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: GENOME SEQUENCING AND ANALYSIS OF SALMONELLA ENTERICA SUBSP. ENTERICA SEROVAR STANLEY UPM 517: INSIGHTS ON ITS VIRULENCE-ASSOCIATED ELEMENTS AND THEIR POTENTIALS AS VACCINE CANDIDATES
Review round: 2
Reviewer: 1

Output:
Basic reporting: The writing is still deficitary.
Experimental design: OK
Validity of the findings: See the general comments
Additional comments: This is a substantially modified version of the manuscript, containing a big amount of changes concerning the scientific writing quality as well as in diverse aspects of the results description and discussion. Respect to writing, it is certain that quality was improved, but it is still rough, so in occasions the messages can be misunderstood or misinterpreted. Below, you can find some concerns from the discussion section, some of which eventually could be due to that, concerns that should be addressed.

A first general concern is about the descriptions of the evidences from literature. Frequently the description does not include the specie or serovar from which the evidence was generated, leading to the reader to think that the evidence could be reffered to the Stanley serovar. The authors should be more rigorous about that. For instance, see lines 416-418.

A great amount of writing concerns were detected. Here you have two of them.
Line 437-439. “The other putative SPI which is associated with intestinal colonization of the host is CS54-encoded virulence factors (shdA, ratB and sinH) were discovered to be close together in S. Stanley UPM 517” Please revise the redaction
Line 439-441 “Also presented was fimbrial biosynthesis-encoded operons (csg, fim, lpf, bcf, saf, stb, stc, std, sth and sti) and was said to contribute to the persistence of bacterial intestinal in the host (Weening et al., 2005)”. Please revise the redaction

Major concerns
lines 291-292. "This is possibly because there were more intergenic regions presented in S. Typhimurium LT2".  Do the authors want to say a higher number of intergenic regions? If it is so, this statement would not necessarily explain why UPM 517 has a higher number of coding sequences than LT2 strain, in spite of the genome of UPM 517 has a slightly lower size than the LT2 one: this explanation does not exclude the possibility that the higher number of intergenic regions, the higher number of genes

353-355. "It is noted that SPI-1 in Salmonella is responsible for encoding of invasion, secretion system and translocation of proteins to host cells from Salmonella's extracellular".

356-359. "SPI-1's major transcriptional regulator, hilA, is responsible for the regulation of these effector proteins (Bohez et al., 2007) and was initially found to be a potential vaccine for broiler as discovered by Bohez et al. (2007) that SPI-1 was able to protect broilers against salmonellosis" So,  the authors state that HilA could be used as a vaccine against S. Stanley. Considering the below, I think that they wanted to say taht a hilA mutant could be a candidate for a vaccine.

line 362-365." According to another study by Matulova et al. (2012), the entire SPI-1 was removed and via oral administration, it was used as a vaccine to 1 day-old chickens. This deletion has revealed that the wild-type Salmonella was able to block colonization, hence protected the poultry from the challenged strain". WT bolcks colonization or the colonization of wt was blocked?

line 365-368. "Dieye et al. (2009) obtained similar finding in their study where 1 day-old chickens had been infected with the strain of SPI-1 mutant (the whole SPI-1 removed) Dieye et al. (2009) obtained similar finding in their study where 1 day-old chickens had been infected with the strain of SPI-1 mutant (the whole SPI-1 removed) with a resistant towards several antibiotics such as nalidixic acid, chloramphenicol and kanamycin, and successfully reduced the colonization of bacteria in the chicken's cecum and spleen.  and successfully reduced the colonization of bacteria in the chicken's cecum and spleen. " Why the authors included the resistant profile in this statement?... it is confusing and unnecessary for stating the point.

370-373. "The binding of an effector protein, sipA, to the actin in modulating the small GTPases of the host cell and regulating the signal transduction pathways subsequently resulted in polymerization of F actin being activated at the bacterial adhesion sites as well as formation of membrane ruffles, hence improving the Salmonella uptake effectiveness". To my undestanding this statement is not referred to serovar Stanley, as no experiments to study this system were conducted in this work. So, the authors should clearly indicate which bacteria they refer and some reference(s) should be included.

