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: IN VITRO ANTICANDIDAL ACTIVITY AND GAS CHROMATOGRAPHY-MASS SPECTROMETRY (GC-MS) SCREENING OF VITEX AGNUS-CASTUS LEAF EXTRACTS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The MS “In vitro anticandidal activity and gas chromatography-mass spectrometry (GC-MS) screening of Vitex agnus-castus leaves extracts”, authored by Ibtisam Mohammed Ababutain and Azzah Alghamdi, describes the chemical characterization and the study of the anticandidal potential of the extracts extracted from leaves of Vitex agnus-castus plant. The protocol used in the chemical characterization is in agreement with literature procedures and the results obtained regarding the biological potential were quite promising. This work could be a relevant contribution in this direction and deserves publication in Peer J. However, there are some points to be addressed by the authors before a resubmission.
Experimental design: The research agrees with the Scope of the journal. The experimental part describes consolidated methodologies and promising results were obtained. However, in some cases the methods are not described with sufficient details or information to replicate. I suggest a detail review.
Validity of the findings: The results obtained are quite promising. However, they need to be better discussed. I suggest adding in the discussion the results obtained for the positive control indicating the potency and efficiency of the tested extracts. This comment applies to the results described in tables 1 and 2.
Additional comments: Major concerns:
#1. It’s important that in the Abstract other relevant information are included, besides those already presented, such as the majority compounds showed in the different extracts (ethanol, methanol and water).
#2. The leaves of plant were washed with tap water and dry for two days at room temperature (lines 102-103). Please, comment more about this. The ambiental conditions of room were control, like the humidity for example.
#3. Regarding the determination of the anticandidal efficiency of leaves extracts (line 150), how this was determined? Please, comment on this.
#4. The result and discussions are presents in a clear and objective way. However, I suggest adding in the discussion the results obtained for the positive control indicating the potency and efficiency of the tested extracts. This comment applies to the results described in tables 1 and 2.
#5. Regarding to lines 193-194 and 295-296, it was described that the extracts could be used as a promising source to develop an alternative anticandidal agent. Has any toxicological study been performed on this extract? Please add more details.

Minor points:
#1.  Make a comparative study between the chemical compounds found in those extracts with other works already described in the literature. Other extracts are used to treat candida infections? Can you add this information on the introduction?
#2. Make a careful review for typos. For example, page 1 line 31 and page 2 line 50.


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

Input:
Title: IN VITRO ANTICANDIDAL ACTIVITY AND GAS CHROMATOGRAPHY-MASS SPECTROMETRY (GC-MS) SCREENING OF VITEX AGNUS-CASTUS LEAF EXTRACTS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Text Corrections:

-Authors need to correct the words "cifrrii" for "ciferrii" in lines: 23 and 116.

-I would suggest to the authors to change or remove the sentence in lines 42-43, “…is possible to use this plant extracts in manufacturing of alternative anticandidal drugs.”
It is important to make sure that there are studies of toxicity, genotoxicity and mutagenicity of this plants to avoid adverse reaction.

-line 47; correct the sentence “…risk to human health. Due to their virulence, ability to survive…” for “…risk to human health, due to their virulence and ability to survive…”

-Table 1 – change de word “identfed” for “identified”

-Table 2 – only the first “ratio” written in the column has an “*”. Put on those two others also.

-Table 3 – In the third column, named as “Peak Area%”, authors must make clear what means “EL, ML, WL”, presumably refers to the extraction’s solvent.
Experimental design: - Authors have performed the determination of Minimum Fungicidal Concentration (MFC), which is an important and complementary result for Minimum Inhibitory Concentration (MIC).

- I miss in table 2 the use of a positive control, authors could use nystatin or fluconazole for example, as used by Oliveira et al.(http://dx.doi.org/10.1590/0001-3765201720170254)

- It is not clear in the section about Gas Chromatography-Mass Spectrometry (GC-MS), lines 155 to 168, that all 3 extracts were injected. Please provide more information.

- Authors claimed they calculated the ratio from MFC/MIC (Levison and Levison, 2009), the given reference says at page 792 that “…For bactericidal drugs, the MBC is usually the same as, and generally, not more than fourfold greater than, the MIC.”
The rations calculated by the authors (Table 2) seems to be not correct. Please recalculate them or provide better explanation.
Validity of the findings: The work has relevance once still lack of knowledge regarding VA-C against human Candida sp, as highlighted in lines 89-91. However, I am not convinced by the results and references provided that "... VA-C leaf extracts were superior to antibiotics in general, ..." as stated in the conclusions, lines 287-288. I suggest to remove this sentence.
Additional comments: Ababutain and Alghamdi, described the activity of Vitex agnus-castus (VA-C) leaves' extracts against three Candida species (Candida tropicalis, albicans and ciferrii), for that, they used three different extracts, from water, methanol and ethanol.

The work is relevant and well written. I can recommend this manuscript for publication if corrections are made, since the results obtained by the authors contribute to the knowledge of VA-C as a bioactive extract against the investigated pathogens.


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: IN VITRO ANTICANDIDAL ACTIVITY AND GAS CHROMATOGRAPHY-MASS SPECTROMETRY (GC-MS) SCREENING OF VITEX AGNUS-CASTUS LEAF EXTRACTS
Review round: 1
Reviewer: 3

Output:
Basic reporting: 	The manuscript is interesting since there is a worldwide trend in the study of natural extracts with biological activity, but as seen in the development of the text, there are many published works on the same topic, that is, this article is not unpublished.
	It should be clarified that candida is not yeast, it is a fungus.
	In materials and methods, I see that the concentration of the extracts was done in an oven and not in a rotary evaporator at low pressures, I think that extracts at 80 ° C can lose compounds or modify their structure, in the article by Amita Pandey, Shalini Tripathi (2014), the methodology is not indicated in the way that the authors of this article describe.
	Furthermore, with oven evaporation, the total evaporation of the solvents is not guaranteed, which leads to think that the antifungal action may be caused by the extract and residues of solvents such as ethanol.
	The way to express the concentration of the mixture of leaf powder and solvent is ambiguous and needs to be corrected.
	On line 111 is the number 33 that I don't make sense of.
	It is not written one μL is 1 μL.
	The list of References is very extensive since they placed the citations of table 3 of the “Compound nature and biological activities”.
	The English language should be improved to ensure that an international audience can clearly understand your text.
Experimental design: no comment
Validity of the findings: 	In the results it is not known if the antifungal effect is due to the essential oil of the VA-C extracts leaves or the solvent used for the extraction since it was not completely eliminated.
	The discussion should be reviewed and summarized as information is repeated.
	It cannot be stated with the result of the GC-MC analysis that the extracts have significant activity such as anti-inflammatory, anticancer, anti-alzheimer, anti diarrhea, anti-diabetic, anti-viral, antioxidant, anti-allergic, nematicide, antibacterial, antifungal, as well as other uses as food, for this you have to do studies.
	 In the text there is no information that leads me to Table 2, in addition this table lacks explanatory information.
Additional comments: Studying extracts with biological activity requires us to read several articles in order to design the methodology that we are going to develop in our research, we cannot use a method with which our results cannot be comparable.


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: IN VITRO ANTICANDIDAL ACTIVITY AND GAS CHROMATOGRAPHY-MASS SPECTROMETRY (GC-MS) SCREENING OF VITEX AGNUS-CASTUS LEAF EXTRACTS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The MS “In vitro anticandidal activity and gas chromatography-mass spectrometry (GC-MS) screening of Vitex agnus-castus leaf extracts”, authored by Ibtisam Mohammed Ababutain and Azzah Alghamdi, describes the chemical characterization and the study of the anticandidal potential of the extracts extracted from leaves of Vitex agnus-castus plant. The protocol used in the chemical characterization agrees with literature procedures and the results obtained regarding the biological potential were quite promising. This work could be a relevant contribution in this direction and deserves publication in Peer J. The authors made the necessary adaptions suggested in the previous review.
Experimental design: The research agrees with the Scope of the journal and the experimental part describes consolidated methodologies and promising results were obtained.
Validity of the findings: The results obtained are quite promising and after the changes made to the text, the results became clearer and more objective.
Additional comments: The authors made the necessary adjustments in the text and I recommend the publication of this MS in the current form.


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: IN VITRO ANTICANDIDAL ACTIVITY AND GAS CHROMATOGRAPHY-MASS SPECTROMETRY (GC-MS) SCREENING OF VITEX AGNUS-CASTUS LEAF EXTRACTS
Review round: 2
Reviewer: 2

Output:
Basic reporting: Authors properly performed all the given suggestions.
Experimental design: Authors properly performed all the given suggestions and clarified doubts.
Validity of the findings: Corrections were made.
Additional comments: As I stated previously, the work of Ababutain and Alghamdi is relevant.

Since all correction were applied and some doubts were clarified, I recommend this work for publication on PeerJ.


