELEVANT SALMONELLA ENTERICA SUBSP. ENTERICA
Review round: 1
Reviewer: 3

Output:
Basic reporting: The English language in the manuscript should be improved to clearly understand what the authors wanted to say. Grammatically it should also be very sound.
Experimental design: I have no issues with the authors experimental design for this study.
Validity of the findings: In materials & methods, the authors mentioned that they used 28 stool samples (line 107) but in the results section they only mentioned about 26 isolates (lines 252,294, 310). They also mentioned 16 isolates dominated in 3 HRM profiles but the numbers they provided did not prove that (line 253-254). It is suggested that they should check with all the numbers and percentages they provided in first paragraph of the results section.
In line 264 while explaining figure 2, they wrote isolates 458 and 378 but in actual figure it says 454.
Figure 4 does not have full legend.
In figure 5 legend, they mentioned about 25 clinical isolates but in the results sections they wrote 26 isolates. They also did not show A and B in their original figure.
Figure 6 was not labeled as A, B and C.
Additional comments: In the discussions, the authors need to provide more explanations about why their methods are better than the existing methods. They also need to write in more detailed way the advantages and limitations of their assay. They also should mention what can be done to improve it further. In the discussions, I would suggest them to describe the scope of their assays.


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: CRISPR 2 PCR AND HIGH RESOLUTION MELTING PROFILING FOR IDENTIFICATION AND CHARACTERIZATION OF CLINICALLY-RELEVANT SALMONELLA ENTERICA SUBSP. ENTERICA
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: not 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: CRISPR 2 PCR AND HIGH RESOLUTION MELTING PROFILING FOR IDENTIFICATION AND CHARACTERIZATION OF CLINICALLY-RELEVANT SALMONELLA ENTERICA SUBSP. ENTERICA
Review round: 2
Reviewer: 2

Output:
Basic reporting: The language is very clear and professional this time.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: The authors have put so much effort to make the manuscript a better one than the previous version. The manuscript is much better understood with all corrections that the reviewers suggested. They also added legends and modified some figures based on reviewers' comments. This version of manuscript is a good read now.


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: INBREEDING DEPRESSION IN ONE OF THE LAST DFTD-FREE WILD POPULATIONS OF TASMANIAN DEVILS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript was clearly written and the figures were well labeled. However, I had some trouble understanding some of the statistics relating to regression of IR to fitness (lines 109-115). I think if you provided the regression equation in the text, this would clear things up.
Experimental design: Overall, the design of the work is solid, but I think there is some opportunity for expanding the analyses. Were all 168 devils that were genotyped (line 84) adults? If not, it would be very interesting to identify parent offspring pairs each year, as this would provide an estimate of male reproductive output. You could even try to understand the accuracy of the estimate by comparing the field estimates of female reproductive output to estimates made from a genetic parentage analysis. In the same line of thinking, even with all adult samples you could examine your data for parent-offspring pairs across years and compare IR between parents with and without surviving offspring.
Validity of the findings: Although the authors seem to have verified their genotyping well (repeated genotyping of the same individual, use of microchecker), it is difficult for me to assess the validity of the regression results provided in table 2. At a minimum, I’d expect to see R2 estimates included in this table.

In addition to this, the comparisons over years is confusing. In the text, the results are a non-significant slope (line 122) between IR and year and Table 2 displays a significant relationship between year and the number of joeys. How much of the marginally significant relationship between IR and number of joeys is due to the effect of year (figure 2)? This should really be statistically addressed by using interactions in your statistical model, random effects, or some other method.

Finally, I am curious why a single genetic statistic – internal relatedness – was used in this study. I would like to see this potentially interesting result built out a little to really sell this as being the whole picture of this population. For example, what about other estimates of inbreeding (Fis, Queller and Goodnight’s R), do they also relate to fitness? What were the drivers of the year-to-year differences in fitness (if abiotic data can be obtained, for example)? These and many other analyses could make this paper much more substantial.
Additional comments: Overall, I think this manuscript has merit, provided the authors clarify their statistical approach, and shore-up the issues noted above. In addition to this, I caution the authors on overselling their inferences. For example, the first paragraph of the discussion includes statements suggesting that devils may be “close to the tipping point” of the inbreeding depression spiral, despite the marginal significance noted in figure 2. This paragraph, and really the intense tone of the entire paper, is somewhat contradictory to the strength of the findings.


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: INBREEDING DEPRESSION IN ONE OF THE LAST DFTD-FREE WILD POPULATIONS OF TASMANIAN DEVILS
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: see general comments to the author
Validity of the findings: see general comments to the author
Additional comments: The introduction sets up the paper by suggesting that inbreeding depression can affect population persistence (which it can in some cases). The paper then tests for an association between heterozygosity and an individual fitness component (number of offspring born to a female). However, it is not discussed whether this fitness component strongly affects population growth in devils; it is thus not known whether the observed inbreeding depression detected is important to population persistence.  This issue is relevant to much of the Discussion also. I suggest trying to address this with methods borrowed from population ecology (i.e., a sensitivity analysis) if at all possible; this would really be a great addition to devil population dynamics and management.