line 382-392. This is a controversial paragraph. First, some references should be included to support that "It was shown in previous research that the effector proteins of the mutant ssaB and sseA strains could not be translocated". Second, which is the "protector" activity of SseA. Third, the authors stated that  "Nonetheless, even without sseA and ssaB, the translocon proteins like sseB and sseD remained available, indicating that sseA has no influence over the translocation of the S. Stanley UPM 517's effector proteins to the host cell." What are the evidences that support the statement... this is highly speculative. Finally, the authors propose removal ssaV gene as a vaccine candidate, but as previously they stated, in the absence of ssaB and sseA effector proteins cannot be secreted, so it is probable that secretory system encoded in SPI2 was not functional. As I stated in the previous report, a question that arises from this is how Stanley could survive in the animal without the protection provided by the effectors secreted by this system. Has this serovar a functional TTSS-2?. This should be discussed.

line 394-396 "The effector protein of SPI-5, SopB/sigD, also aids in the invasion of Salmonella and host cell's membrane ruffling in phosphoinositide phosphatase way and co-regulates alongside the effector proteins of SPI-1 and secreted by T3SS of SPI-1." This is a very confusing redaction.

Line 400-401. “In addition, the virulence factors that were earlier not related to any SPIs, namely mprA and abpB, were expected to be discovered in SPI-5 of S. Stanley UPM 517”. Why the authors expect this possibility?

Lines 428-432. “ORFs in S. Stanley UPM 517 draft genome was found to be homologous to the SPI-13 and SPI-14 in S. Gallinarum. It was showed from the result that both SPI-13 and SPI-14 might have been transferred from a plasmid or phage via HGT. Nonetheless, the functions of SPI-13 and SPI-14 in Salmonella pathogenicity had yet to be thoroughly characterized”. What would be the orfs present in these islands?. It is confusing, because the paragraph is referred to fimbrial genes cluster and as shown in figure 4 this islands, al least the SPI-13, do not included them.

Lines 451-453. “Nonetheless, the virulence role of GipA in hosts may be arguable as a number of countries were reported to still having cases of salmonellosis continuously.” This statement seems to be out of context. The authors should be clearly indicate the serorovarto which they refer to.

Line 467-485. How these genetic elements could contribute to serovar Stanley virulence and the eventual vaccine development?


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: GENOME SEQUENCING AND ANALYSIS OF SALMONELLA ENTERICA SUBSP. ENTERICA SEROVAR STANLEY UPM 517: INSIGHTS ON ITS VIRULENCE-ASSOCIATED ELEMENTS AND THEIR POTENTIALS AS VACCINE CANDIDATES
Review round: 2
Reviewer: 2

Output:
Basic reporting: The use of english language has improved from the original version of the manuscript but there are  still many parts throughout the manuscript that need to be revised. The authors claim that an editing service was used but that is not reflected on the manuscript.
Experimental design: My previous concerns were taken into account. I have no further comments.
Validity of the findings: My previous concerns were taken into account. I have no further 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: ESTROGEN RECEPTOR 1 GENE POLYMORPHISMS (PVUII AND XBAI) ARE ASSOCIATED WITH TYPE 2 DIABETES IN PALESTINIAN WOMEN
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript entitled “Estrogen receptor 1 gene polymorphisms (PvuII and XbaI) are associated with type 2 diabetes in Palestinian women” by Ereqat et al describe the association of ESR1 gene polymorphisms with T2DM in Palestinian women. In this manuscript, the atuhors conducted a cross sectional study of 214 Palestinian women (102 with T2DM and 112 non diabetic controls), aged 45–70 years, characterised for blood glucose, blood lipid, blood pressure and BMI, and genotyped for two ESR1 gene polymorphisms, the PvuII (rs2234693) and XbaI (rs9340799). The manuscript is generally well written, the protocol appears to be appropriate and well-conducted, and the statistical analysis were performed appropriately. However, the following issues need to be addressed and clarified before the manuscript could be considered for publication:

1. The authors highlighted the importance of estrogen and estrogen receptors, as shown in the manuscript (line 59-65, first paragraph of Introduction): “Recently, several reports have revealed the role of estradiol in regulating energy metabolism and highlighting the role of the estrogen receptors which are distributed in many tissues, including the pancreatic islets, skeletal muscle, adipose tissue, and cardiovascular system (Hevener et al. 2018).  It was evident that estrogens exert certain physiological and metabolic disorders including insulin resistance, dyslipidemia, and hypertension (Howard et al. 2003; Lindsay & Howard 2004), which all participate in the pathogenesis of T2DM.” However, the authors did not provide enough information on how and by which mechanism that estrogen and its receptors influenced metabolic disorder. For the mechanism, please see Meyer et al, 2011, Acta Physiol (Oxf), doi:10.1111/j.1748-1716.2010.02237.x, for review.