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: SERUM METABOLITE PROFILING OF A 4-NITROQUINOLINE-1-OXIDE-INDUCED EXPERIMENTAL ORAL CARCINOGENESIS MODEL USING GAS CHROMATOGRAPHY-MASS SPECTROMETRY
Review round: 1
Reviewer: 1

Output:
Basic reporting: The current manuscript describes the metabolomics based 4-Nitroquinoline-1-
oxide-induced oral cancer. The subject is interesting, especially due to the animal model used, which resembles the human oral cancer than the DMBA induced one. The work undertaken is substantial whereas the manuscript is well organized and written in a concise manner. experimentatal
Experimental design: The experimental design concerning the animals is rather adequate. Although there is a sufficient amount of experimental animals they were assigned to rather limited population groups (n=5 is marginal given also the fact that the experimental animal setup is not very difficult). The chemical analysis setup is well designed and executed but in my opinion the statistical treatment is not proper. Some specific comments can be found below
line 142 please refer the instrumental parameters used, the calibration procedure of the instrument as well as the mass accuracy obtained.
line 147 please refer the parameters used for data pretreatment e.g what was the mass accuracy in order to consider a metabolite as the same between samples or the time shift between peaks that should be aligned etc
line 155 no data appear on the statistical analysis e.g. are the the UV or Pareto treated how did the authors choose the number of the latent variables etc
line 172 did the authors use some kind of weighting the metabolite levels according to the body weight?
line 191 in order to validate the model as well as the metabolites up/down regulated further steps should be taken such as permutation testing ROC curves and FDR base tests. Did the authors use some QC samples in order to check the validity of the chemical and statistical analysis? Did they use some margin for the appearance of the metabolites in the samples e.g. if the metabolite is present to >50% (or any other percentage the authors consider arguably appropriate) of the samples?
line 194 please refer how did the authors identify the metabolites e.g. based on accurate mass or employing also the fragmentation patterns from the EI ionization.
line 264 In my opinion these results should be considered along with the diminished food intake of the rats that developed cancer. This could probably differentiate the rats in a more pronounced way.
Validity of the findings: no comment
Additional comments: line 57 function: please specify what kind of function is lost
line 57 healing:maybe this is healed?
line 73 higher please change the word to differentiated
line 77 high resolution not all NMR or MS instruments can be considered as high resolution and definitely not the HPLC. Please rephrase
line 78 metabonomics: please use only one term. It seems that currently metabolomics is the way to go.
line 107 please change to phenobarbital
line 115 please specify what HE is.
line 139 please correct 270oC, 260oC also leave a space before units. It is recommended to use a non breaking space
line 154 PLSDA is a multivariate analysis technique
line 232 please use only one term in order to avoid confusion (metabolomics / metabonomics)


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: SERUM METABOLITE PROFILING OF A 4-NITROQUINOLINE-1-OXIDE-INDUCED EXPERIMENTAL ORAL CARCINOGENESIS MODEL USING GAS CHROMATOGRAPHY-MASS SPECTROMETRY
Review round: 1
Reviewer: 2

Output:
Basic reporting: In this study, PLS DA were used to analyze the GC MS profile of serum using a 4NQO induced oral carcinogenesis rat model and to identify metabolic biomarkers in the development of oral cancer. The data indicates that GC based metabolomic analyses of plasma distinguish between oral premalignant and malignant lesions.

Comments
This is a nicely designed study and the statistical models are appropriate, and the methodology is reliable. The results of the study  are not explained well though. The manuscript could be made more interesting by these suggestions.
In the introduction line 75, the authors should mention the difference between metabolomics and metabonomics in a better way to educate the readers.
Line 72-73 needs a reference for the claim.
Line 80-81 states that 1HNMR combined with PLS-DA can distinguish
oral cancer from healthy serum samples. This sentence is ambiguous and needs to be rewritten.
Line 85-86.  How is GC MS better than NMR or LC MS or other methods? The claims about sensitivity and specificity needs references.
Experimental design: Spell check Line 139

What is the reason for sacrificing the animals at 9, 13, 20, 24, and 32 weeks from the beginning of the experiment? Explain in the methodology.

How were the metabolites identified? To elaborate on my question, was a metabolite classified as “identified” if it was found to be present in at least two samples of the same group? Include the criteria in the methodology.

Mention about the reference standards used to identify the 16 metabolites in the methods
Validity of the findings: Line 187-188 states that the growth of animal cycles on metabolite changes was greatly influenced, so we ultimately selected fourth and fifth sampling time points samples. I do not understand what the authors mean by this. Explain or remove the sentence.
Line 192; is Q2 really 762?
Line 197; what is the fold change? Specify.
Abstract mentions glycocine. Is it glycine?
The major drawback of this study is that there is no independent method to validate these findings. The authors should compare their results to these similar studies in the discussion.
https://doi.org/10.3892/ol.2014.2619
DOI: 10.3390/metabo9070140

The concentration variations of the discriminant metabolites at various stages of oral carcinogenesis compared to controls should be graphed.
Additional comments: The supplemental file with the excel data is not easily understandable.  Descriptions should be given for each data set. Alternatively, the raw data can be uploaded to metaboLights platform if the authors wish to do so. It is fine if they do not want to.


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: SERUM METABOLITE PROFILING OF A 4-NITROQUINOLINE-1-OXIDE-INDUCED EXPERIMENTAL ORAL CARCINOGENESIS MODEL USING GAS CHROMATOGRAPHY-MASS SPECTROMETRY
Review round: 1
Reviewer: 3

Output:
Basic reporting: The work of Ge et al. dedicated to the metabolic profiling of serum in animals treated with known compound that causes oral cancer. The authors divided treated animals to the subgroups in respect to the cancer progression, and compared their metabolic profiles with the healthy control. GC-MS methodology was used to perform metabolic profiling, and the data were analyzed using multi- and univariate analyses, that suggested 16 potential biomarkers for the oral cancer diagnostics.
The scientific premise of the work is scientifically sound. The work is of interest, and mostly written in a clear way. However, I have several concerns regarding the experiments design, and statistical methods, as well as main conclusions from this study.
Experimental design: The experimental design is not fully clear for me. The authors said that 38 animals were included to the study, but all results are based on the analysis of 30 animals. What happened to another 8? Did they die, or were excluded due to other reasons? I also did not really understand how the serum collection was performed. Metabolite turnover is a very fast process, so how fast serum samples were collected and frozen after animal death? I am also concerned about usage of sodium phenobarbital for metabolomics studies. Can the authors provide references in favor of this method?
For the statistical analysis the authors used the Student’s t-test and Wilcoxon-Mann-Whitney for differentiated analysis, however, it is not clear what 2 groups were compared, as before this the authors segregated “diseased” group to 5 sub-groups. Based on their PLS-DA results, I actually would recommend them to perform Dunnett’s test, as there are evidences (based on PLS-DA) that the differences between control group and most severe “scc” group can be stronger. It is also will make more sense considering the division to sub-groups. The results of fold-change between each of 5 groups vs control group can be nicely presented in a heat-map view.
Validity of the findings: If the results section is not detailed enough, the discussion section is very long and vague. I recommend the authors to largely reduce the 1st paragraph of the discussion as it has weak connection to the main goal of their study. Next, arachidonic acid as a fatty acid is not usual metabolite for the method of extraction, derivatization and column that authors used. The methodological set-up of the authors is optimized for polar compounds. Indeed, it is possible to detect FAs under these conditions, but I am concerned about repeatability and preciseness of this. Was arachidonic acid confirmed by analytical standard?
My last major comment is about the PLS-DA role in biomarkers discovery written in both conclusion section and abstract. This is the very well-known fact. It does not mean that we do not have to use it, but I think it is weak to claim as the main conclusion. I think, the biological findings of the authors about alterations in citrate, lactate, pyruvate are much more important and exiting. So it is not “PLS-DA could be used to screen potential biomarkers”, it is “metabolic profiling could be…”
Additional comments: Minor points:
1) Typos (no spaces before references, row 63 – no space before quotation, row 86 – dot, row 139 – degree signs)
2) I am not sure that the statement about novelty of the GC-MS application to oral cancer studies is correct. For example, Yang et al. (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7174902/) used it. It is true that their experimental design was different, but I think it should be corrected. Also, if the authors want to emphasize the role of GC-MS vs other metabolomics approaches (works that used LC-MS for oral cancer: https://www.nature.com/articles/srep31520, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5327379/) they should explain the benefits and limitations of GC-MS better.
3) Row 150, “total chromatogram” should be changed to “total ion current”
4) Available “reference compounds” (row 166) should be listed in the Materials section
5) Please use similar style for text organization (margins for a new paragraphs)
6) Since metabolomics is the main part of this work, I think it is necessarily to add more details about the results, such as how many metabolites were detected in total by GC-MS.
7) Row 198 – citric acid is listed twice.