82: Is there good evidence that the count of active teats is actually a reliable measure of the number of offspring? Can a single offspring result in multiple active teats? What proportion of reproduction estimates are based on the count of active teats?

It is said that the microsatellites are neutral (line 89), and this may well be true. However, I assume it is unknown whether each of these loci is closely linked to loci that are responsible for some of the variance in fitness, in which case their evolution would be affected by linked selection. I suggest just saying that microsatellites were used, and that the analyses assume that they are unaffected by natural selection.

121-122: One of the findings in this study is that IR remained temporally constant. This finding could be sensitive to how the allele frequencies were calculated, so I suggest clarifying this. What year classes of samples were used to calculate IR for all of the individuals? If IR was calculated based on allele frequencies estimated for each year class of individuals separately, then IR may remain roughly constant through time even if heterozygosity has been lost through time. I suggest showing a plot of multiple-locus heterozygosity versus year (which does not account for allele frequencies), and testing if this showed a temporal decline in the study population.

164-166: You would really need to quantify the effect of the observed inbreeding depression for litter size on population growth rate to make this useful for population management. It seems like the data on vital rates needed for a sensitivity analysis (to assess the importance of litter size to population growth) may well available for devils given the intensity of the monitoring program.


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: INBREEDING DEPRESSION IN ONE OF THE LAST DFTD-FREE WILD POPULATIONS OF TASMANIAN DEVILS
Review round: 1
Reviewer: 3

Output:
Basic reporting: No comment.
Experimental design: I made a few suggestions regarding the analysis for the authors to consider.

I think more explanation and detail should be provided regarding how the litter count data were obtained in the field.
Validity of the findings: No comment.
Additional comments: Gooley et al. present a straightforward study investigating inbreeding effects on reproductive data from Tasmanian devils.  In general the study is well done and the paper is well written. I especially appreciated the concise manner in which the paper is written.  I had several comments for the authors to consider that, if addressed, might help with clarity of methods and might provide additional information from the data and analysis. They are listed in order of importance.

1. Line 109-113: Using binomial regression in this fashion seems like a reasonable approach.  However, I think there might be value to considering probability of parturition in a given year and litter size separately. For probability of giving birth you could use a binomial response of 0 = did not give birth and 1 = gave birth to at least 1 offspring.  For litter size, you could cumulative logit regression for the devils that did produce young (response 1-4).  See Hostetler et al. 2012, Oecologia 168:289-300 for an example of a study addressing very similar questions as yours with inbred and outbred Florida panthers. They first modeled probability of giving birth and then modeled litter sizes using the regression approaches I mentioned.

My reasoning for this suggestion is you have many, many 0’s in your dataset as evidenced by Fig. 2.  Do you know anything about the nature of those 0’s?  It seems like what you have modeled might not actually be litter size but rather a combination of probability of breeding/parturition and litter size.  By modeling these separately you would know whether inbreeding influences 1) the probability of giving birth at all and 2) litter size.  Given all those 0’s and the fact that it looks like most females that actually had young had 3 or 4 offspring, it may be that inbreeding is actually affecting the probability of giving birth rather than litter size per se.

2. Lines 79-84: Can you be a bit clearer and provide more detail about the litter size evaluation methods?  It sounds like either you 1) observed young in the pouch or 2) counted the number of active teats.  Can you explain the timing of these young and teat observations relative to the birth of the offspring?  Is there any chance some young had died prior to you observation of the actual offspring in the pouch?  Do the number of active teats accurately reflect litter size and is this unambiguous to assess in the field?  How long can an active teat be detected after the young are born?

3. 109-115: Were each of your 90 datapoints for the regression analysis litter counts for different individuals or do you have multiple litter counts from some females?  If that latter, those data would obviously not be independent and would need to be accounted for with a random effect of individual.

4. Line 125-128: I think it would be worth doing a simple model selection process (e.g., using AIC) to see which of the variables are retained in the model or models with the strongest support. You appear to have strong effects of year and inbreeding but it would be good to see what the best models were.

5. Line 96-100: Did you also estimate the other individual-level heterozgosity metrics that can be estimated using Rhh? Were these also strong predictors of reproductive values?

6. Line 40: I suggest revising and removing the word “rallied”. I suggest simply explaining the research and conservation actions that have been undertaken.