2. Why did the authors select the two ESR1 SNPs, PvuII (rs2234693) and XbaI (rs9340799) for their study? What is the importance of studying these variants? Please described in Introduction.
Experimental design: 1. The description of the study population is not clear. The authors should explain how the participants of this study were enrolled in detail. Are there inclusion and exclusion criteria?

2. Methods of BMI, SBP, DBP, and FBS measurements were not described in the text. The authors should describe those measurements clearly in Methods.

3. The authors should describe the HWE calculation in Methods.

4. The authors used linear regression for multivariate analysis, as described in Methods, Statistical analysis section, line 134-135: “Multivariate linear regression models taking into account age and BMI were performed to assess associations between SNPs and T2DM complications”. Are the parameters distributed normally?

5. In Methods, Statistical analysis section, line 135-136, the authors stated at the bottom of this section that "Comparison of the tested SNPs was statistically significant at P < 0·05." However, the authors tested 2 different SNPs of ESR1 gene. To minimize the chances of obtaining type I error, the authors should perform Bonferroni correction, such that the statistical significance level shall be 0.05/2=0.025.

6. The sample size was rather small. Therefore, please provide statistic power of your sample. "GeneticsDesign" package (Purcell et al, 2003, Bioinforma Oxf Engl, 19:149–150) could be use for power calculation
Validity of the findings: 1. In Result (line 140-141), the authors stated that “...all clinical parameters showed statistically significant increase in T2DM patients (P<0.05)”. From Table 1 one can see that the control group is 11 years younger than the T2DM group. Will the control group developed T2DM eventually? Is the significant differences of SBP and TC between control and T2DM groups related to T2DM or age?

2. The authors did not present any Table related to the “Association of ESR1 PuVII and Xba Polymorphisms with T2DM complications” section (Result, line 177-186). The authors should provide a Table that referred to the result described in this section. The authors should also described what are the CVD risk and T2DM complications that they mention in this section.
Additional comments: 1. Line 59 is missing.
2. Line 84: “A meta-analysis study investigated the new-onset T2DM in postmenopausal women following estrogen replacement therapy,...”, should be “A meta-analysis study investigating the new-onset of T2DM in postmenopausal women following estrogen replacement therapy,...”
3. Line 96-98: Please provide a reference for this statement “Diagnosis of T2DM was based on World Health Organization (WHO) criteria (fasting plasma glucose126 mg/dl and/or currently on treatment for diabetes)”.
4. Line 103-104: “Genomic DNA was extracted from whole blood (300 μl) using genomic DNA purification kit QIAamp...”, should be “Genomic DNA was extracted from whole blood (300 μl) using genomic QIAamp DNA purification kit...”.
5. Line 108-109: “Both primers were modified with over hanged Illumina adaptor sequences at the 5′ ends (italic bolded), should be “Both primers were modified with over hanged Illumina adaptor sequences at the 5′ ends (italic bolded, Table S1)”.
6. Please use consistently “Control group” instead of: “healthy control” (line 34), or “healthy subjects” (line 40, 156, 174) , or “healthy women” (line 233).
7. Line 139: Table 1, with uppercase T.
8. Line 147: P>0.05, with uppercase P.
9. Line 152: typographical error: “PuVII and Xba” should be “PvuII and XbaI”.
10. Line 158: Table 3, not 2.
11. Line 165: Table 4, with uppercase T and without brackets.
12. Line 174: Table 5, with uppercase T and without brackets.
13. Line 189: obese, with lowercase o.
14. Line 201-202: allele frequencies.
15. Line 204: XbaI.