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: SERUM METABOLITE PROFILING OF A 4-NITROQUINOLINE-1-OXIDE-INDUCED EXPERIMENTAL ORAL CARCINOGENESIS MODEL USING GAS CHROMATOGRAPHY-MASS SPECTROMETRY
Review round: 2
Reviewer: 1

Output:
Basic reporting: The  authors have made some cosmetic improvements to the  MS in their revised version.
Specific comments
Change metabonomics to metabolomics in abstract
Lines 87-92 do not make sense; rephrase.
Add ref for Line 292
Line nos 230 and 308; the conclusions about diagnostic potential is far fetched since this is only a chemical induced animal model. I suggest this be removed.
Experimental design: no comment
Validity of the findings: The lack of independent methods for validation of the results still remains a caveat but may be addressed by other  comparative studies.
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: SERUM METABOLITE PROFILING OF A 4-NITROQUINOLINE-1-OXIDE-INDUCED EXPERIMENTAL ORAL CARCINOGENESIS MODEL USING GAS CHROMATOGRAPHY-MASS SPECTROMETRY
Review round: 2
Reviewer: 2

Output:
Basic reporting: I would like to thank the authors for their attempts to improve the manuscript according to my and other reviewers suggestions. I think the work has benefited by these changes.
Experimental design: However, I still miss some information:
1)	Thank you for clarifying experiment design and providing the exact number of animals per each group. But why PLS-DA image itself does not include all these animals? Do I miss something when see 25 samples (dots) only on the plot in the figure 2?
2)	I do not insist on Dunnett’s test (although it is very conventional too), but it is still not clear which two groups were compared by M-W test? Please provide the description and N for each tested group.
Validity of the findings: Conclusions were improved.
Additional comments: Minor points:
end of introduction: appliedto -> applied to
thought the text: space between words and references
if arachidonic acid was confined by external standards, it has to be included to the Method section (so far only citric acid is listed as used external standard)


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 AND MOLECULAR CHARACTERIZATION OF MUTATIONS IN NUCLEOCAPSID PHOSPHOPROTEIN OF SARS-COV-2
Review round: 1
Reviewer: 1

Output:
Basic reporting: The article is well written.
The literature should provide a more detail discussion about other mutations found in the genomic sequences of SARS-CoV-2. for example see Korber et al., 2020 and Pachetti M. et al., 2020
Article structure, figures and table are well presented.
The manuscript is not self-contained. The results are relevant to the hypothesis.
Experimental design: The manuscript is within AIMS and Scope of PeerJ.
Research questions and details are relevant and well defined.
The investigation was performed abiding to a very good technical standard.
Methods are described very well.
Validity of the findings: The findings are interesting and novel. All the data have been provided and the conclusions are well presented and clear.
Additional comments: The manuscript "Identification and molecular characterization of mutations in Nucleocapsid Phosphoprotein of SARS-CoV-2" was conducted  to identify the variations present in N protein of SARS-CoV-2. the authors analysed 4163 reported sequence of N protein from United States of America (USA) and compared with first reported sequence from Wuhan, China. Our study identified 107 mutations that reside all over the N protein. Further, the authors show the high rate of mutations in intrinsically disordered regions (IDRs) of N protein. The study show 45% residues of IDR2 harbour mutations. The RNA binding domain (RBD) and dimerization domain of N protein also have mutations at key residues. The authors  further measured the effect of these mutations on N protein stability and dynamicity and
our data reveals that multiple mutations can cause considerable alterations. Altogether, the data strongly suggests that N protein is one of the mutational hotspot proteins of SARS-CoV-2 that is changing rapidly and these mutations can potentially interferes with various aspects of N protein functions including its interaction with RNA, oligomerization and signalling events. Overall, he article is well written. Article structure, figures and table are well presented. The results are relevant to the hypothesis. The findings are interesting and novel. All the data have been provided and the conclusions are well presented and clear.
To provide a little more context to their findings, the authors should provide a more detailed discussion about other mutations found in the genomic sequences of SARS-CoV-2, including Korber et al., 2020 and Pachetti M. et al., 2020


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 AND MOLECULAR CHARACTERIZATION OF MUTATIONS IN NUCLEOCAPSID PHOSPHOPROTEIN OF SARS-COV-2
Review round: 1
Reviewer: 2

Output:
Basic reporting: The pandemic caused by SARS-CoV-2 has steered the scientific community globally to explore for strategies to combat mortality and morbidity caused by COVID-19. To tackle the situation there is pressing need to understand the evolutionary forces that shape the fitness of involved biomolecules. Therefore, this study holds importance as it presents the identification and characterization of mutations in a structural protein namely Nucleocapsid Phosphoprotein (N protein) that interacts with SARS-CoV-2 genome and mediates its packing.
Utilizing bioinformatics approach authors identified 107 mutations in N protein, categorized them based on location and used DynaMut web server to access their effect on the stability and conformation of the protein.
Introduction highlights the importance of N protein in the viral life cycle. It will in interest of authors to also mention articles that highlight the importance of N protein as a potential therapeutic target. The specific suggestions are mentioned along with other comments.
The article is well structured, English is clear, unambiguous, and professional. There are some minor issues that needs to be corrected. The details of which are provided along with other comments.
Experimental design: Overall, the article is well within the scope and it will be of interest to the readers of the journal. The article is well organized into sections and sub-sections. However, to be comprehensive recent therapeutic needs should be included.
To increase the impact of the manuscript, phylogenetic analysis can be incorporated and the evolution of N protein can be discussed. Is it directed towards more flexibility or rigidity and its implications? How these changes shape virus fitness and its virulence?
Validity of the findings: The authors performed sequence alignment to identify mutation in N protein and then subjected them in Dynamut webserver to predict the effect on protein stability.

Authors can discuss how these changes in turn effect viral nucleic acid or related functions. Authors can also highlight how their work fulfills the knowledge gap in this area.
Additional comments: General Comment: Overall, the study is interesting and a useful addition to the area. The current study reveals mutations occurring in N protein and its impact on the protein stability, which is not presented in previous research. However, there are certain things that can be addressed to bridge the knowledge gap. A revision is recommended for improving the manuscript.

Authors show top 3 mutations that show rigidification in structure and another top 3 mutants that show gain in flexibility (Figure 2). These mutations have contrasting effects. How this will affect the function of N protein (RNA packaging or other function) needs to be discussed. What benefits the viral genome, making N protein more flexible or rigid? These effects can be discussed in regard to specific regions of N protein - whether IRD, RBD, or Dimerization domain where mutation is present.

A phylogenetic analysis could be performed with the available sequences using the initial sequence (from Wuhan, China) as root to see how the sequences have evolved over time. Are there any specific residues that are selected over time? What is the effect of these selections on N protein conformation?

Introduction: There are some important articles regarding N protein that can be referred. For instance, the abundant expression of N protein during infection and it being a highly immunogenic and a potential vaccine target.
References:
Ahmed SF, et al. Preliminary Identification of Potential Vaccine Targets for the COVID-19 Coronavirus (SARS-CoV-2) Based on SARS-CoV Immunological Studies. Viruses. 2020 Feb 25;12(3):254. doi: 10.3390/v12030254. PMID: 32106567; PMCID: PMC7150947.
Tai W, et al. Characterization of the receptor-binding domain (RBD) of 2019 novel coronavirus: implication for development of RBD protein as a viral attachment inhibitor and vaccine. Cell Mol Immunol. 2020;17(6):613-620. doi:10.1038/s41423-020-0400-4
Zeng W, et al. Biochemical characterization of SARS-CoV-2 nucleocapsid protein. Biochem Biophys Res Commun. 2020 Jun 30;527(3):618-623. doi: 10.1016/j.bbrc.2020.04.136. Epub 2020 Apr 30. PMID: 32416961; PMCID: PMC7190499.
Shang B, et al. Characterization and application of monoclonal antibodies against N protein of SARS-coronavirus. Biochem Biophys Res Commun. 2005 Oct 14;336(1):110-7. doi: 10.1016/j.bbrc.2005.08.032. PMID: 16112641; PMCID: PMC7092910.
Liu SJ, et al. Immunological characterizations of the nucleocapsid protein based SARS vaccine candidates. Vaccine. 2006 Apr 12;24(16):3100-8. doi: 10.1016/j.vaccine.2006.01.058. Epub 2006 Feb 8. PMID: 16494977; PMCID: PMC7115648.