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: INBREEDING DEPRESSION IN ONE OF THE LAST DFTD-FREE WILD POPULATIONS OF TASMANIAN DEVILS
Review round: 2
Reviewer: 1

Output:
Basic reporting: I wonder if something is missing from the introduction. Perhaps this is obvious to the authors, but it took me until the middle of the second round of reviewing to think that maybe the reason this population does not have DFTD is because of its extreme isolation. If this is indeed the case, I suggest the authors put this information up front in the introduction, as it really does speak to the likelihood of increasing severity of inbreeding depression. To me, this is a much more compelling line of thought than the wild vs. captive inbreeding depression effects currently put forth in the text.

Line 33: “in areas infected by an infectious clonal cancer” the 2 x infect* is a little confusing to read. Please consider rephrasing.

Line 103: Should be ‘Sambrook’ instead of ‘Sanbrook’
Experimental design: The questions are clearly stated and analyses conduct are robust. One minor thing is that I do not understand the format used for providing the regression equations. Shouldn’t there be coefficients with subscripts to denote individuals and error terms in the equations?

Secondly, and perhaps more importantly, I am still a little unsure about how to separate the year effects from the IR effects (displayed in Table 2). The year-to-year effects seem much stronger than the IR effects, which is troubling because the authors did not see increasing inbreeding over time (lines 203-204). How much do the environmental effects influence fitness? Including year as a random effect in the model may allow the authors to tease these the IR and environment effects apart.
Validity of the findings: Given the relatively weak relationship between IR and fitness, I am not sure it makes much sense to supplement the population, as that is not without risk. I suggest this be toned down in the paper.
Additional comments: I am overall happy with the revisions the authors have made to their manuscript.


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: INBREEDING DEPRESSION IN ONE OF THE LAST DFTD-FREE WILD POPULATIONS OF TASMANIAN DEVILS
Review round: 2
Reviewer: 2

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


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

Input:
Title: COMBINATION OF FERRIC AMMONIUM CITRATE WITH CYTOKINES INVOLVED IN APOPTOSIS AND INSULIN SECRETION OF HUMAN PANCREATIC BETA CELLS RELATED TO DIABETES IN THALASSEMIA
Review round: 1
Reviewer: 1

Output:
Basic reporting: The study was well designed and performed meeting the criteria of the journal.
Experimental design: This a nice designed and performed study using quite difficult methodologies.
No external validation of the method was performed.
Validity of the findings: The study was well designed and performed but as no external validation was performed the validity of the findings is only on researchers responsibility.
Additional comments: In this study Rattanaporn et al. aimed to investigate the effect of the interaction of FAC on cytokines, IL-1β and TNF-α, in 1.1B4 human pancreatic β-cell line. The authors add new experimental data in the pathophysiology of DM development in patients with Thalasemia. As no direct measurements in humans as well as no external validation was performed, the discussion-conclusion (last two paragraphs) might be editing in this context.


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: COMBINATION OF FERRIC AMMONIUM CITRATE WITH CYTOKINES INVOLVED IN APOPTOSIS AND INSULIN SECRETION OF HUMAN PANCREATIC BETA CELLS RELATED TO DIABETES IN THALASSEMIA
Review round: 1
Reviewer: 2

Output:
Basic reporting: Iron overload may lead to dysfunction of pancreatic β-cell function and results in Insulin deficiency or resistance in β-thalassemia patients. Earlier studies have also investigated that elevated cytokines in pancreatic β-cells may damage the cellular integrity. In this manuscript, author have investigated iron supplementation together with cytokines to human pancreatic cell line and analysis of cellular anti-oxidant signaling and apoptotic cell death. This manuscript is well conceptualized and written, studies conducted with high glucose medium provides a physiological DM condition to test the hypotheses.
Experimental design: Authors need to conduct some additional experiments to improve the outcomes of this study. Some examples where the authors could provide more insight includes-
1.	Figure 1- Given that FAC mediated loss of cell viability leading to apoptotic death, it is important to include the expression of some apoptotic regulatory proteins to validate the data.
2.	Authors should include the flow plots for apoptotic death.
3.	To support the fact that FAC treatment leading to ROS elevation, authors need to conduct experiments with ROS quencher (N acetyl cysteine-NAC) and correlate the specificity.
4.	Figure 2- It is not clear in the text about interpretation for no effect of TNF and IL-1b with FAC treatment has no effect on any readouts. In contrast with high glucose (Figure 3) similar treatment has significant effect?
5.	Figure 3- What is the levels of Insulin mRNA with FAC alone treated cells incubated in high glucose medium?
Validity of the findings: Authors should test some of the experiments with more than one cell line to propose a broad effect of FAC. Experiments conducted with high glucose concentrations shows significant apoptotic death compared to normal medium in untreated cells but no change in cell count and ROS? Authors should explain these rationals in the text.
Additional comments: This study will highlight the combinatorial effect of iron overload with cytokine elevation during pathogenesis of DM associated thalassemia. Still authors need to add some more data that will further enhance the outcomes of this manuscript.