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: ESTROGEN RECEPTOR 1 GENE POLYMORPHISMS (PVUII AND XBAI) ARE ASSOCIATED WITH TYPE 2 DIABETES IN PALESTINIAN WOMEN
Review round: 1
Reviewer: 2

Output:
Basic reporting: The submitted manuscript aimed at investigating the possible role played by two ESR1 gene polymorphisms in conferring Palestinian women susceptible for T2DM. the study population comprised 104 patients previously diagnosed with T2DM, and 112 control healthy females.
The results of the manuscript are interesting. However, extensive revisions are needed in order to make it publishable.
Experimental design: The study is a case control study, although not stated in the methods section.
Selection criteria for the cases and controls is note stated. A number of pieces of information needs to be introduced, including the type of study, sampling method, inclusion criteria, and exclusion criteria.
The cases and controls were not matched in terms of age, which is a major risk factor for T2DM??
Were the cases recruited from one UNORWA clinic? Which one?
Were all cases recruited from the same governorate/city??
How were controls recruited? Are they healthy only in terms of T2DM?
Did the authors confirm the diagnosis of cases? If yes, then how?
Did the authors confirm the controls are not diabetic??
What data was collected from the patients and/or their records in the primary health care clinic? How?
Did the authors take the permission of UNORWA to recruit the cases from its clinics?

The commercial names and manufacturers should be stated for all kits used in the experiments (example line 103, 112, 118).

What methods were used for determining the FBG, CL, LDL, HDL, TG levels??

The statistical analysis looks sufficient.
Validity of the findings: In order to be valid, the authors should discuss their findings as follows:

An important requirement for studying allele frequencies in population genetics is a large sample size. Therefore, the authors should discuss how their results would be valid in light of their sample size before drawing important conclusions.
Over-dominance (or heterozygous advantage) is a way by which allele frequencies become different from those expected by Hardy Weinberg equations However, the authors report that the frequencies are in HW equilibrium (line 147). Moreover, how do the authors explain their predicted model? In other words, how can polymorphic ESR1 alleles give an advantage for heterozygotes over wildtype and mutant homozygotes as predicted by your results of overdominance?
Additional comments: Intensive English language revisions should be carried out.
In the abstract section, the background and methods should be rewritten in order to reflect the justification of the study and clarify the methods used in the study.
The Introduction section needs to be rewritten in a more focused and concise way. The sentences are some times very long and not clear. For example, in line 78-82 the sentence “A recent study conducted on three refugee camps in the West Bank, revealed that the overall prevalence of obesity and overweight was 63.1% while the prevalence of MetS among obese and overweight was 69.4% with no significant differences between men and women (Damiri et al. 2018) indicating that they are at high risk of both cardiovascular diseases and type 2 diabetes”

The references are sometimes old.


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: ESTROGEN RECEPTOR 1 GENE POLYMORPHISMS (PVUII AND XBAI) ARE ASSOCIATED WITH TYPE 2 DIABETES IN PALESTINIAN WOMEN
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: Some part of the revised Introduction section is too long and needs to be shortened in a more focused and concise way (line 60-73).


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: ESTROGEN RECEPTOR 1 GENE POLYMORPHISMS (PVUII AND XBAI) ARE ASSOCIATED WITH TYPE 2 DIABETES IN PALESTINIAN WOMEN
Review round: 3
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The revised Introduction is acceptable.


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

Input:
Title: TOXIFY: A DEEP LEARNING APPROACH TO CLASSIFY ANIMAL VENOM PROTEINS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The authors present a novel software package TOXIFY, for inferring the probability of being a venom protein which is 20X faster and uses less memory than previously published methods. Toxify is a nice technical tool. Professionally written. The motivation and results are clearly presented.
Importantly, the authors share the tool and code used in an easy to use CMD interface/tool that is commendable.
Experimental design: 1. The design is valid and well presented. However, The authors used TOXIFY in tandem with TOXCLASSIFIER. Testing with respect to other ML method is needed to better understand the performance in a more general sense. It is needed for supporting the conclusion stated in the abstract (Lines 15-16). At present, this statement should be written more precise.

2. The degree of sequence redundancy in the all the training, test and the showcase were not mentioned. It is relevant to the final performance
Validity of the findings: The findings seem appropriate. However, the datasets available on the GitHub page do not match the numbers in the article. Specifically, in the Github, the training set holds 12,462 rows/sequences, with 367 in the benchmark test data. The article states other numbers (4,808 and 32,391 for positive and negative proteins. Authors should clarify the discrepancy in numbers.
Additional comments: Few minor comments for improving the presentation
a.	Line 28, Numerous studies support this claim and citations are missing (e.g. doi: 10.3390/toxins9110350).
b.	Lines 33-34: need a small revision, as SVMs can capture nonlinear relationships using kernels, e.g. RBF kernels, and/or feature transformations of the input space.
c.	Line 171: The concept of transforming sequences into a rich set of features rather than the slow BLAST alignment was presented. Citation is missing (e.g.  doi.org/10.1093/bioinformatics/btv345 and others.
d.	Table 1. Please explain the asterisks
e.	Lines 166-168 need a reference.