Page 3, Line 76-77: It will be appropriate to refer these recent studies as well. It discusses about N protein contribution in the formation of helical ribonucleoproteins during the packaging of the RNA genome, regulation of viral RNA synthesis during replication and transcription and modulation of metabolism in infected individuals.
Cong Y, et al. Nucleocapsid Protein Recruitment to Replication-Transcription Complexes Plays a Crucial Role in Coronaviral Life Cycle. J Virol. 2020 Jan 31;94(4):e01925-19. doi: 10.1128/JVI.01925-19. PMID: 31776274; PMCID: PMC6997762.
Astuti I, Ysrafil. Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2): An overview of viral structure and host response. Diabetes Metab Syndr. 2020 Jul-Aug;14(4):407-412. doi: 10.1016/j.dsx.2020.04.020. Epub 2020 Apr 18. PMID: 32335367; PMCID: PMC7165108.

Some additional minor changes that will improve the manuscript are as as follows:
Page 3, Line 80: An appropriate reference for the statement is required.
Page 3, Line 81-86: To make text consistent either use N- and C-terminus or N and C terminus. Also, keep it uniform throughout the manuscript.
Page 3, Line 82: In the sentence “The domain present towards the N- terminus also known and RNA binding domain...”, replace it with “The domain present towards the N-terminus is also known as RNA binding domain…”.
Page 3, Line 95: Remove extra spacing “NCBI-virus- database”
Page 3, Line 97: In the sentence, “…..sequences of N protein are being deposited from USA.” It will appropriate to change it to “…..sequences of N protein were deposited from USA.”
Page 4, Line 103, 113, 131, 166: “programe” needs to be changed to “program”.
Page 4, Line 114: Please include spacing in between 6VYO and the reference.
Page 4, Line 123, 124, 135, 145, 150, 156, 157, 159: Please be consistent in using either N protein or N-protein. Also, keep it uniform throughout the text.
Page 6, Line 177-178: The text is redundant as it is already mentioned in page 4, line 117-118.

Discussion
Page 6, Line 199-211: The text presents almost the same information as presented in the introduction. It will better if this portion is removed entirely. Also, abrupt bringing of spike glycoprotein in picture might confuse the reader. Instead, authors can directly discuss their findings, the knowledge gained and its future application.
Page 7, Line 215: It will be appropriate to change “primary” to “primarily”.
Page 7, Line 233-236: It will be more informative if authors can mention specific residues of N protein to which these drugs interact and what mutations are present in those positions that author have identified.

Figures and Table Legends
Page 8: Line 250-253: The text looks redundant and needs rephrasing, as the same information is already described in page 3, line 81-86 and page 4, line 125-129.
Suggestion: The N protein comprising of 419 residues is shown. The RNA binding domain, dimerization domain, intrinsically disordered regions including IRD1, IRD2, and IRD3 are labelled.

Figure 2: It will more appealing to the readers if the panel labeling is done inside, instead of outside. It is suggested to move mutant name inside the panels. Also, author can label N- and C-terminus in figure panels. Also, keep all structures in the same orientation - Panel A seems to be in different orientation as compared to others.

Figure 3: Similarly, to Figure 2, it is suggested to move mutant name inside the panels. Also, measure atomic distances and label in corresponding panels. Legend should clearly mention what kind of interaction different dots indicate. For instance, the polar interactions are depicted in red dotted line, hydrophobic interaction in green. Only show important interactions and corresponding distances. It can be done by downloading PyMOL sessions instead of direct images from DynaMut web server. The interatomic distances can be measured in PyMOL.

Page 8, Line 270-273: Table 1: The following sentences seem redundant as author has already discussed it earlier in the text, “These mutations are present in the N protein of SARS-CoV-2 reported from USA. The first reported sequence of N protein from Wuhan, China was used as wild type sequence for this analysis. The table shows only those residues that have variation, rest of the sequences are identical among all samples.”. Suggestion is to remove it or rephrase.

The same text is repeated from Table 1 legend. Removal of these sentences is recommended in the following:
Page 9, Line 275-279: Table 2
Page 9, Line 282-285: Table 3
Page 9, Line 288-291: Table 4
Page 9, Line 294-297: Table 5

Another minor suggestion is to use consistent scheme for tables, either use smaller or capital letter in the header. In some places author has mentioned wild type residue while in other it is Wild type residue. Also, S.No in table 4 is S.No. in other tables.


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

Input:
Title: IDENTIFICATION AND MOLECULAR CHARACTERIZATION OF MUTATIONS IN NUCLEOCAPSID PHOSPHOPROTEIN OF SARS-COV-2
Review round: 1
Reviewer: 3

Output:
Basic reporting: 'no comment'
Experimental design: 'no comment'
Validity of the findings: 'no comment'
Additional comments: In this work, Azad Gajendra K performed sequence analysis of 4163 sequences of N protein of SARS-CoV-2 from United States of America to identify the variations present in it. This study identified 107 mutations those are distributed all over the N protein. The analysis also finds mutations in RNA binding domain (RBD) and dimerization domain of N protein. The effects of these mutations were predicted using DynaMut programe and concluded that some mutations could possibly hamper N protein functions. Overall, this is reasonable analysis that would advance our understanding of variations in SARS-CoV-2 genome. However, some changes could further improve the quality of the manuscript.

1)	What is probability of mutations- representing it in logo plot would be helpful.
2)	Show mutations (structural representation) in carton and surface diagram of crystal structure and highlighting mutated residues in one color for overall distribution of mutations in the protein.
The structure representation is not properly presented. It should be like that, for example a figure ligand be like this:
“Cartoon and surface representation of crystal structure of the ---- with mutations shown in red spheres (PDB ID:…..).  All structural representations are made using …… software (PyMOL ?  https://www.pymol.org)

3)	Figure 3 is really hard to interpret. Show side chain as a stick representation without any hydrogens (in structural figure generating program do- hide hydrogens-all) for better and clear figure generation. These current figures are outcome from DynaMut programe but should be generated using program PyMOL which is simple and can generate descriptive figures.


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 AND MOLECULAR CHARACTERIZATION OF MUTATIONS IN NUCLEOCAPSID PHOSPHOPROTEIN OF SARS-COV-2
Review round: 2
Reviewer: 1

Output:
Basic reporting: Well presented
Experimental design: Well defined
Validity of the findings: Novel and clear
Additional comments: The author has revised the manuscript carefully and resolved all the raised concerns. The work is appreciable. The revised manuscript in the current form seems suitable for publication. It will be of interest to the readers of the journal.


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

Input:
Title: IDENTIFICATION AND MOLECULAR CHARACTERIZATION OF MUTATIONS IN NUCLEOCAPSID PHOSPHOPROTEIN OF SARS-COV-2
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: In this revision, Author could have added reviewers suggested changes. I have suggested some structural figures changes but ignored. Also, I suggested to add weblogo diagram and similarly one reviewer suggest to add phylogenetic analysis but that is also ignored in the revision. This changes could immensely improve the quality of the 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: IDENTIFICATION AND MOLECULAR CHARACTERIZATION OF MUTATIONS IN NUCLEOCAPSID PHOSPHOPROTEIN OF SARS-COV-2
Review round: 3
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: In the current version, the incorporation of weblogo is a valuable addition. However, there are concerns regarding the logo and some suggestions for improvement of manuscript.
1)	Page 5, Line 138: The author needs to describe how the weblogo was generated. If referring to another study, briefly describe the procedure that author followed providing the details how many sequences used. Author should provide the multiple sequence alignment file or the set of N-protein sequences that was used to generate weblogo as supplementary data.

2)	Figure 1, Panel D: The presented weblogo needs to be updated. Is it generated from multiple sequence alignment of N-protein? The current weblogo is not representing the relative frequency of each amino at that position.
In the present context, the weblogo should basically be a graphical representation of an amino acid multiple sequence alignment of N-protein. As author has mentioned in the figure legend the overall height of the stack indicates the sequence conservation at that position, while the height of symbols within the stack indicates the relative frequency of each amino at that position. But relative frequency is not observed.

3)	Figure 1, Panel B and C: There is a suggestion to improve the quality of these figures. The author can download UCSF Chimera, which is free for academic research. It is easy to use and provides high resolution images. Author can render these structures in Chimera, use white background as in other figures and label residues shown in sticks. This will improve the clarity and quality. Also, mention the PDB ID in legend.

4)	Figure 3 and 4 panels: The DynaMut webserver provides either PyMol sessions or high-resolution images of predictions. The analysis of prediction using the PyMol session file and mapping the interatomic distances would have useful if author had license to PyMol. Although, the free source code is available but requires compilation of libraries and can be difficult. Author can try Pre-compiled Open-Source PyMOL, if well versed with command line.
https://pymolwiki.org/index.php/Windows_Install
https://www.lfd.uci.edu/~gohlke/pythonlibs/#pymol-open-source
Author seems to have used image files of prediction because of the limitations.


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 AND MOLECULAR CHARACTERIZATION OF MUTATIONS IN NUCLEOCAPSID PHOSPHOPROTEIN OF SARS-COV-2
Review round: 3
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Author have done some suggested changes, Accept the manuscipt. However this is strange that author is not able to download and follow instructions on how to use freely available software to scientific community.