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: TOXIFY: A DEEP LEARNING APPROACH TO CLASSIFY ANIMAL VENOM PROTEINS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The basic reporting in this article is good.
Experimental design: I have issues with the experimental design in sections 3.3.2, and 3.3.3 as detailed in General Comments below.
Validity of the findings: Addressing the experimental design issues I have raised will increase the confidence of the reader in the validity of the findings.
Additional comments: Review of PeerJ-34316-v0

Disclaimer
Please note that I am a molecular biologist and toxinology researcher with very limited knowledge of deep learning and associated fields, and this review is confined to those aspects I am capable of addressing.

General comments
This article describes the development and testing of TOXIFY, a deep learning algorithm aiming to classify toxins or non-toxins based on their primary structures. The problem addressed is a worthwhile one, and the manuscript has been carefully prepared. From the data presented, the authors have produced an algorithm displaying considerable increases in both accuracy and speed compared to both traditional annotation methods and previous machine learning approaches. My main issues with the manuscript relate to how the algorithm has been tested, and how those tests are presented in the manuscript. These are listed below. I recommend the manuscript is suitable for publication after revisions to address these issues and/or the authors have responded to these issues raised.
•	Section 3.3.2: I am not sure this section demonstrates anything meaningful. Am I right in thinking that all it shows is that Toxify identifies more ORFs as toxins than BLAST searches do? If so, this may or may not be a desirable feature, depending on whether these many thousands of additional sequences actually represent toxins or not. From what is presented, there is no reason to think that this is the case except the assertion on lines 157–159 that “Because many additional venoms are known to be present in these taxa beyond the number indicated by BLAST searches, this highlights TOXIFY ’s greater predictive power compared to BLAST-based approaches.” A more convincing demonstration would test the ORFs in this way and then compare the results to some kind of empirical classification of if the protein sequences were venom or non-venom, most ideally if they can be detected in venom by mass spectrometry. Surely this would be possible for one of these or another well-characterised species?
•	Section 3.3.3: I also have several problems with this section: (a) that the test consists of a single ‘positive’ sequence and no negatives; and (b) that this sequence appears to belong to a protein superfamily (inhibitor cystine knots) that is represented not just in asilid venoms but also the venoms of numerous other animal groups, particularly spiders and cone snails. This means that although this test does indeed support the applicability of Toxify to ‘other venomous animals’ not included in the training group, the scope of this may be considerably more limited than what is inferred. How would Toxify perform on the numerous other protein families recently discovered in asilid venom? Ideally, I would like to see the study measure (or at least discuss the issues surrounding) how ‘novel venomous taxa’ vs. ‘novel venom protein families’ affect Toxify’s performance.
•	Table 1: This table compares the performance of ToxClassifier and Toxify on the negative and positive datasets described on lines 79–81: “a positive dataset with all 274 verified venom protein sequences and a negative dataset with 274 randomly selected and verified non-venom protein sequences.” Tested on this dataset, Toxify is in general more happy to classify things as venom proteins, and has a substantial increase in true positive (TP) and decrease in false negative (FN) with only very slight increase in false positive (FP) and decrease in true negative (TN). However, the ToxClassifier algorithm seems to have been designed and tested on quite different datasets. The negative dataset used to generate Table 1 seems to more closely resemble the ‘easy’ dataset used in the Gacesa et al. 2016 article (10.7717/peerj-cs.90), whereas the ‘moderate’ dataset in this 2016 study consisted of sequences with blast homology to verified toxins but that were verified non-toxins, more similar to that described in 3.3.1. How would Toxify compare to ToxClassifier on this more difficult type of dataset?

Other minor comments
•	Lines 79–81: Can you tell us more detail of how the sequences in the negative test dataset were verified as non-venom?
•	Lines 88–99: Why can we not tell the exact number of sequences of each type (TP, TN, FP, FN)?