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: DHA CONTENT IN MILK AND BIOHYDROGENATION PATHWAY IN RUMEN: A REVIEW
Review round: 1
Reviewer: 1

Output:
Basic reporting: The Manuscript 48138v1 is a review on DHA content in milk and biohydrogenation pathway in rumen. The manuscript is a review but it is difficult to understand exactly what is the objective of the review? It is difficult to find a reflection or input from the authors in each of their results and discussion paragraphs.
The review lack of structure and it is difficult to understand its rationale. For example, suddenly Fat-1 gene appears in the end of the document, but this is not explained in the introduction?  It just seems that authors just enumerated results from different documents but they do not discussed them with profound criticism. The manuscript should be revised for its English style.
Experimental design: Line 72 should say Methods or Methodology
Lines 73-79 provide range of dates or months you used for your search, provide data bases used i.e., scopus, web of knowledge, google scholar?
What do you mean by qualitative and quantitative articles?
Validity of the findings: Line 72 should say Methods or Methodology
Lines 73-79 provide range of dates or months you used for your search, provide data bases used i.e., scopus, web of knowledge, google scholar?
What do you mean by qualitative and quantitative articles?
Additional comments: The Manuscript 48138v1 is a review on DHA content in milk and biohydrogenation pathway in rumen. The manuscript is a review but it is difficult to understand exactly what is the objective of the review? It is difficult to find a reflection or input from the authors in each of their results and discussion paragraphs.
The review lack of structure and it is difficult to understand its rationale. For example, suddenly Fat-1 gene appears in the end of the document, but this is not explained in the introduction?  It just seems that authors just enumerated results from different documents but they do not discussed them with profound criticism. The manuscript should be revised for its English style.
Specifics:
Abstract
Lines 27-29 please re write and improve English structure,
Introduction
Line 49, delete, why will you have subtitles in the introduction?
Lines 42-48 please update references; there is a continuous debate on the pros and cons from consuming milk fatty acids
Line 72 should say Methods or Methodology
Lines 73-79 provide range of dates or months you used for your search, provide data bases used i.e., scopus, web of knowledge, google scholar?
What do you mean by qualitative and quantitative articles?

Tables 1 and 2. What is milk fatty acid (%), is it milk fat content? Please change accordingly
Check the use of decimals in your tables, also if control diets do not have fish oil or algae then use a “–“ which means the same and use it in all places where you do not have data. For decimals use 3 digits, for example 345, 3.45, 0.34


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: DHA CONTENT IN MILK AND BIOHYDROGENATION PATHWAY IN RUMEN: A REVIEW
Review round: 1
Reviewer: 2

Output:
Basic reporting: The review provides great insights about DHA metabolism and synthesis in animal organism. In addition, provides information about the use of DHA-rich products in dairy cows nutrition and its excretion in milk, and other tools to increase DHA amount in animal products. This subject is important nowadays and has been greatly studied, since human health can be improved by such findings. Therefore, I support the further publication of this review, after revision is carefully done. Hence, I recommend major revision.
Language is clear, with minimal errors that can be easily corrected. The introduction part is well-written, concise and clear.
Review is supported by adequate references, although some of them should be revised.
Some parts must be improved, as I stated specifically in the pdf attached.
Information provided in the tables should be revised and clarified by explaining how calculations were done. Please, revise the concepts of "milk fat content" and "milk fatty acid content".
Experimental design: Review brings important information, although some should be revised as stated.
Sources are adequately cited, with exception of some that I recommended to be revised.
Review is organized logically in adequate subsections and paragraphs.
Validity of the findings: One of the main problems is the information shown in the tables 1 and 2, where DHA content in milk is calculated based on the papers cited. It appears to be a confusion with "milk fat content" and "milk fatty acid content". Some inferences are made based on this results, so they must be revised before publication. I believe the calculus can not be done on this way to be published. However, if the author provides a feasible explanation and show in details below the table how the calculation was done, this data may be approved.
Additional comments: Review brings relevant information and after major revision. Please, read comments in the pdf attached.


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: THE RELATIONSHIP BETWEEN ANTI-MÜLLERIAN HORMONE (AMH) LEVELS AND PREGNANCY OUTCOMES IN PATIENTS UNDERGOING ASSISTED REPRODUCTIVE TECHNIQUES (ART)
Review round: 1
Reviewer: 1

Output:
Basic reporting: The article is well written and well divided.
Nevertheless, the article does not bring anything new to the subject that has been widely discussed in the last years.
The objective of the study was to investigate the relationship between Anti-Müllerian
hormone levels and pregnancy outcomes in patients undergoing in-vitro fertilization or intra-cytoplasmic sperm injection (ICSI), although, it does not match (not reflect) the title of the manuscript.
Experimental design: The biggest problem is the selection of patients.

The age of the patients (> 40 years) can in itself negatively influence the assisted reproduction outcomes, ends up becoming a study bias.

What was the total dose of gonadotropins used? Did all patients use the same amount, regardless of age? Outcomes should not be compared if different protocols or different amounts of gonadotropins were used.

What were the infertility causes of the patients? Ovulatory factors, endometriosis and severe male factor also negatively influence reproduction outcomes, influence AMH serum values and therefore are biases in the study. Did all patients have both ovaries?

The number of patients is another very important issue. The analysis of 50 or 42 patients is insufficient to analyze whether a serum measurement can be used as a biomarker.
Validity of the findings: It is very difficult to assess whether the results are valid in the face of so many biases found at the study.

You need to adjust the number of patients and especially the patient inclusion criteria.
Additional comments: No additional comments


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

Input:
Title: THE RELATIONSHIP BETWEEN ANTI-MÜLLERIAN HORMONE (AMH) LEVELS AND PREGNANCY OUTCOMES IN PATIENTS UNDERGOING ASSISTED REPRODUCTIVE TECHNIQUES (ART)
Review round: 1
Reviewer: 2

Output:
Basic reporting: Clarity is needed on lines 61-67. Is the intent in this paragraph to indicate that GnRH agonist induced gonadotropin levels correlate with antral follicle counts? Is this referring purely to endogenous gonadotropin in circulation?

Please include the trade names and catalogue numbers for the immunoassays for the purpose of comparisons to other similar studies in the literature.
Experimental design: The sample size of 42 analysable patients is very small for a study with correlative analysis and may be underpowered.

Please clarify what is meant by “non-parametric Pearson correlation”. Typically, Pearson correlation is parametric and Spearman-rank correlation is non-parametric.
Validity of the findings: Lines 167-174 if the p-value of the correlative analysis is greater than 0.05, then it indicates that there is no evidence of an association and that the slope of the line has arisen through random variation within the data. It is not appropriate to refer to these results as “weak correlations” or “weak negative correlations”.

On lines 120-121 the level of significance is indicated to be p < 0.05 but on line 181 it refers to a p-value of 0.065 as significant at the 10% level. This lack of consistency should be addressed.
Additional comments: The study by Umarsingh et al investigates whether AMH levels predict pregnancy outcomes in patients undergoing ICSI. Consistent with other studies the results show that AMH is not correlated with pregnancy outcome. At 42 participants, this is a very small study for correlative analysis and may explain why a large number of non-significant results were obtained, as there may have been insufficient statistical power. However, it is important that the authors do not refer to correlative analysis results with non-significant p-values as weak correlations (see comments below).


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

Input:
Title: THE RELATIONSHIP BETWEEN ANTI-MÜLLERIAN HORMONE (AMH) LEVELS AND PREGNANCY OUTCOMES IN PATIENTS UNDERGOING ASSISTED REPRODUCTIVE TECHNIQUES (ART)
Review round: 2
Reviewer: 1

Output:
Basic reporting: 1. Reviewer’s original comment: 1. Clarity is needed on lines 61-67. Is the intent in this paragraph to indicate that GnRH agonist induced gonadotropin levels correlate with antral follicle counts? Is this referring purely to endogenous gonadotropin in circulation?

Author’s response: Yes, it refers to endogenous gonadotropin circulation. As woman age, they have fewer oocytes (primordial follicles) remaining and they have fewer antral follicles. Antral follicle counts are a good predictor of mature follicles that can be stimulated in the ovary when stimulation medication is administered. Counting antral follicles using an ultrasound can be a subjective process. An ideal antral follicle count depends on the age of the woman; older women are not expected to have the same antral follicle

Reviewer’s response: Clarity is still needed in this paragraph, as no changes have been made. The intent of the authors is not clear and it needs to be redrafted. The authors response mentions antral and mature follicle counts which do not feature in the paragraph. It is not clear whether the authors are referring to endogenous gonadotropin or gonadotropin administered during stimulation cycles.

2. Reviewer’s original comment: Please include the trade names and catalogue numbers for the immunoassays for the purpose of comparisons to other similar studies in the literature.