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: TOXIFY: A DEEP LEARNING APPROACH TO CLASSIFY ANIMAL VENOM PROTEINS
Review round: 1
Reviewer: 3

Output:
Basic reporting: The basic report is good.
Experimental design: The purpose of the experiment is clearly explained. The data used is presented adequately.
Validity of the findings: I have some comments about the representation of the algorithm I included in the general comment.
Additional comments: I am statistician and including some equations explains how   TOXIFY algorithm  tackles the classification of toxic sequences will enhance the work and make the comparison with existing algorithm more visible.


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: TOXIFY: A DEEP LEARNING APPROACH TO CLASSIFY ANIMAL VENOM PROTEINS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The basic reporting in this manuscript is now good.
Experimental design: The experimental design seems appropriate to the scope of the study.
Validity of the findings: I think the validity and context of the findings is substantially improved in this revised version.
Additional comments: Thankyou for carefully considering the revisions and comments and addressing them. I am satisfied that the points I raised in response to the first submission of this manuscript have been sufficiently addressed and recommend that the article is now suitable for publication.


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

Input:
Title: COVARIATIONS BETWEEN PERSONALITY BEHAVIORS AND METABOLIC/PERFORMANCE TRAITS IN AN ASIAN AGAMID LIZARD (PHRYNOCEPHALUS VLANGALII)
Review round: 1
Reviewer: 1

Output:
Basic reporting: See my comments to the authors for details.

-English needs to be improved
-Literature should be updated
-Article structure is good
-Framework needs clarification
Experimental design: See my comments to the authors for details.

-original research within aim and scope of journal
-Framework needs clarification
-rigorous investigation
-methods are detailed
Validity of the findings: See my comments to the authors for details.

-rationale and benefit to literature needs improving
-data collected is good but statistics need revision
-single results are discussed but lacking a broader vision
Additional comments: Peer J Reviewing Manuscript 31219v1

In this manuscript, the authors aim to test the covariance between behavioral traits, metabolism and performance in a squamate lizard species, within the pace-of-life syndrome (POLS) framework.

This is a timely study on a non-model species, on a topic that has recently received ample attention in the behavioural ecology literature.

However, the study could benefit from more appropriate statistical analyses, a more complete theoretical framework, and a broader point of view in the discussion. Furthermore, the writing could be improved in some instances. I feel that all these substantial issues need to be addressed before considering the study for publication.

Here I detail my suggestions to improve the manuscript:

1.	Throughout the manuscript, the authors claim to test traits associations between behaviours and metabolism hypothesized in the POLS framework. However, they miss the crucial point of the POLS: POLS theory is based on life history theory. It is the association between differences in behaviours/metabolism and differences in life history traits that should explain the maintenance of individual (behavioural) variation through life-history trade-offs. This is explained in the paper outlined the POLS theory (Réale et al. 2010, cited in the ms) and updated in the “Topical Collection Pace-of-life syndromes: a framework for the adaptive integration of behaviour, physiology and life history”, published in Animal Behavior in 2018. I suggest the authors to re-frame entirely their paper: either they actually measure life history traits, and keep the POLS framework, or they reconsider their claims and talk about testing relationships between traits involved in the POLS, but focus less on the POLS as they are actually not testing it.
2.	Following this, the broad question behind the study should be clarified. One of the main questions that the field of behavioural ecology is trying to answer is: why is individual behavioural variation maintained? The association between behavior and other traits (e.g. like in the POLS) is one explanation. The introduction needs to be reworked, for now it reads as a general introduction describing what is personality, and then it jumps to describing associations with metabolic traits and performance capacity, but the general question and the implications are not clear.
3.	Reading the abstract and the introduction I was under the impression that the paper would be about different populations in different ecological conditions, since the authors put emphasis on comparing POLS patterns across different ecological contextS. However, none of this is done. I suggest to keep this idea for the discussion, as it’s certainly valid and one of the main questions for the POLS field, but keep it purely speculative when they compare their species with other studies in different conditions, otherwise it’s misleading.
4.	Including the framework developed in the latest papers from the Topical collection 2018 above mentioned would benefit the paper, both for the introduction and the discussion. There are several empirical papers too that could serve as example for the data analyses and how to frame the questions.
5.	In regard to the behavioural measurements, from what I understand from the text, the authors did not take multiple measurements per behavior or when they did, they only kept the maximum value. “Personality” is defined as consistent among individual behavioural variation, and requires taking multiple measures per individual of the same trait in order to separate the among individual or repeatable component. Do the authors have previous data to show that their measured variables are repeatable, and thus can justify using only one measure per trait?
6.	The statistical analyses are very confusing, with the high number of models described. The most straightforward analyses to do, if individuals have repeated measurements, would be to run a multivariate model in order to estimate the among-individual matrix of correlations. In any case I suggest to give some structure to the statistical analyses and the results, it’s a lot of information now with no specific order.
7.	The discussion could benefit from an opening paragraph where the authors recap the aim of the study and its main findings instead of starting abruptly with discussing one by one the results.
8.	The discussion is well detailed for each single finding but lacks a larger vision. I suggest adding at least one other final paragraph where the authors discuss the implications of their study, embedded in the general theoretical framework.