Author’s response: The trade names of the compounds/drugs procured are listed in manuscript in Materials and Methods. However, for clarity the catalogue numbers are as follows:
• B84493 Access Sensitive Estradiol Reagent, 100 Determinations, 2 x 50 tests.
• B13127 Access AMH (Anti-Mullerian Hormone) Reagent, 100 Determinations, 2 x 50 Tests.
• 33520 Access hFSH Reagent, 100 Determinations, 2 x 50 tests

Reviewer’s response: These reagent details listed above have not been included in the methods section of the manuscript. The readers of the final article will need to know where the reagents came from if they want to replicate this study or compare it with others. The methods section of the manuscript refers to a manual 96-well plate format AMH Gen II ELISA (referencing Kumar et al. 2010) whereas the regent listed above is for an automated immunoassay. These two immunoassays have different properties. Which one was used? (state this in the manuscript)
Experimental design: Reviewer’s original comment: Please clarify what is meant by “non-parametric Pearson correlation”. Typically, Pearson correlation is parametric and Spearman-rank correlation is non-parametric.

Author’s response: Yes, we agree with reviewer and amended the manuscript likewise to Pearson correlation to avoid the confusion (L 116) with the readers.

Reviewer’s response: The manuscript methods section still reads “non-parametric Pearson correlation”. Pearson correlation is parametric. The authors have not made the change they claim above.
Validity of the findings: Reviewer’s original comment: 1. Lines 167-174 if the p-value of the correlative analysis is greater than 0.05, then it indicates that there is no evidence of an association and that the slope of the line has arisen through random variation within the data. It is not appropriate to refer to these results as “weak correlations” or “weak negative correlations”.

Author’s response: Yes, we agree with reviewers’ comment and completed necessary amendments as “not statistically significant” in the text (L 178)-red font

Reviewer’s response: If the p-value is >0.05 then there is no evidence of correlation. The manuscript still refers to these results as “correlations” in the results section. The requested changes have not been made, nor addressed with a rebuttal.
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: THE RELATIONSHIP BETWEEN ANTI-MÜLLERIAN HORMONE (AMH) LEVELS AND PREGNANCY OUTCOMES IN PATIENTS UNDERGOING ASSISTED REPRODUCTIVE TECHNIQUES (ART)
Review round: 3
Reviewer: 1

Output:
Basic reporting: -
Experimental design: -
Validity of the findings: -
Additional comments: My prior concerns have all been addressed


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

Input:
Title: SUPRASPECIFIC UNITS IN CORRELATIVE NICHE MODELING IMPROVES THE PREDICTION OF GEOGRAPHIC POTENTIAL OF BIOLOGICAL INVASIONS
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No Comment
Additional comments: Authors of this manuscript use supraspecific units for three invasive species to understand their invasive potential using correlative niche modeling. Authors explore an uncommon way of estimating ecological niches as minimum-volume ellipsoid, a kind of envelope method, than that of using a complex algorithm Maxent. The idea of modeling supraspecific units to improve the modeling potential is good. However, I have some suggestions to improve the manuscript.

1. Instead of using only envelope methods, it would be interesting to compare the similar modeling framework with complex modeling algorithms such as maxent.

2. Authors perform models for supraspecific entities such as U0, U1, U2 and UT. But the explanation only written in text is not very clear, If you can explain these in some cartoon figures, it will be easier for the reader. Because later, in the manuscript, in the results section, there is sudden mention of entity U5, which was not clearly mentioned earlier in the manuscript.

3. Figures need a lot of improvement. Map legend is almost not readable in figure 2. Also the black dots and green dots are not visible in the map. Also, figures presented in the manuscript are only of one species. I was expecting all the species figures in the main manuscript. Because part of the discussion section is devoted to comparing how this modeling framework differs based on species. But to refer to these, one has to go to supplementary material

4. Also there is no mention of how many occurrences were used for each supraspecific modeling entity.
You can certainly add a table for such details.


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: SUPRASPECIFIC UNITS IN CORRELATIVE NICHE MODELING IMPROVES THE PREDICTION OF GEOGRAPHIC POTENTIAL OF BIOLOGICAL INVASIONS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript is well-written; the literature is appropriated and timely; the submission is 'self-contained'.
Experimental design: It is an original primary research, well defined, relevant and meaningful. Methods are well described.
Validity of the findings: Underlying data were provided; conclusions are linked to the aims.
Additional comments: I am very happy by invitation to review the manuscript by Castaño-Quintero et al., that proposes using data from closely related species to better characterize fundamental niche of a given focal species and to investigate potential for biological invasions. The results support the proposition that, based on the assumption of ecological niche conservatism, integrating data from sister species (or from deeper evolutionary units) in niche modeling can improve predictions for invasive species. No doubt, this is a relevant and timely research issue, which approach can be applied to anticipate biological invasions and prevent their consequences. The text is well-written, and the methods are very robust to test their aims.  I have only minor comments related to some points that I think should be clearer.

1. May be it will be interesting to indicate (in the supplementary material) which are the supraspecific entities for each species U0; that is, which are the U1, U2, …I only saw how many species there were in the Figure 1, but you could include a table (for instance) with information of these species and the number of occurrence records for these entities in supplementary material.
2. Some objective explanation for retaining only the first three components from PCA? In the case of terrestrial variables, they only explained 88% of the variance. Why not investigate scree plots or apply Kaiser’s criterion? I am not sure (don’t remember now), but I think the NMTML package from De Andrade et al., 2020 you cited have some approach to select PCA components.
3.  Resolution of some figures should be improved. Figure 2, for instance, I couldn’t see the occurrence records.
4.  Page 31, complete the sentence “… and the green dots where the species.?”. See line 547.


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: SUPRASPECIFIC UNITS IN CORRELATIVE NICHE MODELING IMPROVES THE PREDICTION OF GEOGRAPHIC POTENTIAL OF BIOLOGICAL INVASIONS
Review round: 1
Reviewer: 3

Output:
Basic reporting: This  manuscript is well written, easy to read, and presented clearly with a few exceptions (which are noted below).  It is very concise, and would benefit from expanding both the Introduction and the Discussion a bit, in order to help situate their contribution in both applied and theoretical ecological contexts.

All the maps in the main document are difficult to read. I recommend enlarging them and/or trying different color schemes with higher contrast.
Figure 2. The legends on A and C , and the axes on B and D are too small to be legible.
Experimental design: The methods used are sound. The only aspect I question is , where is the evidence that niche traits are conserved among the “closely related” species used in this study? I think that should be clearly stated since this underlies the premise that is fundamental to their study, namely that niche traits are closely conserved, thus the addition of sister species should improve/expand the delineation of the fundamental niche and improve modeling of the realized niche.  Also, while descriptive data and R code to reproduce the PCAs are included, but raw data (or any data) used to make the models are not.
Validity of the findings: no comment
Additional comments: This  manuscript is well written, easy to read, and presented clearly with a few exceptions (which are noted below).  It is very concise, and would benefit from expanding both the Introduction and the Discussion a bit, in order to help situate their contribution in both applied and theoretical ecological contexts. For example, in the Abstract the authors state that “This approach could be used to model species with invasive potential but that have not yet invaded new regions.” I agree with this and it is a pity that they do not make more explicit ties to this idea in their paper (especially in the introduction and Discussion). This paper would be considerably strengthened if they spend more time on contextualizing their research in the introduction with respect to 1) the theoretical and methodological problem of realized niches being used in ENMs to delineate the fundamental niche, and, 2) how this leads to the failure to predict alien species invasions or the full extent of alien species distributions which in turn has negative ramification for early detection and eradication.
The methods used are sound. The only aspect I question is , where is the evidence that niche traits are conserved among the “closely related” species used in this study? I think that should be clearly stated since this underlies the premise that is fundamental to their study, namely that niche traits are closely conserved, thus the addition of sister species should improve/expand the delineation of the fundamental niche and improve modeling of the realized niche.  Also, while descriptive data and R code to reproduce the PCAs are included, but raw data (or any data) used to make the models are not.

I list some specific points below:
L 71-72 Clearly Define fundamental niche for readers unfamiliar with the concept.  It is already stated here implicitly. Also, it would be more consistent to refer to “existing” niche as the “realized” niche which is the standard term in the literature. (e.g. Soberon & Peterson, 2005, Barve et al., 2011)
L117-119- it would be helpful to briefly describe how these phylogenies were obtained
L133 I would rephrase this. You are not testing hypotheses of which areas have been historically accessible, rather you are making an assumption (which I am not arguing with) that ecoregions represent historically accessible areas to the organisms.
166-169-  it is not clear how these atypical environmental conditions/sink populations are identified, please elaborate. A citation would be nice.
Line 169- please elaborate what the cov. rob function does
Lines 171 and 172-  do you mean “weighting”? The use of the word “weighing” does not make sense in the context presented.
L197-199- this is unclear
236-243. This is plausible. However, how do we know the findings are not simply a statistical artefact of the sister species occupying the same geographic areas as the focal species?

General comment- you can state “all statistical analyses were carried out in R3.4.4, once in the manuscript.
All the maps in the main document are difficult to read. I recommend enlarging them and/or trying different color schemes with higher contrast.
Figure 2. The legends on A and C , and the axes on B and D are too small to be legible.

References:
SOBERÓN, JORGE, and A. TOWNSEND PETERSON. "INTERPRETATION OF MODELS OF FUNDAMENTAL ECOLOGICAL NICHES AND SPECIES’DISTRIBUTIONAL AREAS." Biodiversity Informatics 2 (2005): 1-10.
Barve, N., Barve, V., Jiménez-Valverde, A., Lira-Noriega, A., Maher, S. P., Peterson, A. T., ... & Villalobos, F. (2011). The crucial role of the accessible area in ecological niche modeling and species distribution modeling. Ecological Modelling, 222(11), 1810-1819.


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: SUPRASPECIFIC UNITS IN CORRELATIVE NICHE MODELING IMPROVES THE PREDICTION OF GEOGRAPHIC POTENTIAL OF BIOLOGICAL INVASIONS
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: This version of the manuscript is greatly improved. The authors included additional information in the Materials and Methods and in the supplementary material, which make the manuscript much clearer; changes done in the Introduction and in the Discussion provided a coherent narrative throughout the text. The Figures were also improved. I have no more suggestions to do.


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: SUPRASPECIFIC UNITS IN CORRELATIVE NICHE MODELING IMPROVES THE PREDICTION OF GEOGRAPHIC POTENTIAL OF BIOLOGICAL INVASIONS
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors addressed all my comments satisfactorily.
Experimental design: no comment.
Validity of the findings: no comment
Additional comments: Congratulations on your manuscript! It will be a nice contribution to species distribution modelling.


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: BINGLESEQ: A USER-FRIENDLY R PACKAGE FOR BULK AND SINGLE-CELL RNA-SEQ DATA ANALYSIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Basic
1. The Materials & Methods section contains far too much commentary that is more appropriate for the Results & Discussion section. Examples include lines 85 to 92 and all of the Related Applications subsection.
2. The phrasing is at times awkward and grammatically confusing. Examples include lines 16-17, 113-116, and 150-152. Careful proofreading and editing for clarity would improve the manuscript.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: General
1. The application itself is well designed and offers a great tool for RNAseq analysis.
2. Some minor application improvements could be made, including:
- Adding the ability to select other genomes outside of the mouse and human ones included for gene enrichment analysis
- Using plotly (via ggplotly) or another package to provide interactive plots in some sections to facilitate exploration of results.
- Adding a filterable gene data column (right now gene ids are provided as row ids)
- More information on longer loading screens, e.g. showing cluster progressing during differential expression analysis of scRNAseq data.


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

Input:
Title: EXPRESSION OF THE NEUROTROPHIC TYROSINE KINASE RECEPTORS, NTRK1 AND NTRK2A, PRECEDES EXPRESSION OF OTHER NTRK GENES IN EMBRYONIC ZEBRAFISH
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1. Overall, this manuscript is nicely written and data provided appear to be of high quality.
Although the paper is generally very well written, lines 213-214 could use some attention with respect to grammar. (Our data…., we found….).

2. Very minor point: The last sentence in the abstract is rather speculative, and might be best identified as such (We speculate rather than we suggest).

3. This study focuses on gene expression rather than genes themselves. Therefore it would be more accurate for the abstract (purpose) to state "Because expression of multiple genes", rather than stating "Because multiple genes were not found"

4. A minor point, but the first results sentence in the abstract uses a different tense than the remainder of that section. (find vs. found). Pointing out in case this is unintentional.
Experimental design: 1. The objective of the study was to learn which neurotrophin receptors are expressed during very early neural development in zebrafish. The methodology was exclusively in situ hybridization.  Although not necessary, if antibodies are available for any of the studied genes, use could strengthen the conclusions.  Based on a quick search within zfin.org, this appears to be the case for ntrk1 and ntrk2.
Validity of the findings: 1. The authors define spatiotemporal expression patterns. They conclude that only ntrk1 and ntrk2a are expressed at 16.5 hpf, whereas others become expressed by 24 hpf. They do a good job of relating and comparing their findings to past work.

2. Although this reviewer agrees that the spinal expression probably represents expression in RB neurons, the authors should describe what evidence this is based on.  If none, simply changing the language to state that the expression pattern is consistent with that of RB neurons is advised. If the authors feel that certainty is critical for their conclusions, additional markers should be used to demonstrate expression in RB neurons.

3. Overall the work could be strengthened by using alternative markers that support conclusions such as expression in cranial ganglia at 16.5 hpf, or in RB neurons at 24 hpf. Alternatively, softer language when making conclusions could circumvent the lack of direct evidence.
Additional comments: 1. This may be beyond the scope of the study, but the results and conclusions would be more interesting and valuable if expression were narrowed to specific cell types or regions. For instance, in the hindbrain (ntrk3b), which rhombomeres or cell types express?  This is not necessary, but would again add more value to the study.

2. One thing that could be more clearly stated was whether expression patterns listed on table 2 for BDNF, NGF, and NT-3 extracted from the literature, or were these experiments done by the authors?  The legend states “known expression patterns”, but this might be more explicitly stated to avoid confusion (along with citing those studies within the figure or legend if desired by the authors).

3. If the authors are looking to strengthen the discussion, the first paragraph (line 232) could be strengthened by more specifically pointing out at what stages these neurons are undergoing specification, differentiation, axon outgrowth, etc.  The statement that earliest specification occurs at 16.5 hours seems unsupported as written.

4. In the abstract, it could be more accurate to use the word functionality rather than specificity.  Does testing these at 24 hpf truly demonstrate that they are specific, or just that they can hybridize and produce signal?

5. Overall this is a nice study with quality images and reporting. I hope you will find some comments that you feel improve your paper.  -JH Hines


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

Input:
Title: EXPRESSION OF THE NEUROTROPHIC TYROSINE KINASE RECEPTORS, NTRK1 AND NTRK2A, PRECEDES EXPRESSION OF OTHER NTRK GENES IN EMBRYONIC ZEBRAFISH
Review round: 1
Reviewer: 2

Output:
Basic reporting: Major concerns:
1. The authors have described literature across species for Trk signaling, and raised the critical point about splice isoforms that their study addresses. However, it seems the authors have missed opportunities to discuss key recent literature in zebrafish that is relevant to their question. Indeed, they say that zebrafish “may provide a potentially useful model”, rather than discussing the literature showing just that – this would be a compelling assertion. For instance, Sahu et al. Sci Rep 2019 describes both morphant and mutant ntrk2b zebrafish and effects on particular neurotransmitter systems (perhaps there other knockouts/knockdowns to discuss?). The authors should also contrast their work with another recent study, Anand & Mondal J Neuro Research 2019.

2. The authors have done a commendable job describing their methodology, but in some places it can be condensed, for instance when describing standard kit directions. Additionally, much of the in situ hybridization protocol seems derived from the Thisse & Thisse 2008 Nature Protocols paper. The Methods section can be condensed if this is the case to highlight any differences the authors made from the “standard” protocol. At minimum, the Thisse & Thisse paper should be cited.

3. The authors state that raw data are included in the form of micrographs. However, it sounds as though they have produced novel in situ probes, which require additional validation to show specificity via sense probes (see below). I think it is most suitable to show sense probe staining in the paper; however, it is at minimum necessary to include in the supplemental data.

Minor comments:
1. I was confused by this sentence in particular in the discussion (lines 246-249): “Ligands and receptors are expressed in different cells so overlapping expression does not necessarily mean overlapping function..” and the surrounding paragraph was confusing. I believe the authors are trying to make a circumspect statement about potential BDNF signaling in the telencephalon via ntrk2b, but the language is confusing. Furthermore, the statement suggests that there cannot be autocrine signaling where there is at least some literature precedent for in neurons (McWilliams et al Cell Rep 2015) and particularly in synapses (Harward et al Nature 2016).

2. There are several uses of “data” as a singular noun, particularly in figure captions. Please revise to “data are”.
Experimental design: Major comments:
1. The authors show multiple orientations and magnifications of the expression patterns, which is useful to validate their findings and serve as a reference for the community. However, I am confused about why the magnified images do not always correspond to wide-field. For instance, Fig 1A shows the entire animal and one purple patch of staining, but 1D shows the same animal at higher magnification with two sets of cranial ganglia. There are also differences in 1B and E, 3 A and C, and 5B and D. Why do they appear different? Are they different animals?

2. On a related note, the proportion of embryos that show each staining pattern is not clear. The authors say “The data is [are] representative of results of 2-3 rounds of in situ hybridization with at least 25 embryos per round”. However, do all embryos show the exact same pattern? Even with well-established probes, staining is often variable and dependent upon length of incubation with NBT/BCIP. The authors could express this as a fraction of animals with that pattern vs other patterns, no staining, nonspecific staining etc. In general, more discussion of variability would be helpful to understand how distinct each expression pattern is.

3. For many of the images, the microscopy contrast is appropriate to see anatomical structures, but others (particularly at 16.5 hpf) are harder to discern. Labeling additional landmark structures that were used to identify the regions with staining would be helpful (e.g. letter abbreviations for structure in addition to the arrows/arrowheads pointing out the staining.)

Minor comments:
1. I assume the wide-field and magnified images corresponds to 4x and 10x, as described in the methods. However, scale bars would be very useful in the figure itself.

2. The authors explain the reasoning for hydrolyzing the probes in the discussion (more could be in the methodology), but note in the methods that ntrk2a was not hydrolyzed. Was there a particular reasoning for this? Is there an expected difference in expression patterns?
Validity of the findings: Major concerns:
1. As noted above, the authors seem to have developed a set of novel probes, based on their reporting of primer sequences. However, the authors do not perform the standard control for whole-mount in situ hybridization, in which DIG-labeled sense probes are used alongside the antisense probes for comparison. Even though their staining does appear clear and as predicted in particular subsets of neurons, the sense control should be included to validate these new probes, both for the paper and for the community.

2. Alternatively, a functional assay demonstrating at least one of their predictions would really underscore the validity of their expression studies. While a CRISPR mutant would take a long time to develop, the authors could instead pharmacologically antagonize Trk receptors with acute treatment based on the window of expression. The authors note that Trk/p75 genes are necessary for neuron survival – do the expected neurons persist following receptor antagonism in zebrafish? This might address the cross-talk question nicely.

3. The authors state that in the discussion that their hydrolyzed, full-length probes would have the ability to detect multiple splice variants in an unbiased manner, unlike previous probes based on cDNA fragments. While I agree with that idea, I don't understand how this would produce a more conservative expression pattern, instead of broader to encompass different variants. Did the authors try a side-by-side with the other probes? Could it be differences in the protocol, NBT/BCIP timing, etc. rather than the probes?

Minor comment:
The authors describe potential signaling mechanisms based on the expression patterns and appropriately note the limitations of their study for functionality within the discussion. Though they summarize previous experiments in Table 2, they could experimentally compare their receptor staining patterns to ligand expression with co-staining. However, at minimum they need to cite the papers in the table itself.
Additional comments: Overall, this study has potential to be valuable to the zebrafish community. More controlled experiments should be done to validate specificity and variability of the expression probes and, ideally, show some functional implication for the expression patterns. Suggested text changes include discussion of other relevant literature and some re-working of text and figures.


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

Input:
Title: EXPRESSION OF THE NEUROTROPHIC TYROSINE KINASE RECEPTORS, NTRK1 AND NTRK2A, PRECEDES EXPRESSION OF OTHER NTRK GENES IN EMBRYONIC ZEBRAFISH
Review round: 1
Reviewer: 3

Output:
Basic reporting: Overall the manuscript is well written and clearly states what was previously known about these neurotrophin receptors. Figures likewise are well labelled with various arrows and arrowheads helping to orient the reader to the structures of interest.

There were a few minor typos I found.
In line 92 beginning should be used rather than begin
In line 215 there is an unnecessary and between 24 hpf and (Figure 5)
In the figure legend for Figure 1 there is a space breaking up “cranial”
Experimental design: As one of the key novel findings of this paper is the 16.5 hpf expression data I think it would be helpful to have a little bit more explanation in the introduction as to why this time point was chosen and what knowing expression at this time point adds to the big picture.

While overall I thought the methods were well written and provided a lot of useful details I felt there were a few things had could be added.

I did not see it mentioned in the methods how the animals were originally fixed. This information should be added.

While it is clear later in the manuscript it would be helpful to explicitly state in the probe synthesis section of the methods that these probes originated from full length cDNA.

The authors note that the ntrk2a probe was not hydrolyzed while other the others were. Clarification as to why this was would be helpful.
Validity of the findings: I felt overall the figures clearly showed the expression that was mentioned in the manuscript. However, there were a few instances that the stated expression was not clear from the included figures. Also while I really liked table 2 I think some additional information and formatting could make it more useful.

Based on the images provided it is not clear to me that ntrk3a is localized to the Rohan beard neurons. There is some purple in the tail area but it does not seem localized to the spinal cord but rather a haze throughout the tail. Is there a clearer image of this expression?

For ntrk3b it is mentioned in the discussion and table 2 that it is expressed in the lateral line primordium. However, this is not mentioned in the results or clearly shown in the figures. Are there figures that better show primordium expression?

Table 2 does not seem to include complete expression patterns for the genes. For example, Rohan beard neuron expression is not mentioned. Also for genes expressed in multiple tissue types it would be helpful to have it specifically noted which of those tissues the different ligands are expressed in.
Additional comments: Overall this was an interesting manuscript providing new insight into expression of neurotrophin receptors at an important developmental time point that had not previously been investigated.


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

Input:
Title: EXPRESSION OF THE NEUROTROPHIC TYROSINE KINASE RECEPTORS, NTRK1 AND NTRK2A, PRECEDES EXPRESSION OF OTHER NTRK GENES IN EMBRYONIC ZEBRAFISH
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have nicely addressed comments and improve the manuscript.
Experimental design: No additional comments.
Validity of the findings: Although all reviewers raised several potentially interesting extensions or areas of investigation, the authors did a nice job focusing on the areas needed to solidify their results and interpretations.  For example, the addition of controls and validations such as sense probes improves the manuscript. I have no additional comments.
Additional comments: No additional comments


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

Input:
Title: EXPRESSION OF THE NEUROTROPHIC TYROSINE KINASE RECEPTORS, NTRK1 AND NTRK2A, PRECEDES EXPRESSION OF OTHER NTRK GENES IN EMBRYONIC ZEBRAFISH
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors have improved the manuscript in this revision by including some key papers for comparison and improving their figures and tables for clarity.

There are some typos in the following lines:
120, missing period
145, capital A
159-160, line break
273, "ntr2a"
Experimental design: The sense probe data has greatly strengthened this manuscript, and I commend the authors for tackling the difficult though necessary experiments to validate their probes.

The new supplemental figure is well done, and I wonder if the authors could refer to it more, specifically in the results and even in the figure legends. For instance, in Figure 3, they say “expression is not detected” at 16.5 hpf when there is purple stain visible in the image (this nonspecific staining is what confused me the first time). Could they explicitly note non-specific staining with a reference to the supplemental figure? Given that a key takeaway is defining which genes are NOT expressed at 16.5 hpf, it would be good to make this clear. I am not sure it is as obvious to a reader what counts as "real" expression.
Validity of the findings: As noted above, the authors have heroically validated the specific expression observed. The points made in the discussion that relate to the data are more clear in this revised manuscript.

The one sense control that gives me pause is ntrk3b. The antisense and sense staining look similar in the new supplemental figure, and the antisense looks different from the image in Figure 6B. While not directly relevant to the takeaway message, it does relate to the bigger question about variability. The authors’ points about differences in staining being due to methodology, rather than variability in expression, are well taken. I agree that reporting fractions could be misleading. However, I still think some mention beyond "representative" is useful, especially if the microscope images are less clear than the investigators’ observations. It might save the PI time in the future fielding questions about these probes if the consistent vs. inconsistent staining and/or approximate (narrative, not numerical) frequency of background is noted somewhere in the manuscript, perhaps in a supplemental section to accompany the figure.
Additional comments: No additional comments


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

Input:
Title: EXPRESSION OF THE NEUROTROPHIC TYROSINE KINASE RECEPTORS, NTRK1 AND NTRK2A, PRECEDES EXPRESSION OF OTHER NTRK GENES IN EMBRYONIC ZEBRAFISH
Review round: 2
Reviewer: 3

Output:
Basic reporting: The revisions to the manuscript have addressed my concerns and put the findings in better context.

The only minor change I would like to see is a reference to the new Supplemental Figure 2 somewhere in the text of the manuscript. Perhaps it could be mentioned in the methods section that sense probes were examined to validate the specificity of the antisense probes.
Experimental design: My concerns have been addressed
Validity of the findings: My concerns have been addressed
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: THE ECOMORPHOLOGY OF THE SHELL OF EXTANT TURTLES AND ITS APPLICATIONS FOR FOSSIL TURTLES
Review round: 1
Reviewer: 1

Output:
Basic reporting: This study is based on extensive statistical and morphometric analyses. Since I am not an expert in the methods employed, I find myself unable to competently review this submission.
Experimental design: NA
Validity of the findings: NA
Additional comments: NA