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: COVARIATIONS BETWEEN PERSONALITY BEHAVIORS AND METABOLIC/PERFORMANCE TRAITS IN AN ASIAN AGAMID LIZARD (PHRYNOCEPHALUS VLANGALII)
Review round: 1
Reviewer: 2

Output:
Basic reporting: This manuscripts presents an interesting, well-described study of personality, physical performance, and pace-of-life related variables in a lizard species. The experiments and analyses are explained well, and though I know that the theoretical bases for their questions are strong, the authors need make their logic and research questions more accessible to the reader.

The authors present the background to their study well in many ways. The scope is comprehensive, and the flow is, with some exceptions, logical. However, there are a number of shortcomings in the authors’ overall reporting.

In the introduction, the authors need to present the literature with more clarity, unpacking the concepts and findings that have come before them, where necessary. While the authors cite widely, the meaning of particular references is not always obvious, and this will make the logical flow of the manuscript hard to follow for many readers. In some cases, the authors ought to fill in with more content. Please see the section on general comments for line-by-line evaluations.

There is a stylistic choice that I would recommend to the authors, but I won’t insist on. Using acronyms such a SMR, SVL, TL, TER, and TRB may not be the best choice. While these acronyms fairly faithfully represent what was actually measured, they are not widely known acronyms, and due to there being four of them often used in the same context, keeping them straight is often confusing. The authors relate each of these terms to more general traits, e.g. TL is Exploration Intensity and TRB is Risk-taking. I’d recommend that the authors try using these terms instead of acronyms, and capitalization can help make these terms stand out to the reader as important variables of interest.

On the other hand, SVL, a comparatively minor variable, probably deserves to have its acronym kept, but it also is not immediately clear why SVL was measured, or what precedent there is for this measure and this correction (I don’t think there was a reference). The authors ought to think about what their most important variables are, and how to make their meaning and purpose clear to the reader, throughout the paper.

In general, the presentation of the figures was good, and the content they presented was necessary. I have comments about some of the results presented in the tables and figures.

I appreciate that the authors are non-native English speakers and that they have sought out external assistance in writing their manuscript. In general, the language is satisfactory, though I encourage the authors to continue to work with these partner(s) in future revisions of the manuscript.

The excel spreadsheet supplied appears to contain all the raw data, and could be used to replicate the authors’ results.
Experimental design: The experimental design was the strongest part of this manuscript. The authors present their methods clearly and comprehensively. Their methods ought to be sufficient for another researcher to replicate this work.

The research question is exploratory, but it is vague and not treated as such. Lines 105 - 106: “Support for the pace-of-life hypothesis is expected to be confirmed if all traits are found to be positively correlated.”

The authors did not merely correlate all their variables and draw conclusions from that. They built models, and not kitchen-sink models which are similar to correlations, but models with specific variable included, depending on the outcome variable. This implies a confirmatory framework, but the authors do not give us specific hypotheses in their intro, only that restated above. The authors need to either acknowledge that their analysis is exploratory and explain their process of exploration, or state their hypotheses more clearly so that the confirmatory framework can be used. Whichever direction the authors choose to take, they ought to further their considerations of how this work fits in/into the current literature and its gaps.

Issues arose with some of the authors’ measures – I’m referring to TER and TRB, which needed to be transformed into binary outcomes in order to be analyzed. This wasn’t what the researchers intended, and one could argue that this adds researcher degrees of freedom, but the transformation to binary is reasonable, and I don’t have a fundamental issue with it. See general comments for more.
Validity of the findings: The authors present results that are on the margin of the standard 0.05 significance level, and unfortunately for them, they are not on the lucky side of this margin. Nevertheless, the authors’ discussion is premised on positive results. This makes it difficult to evaluate the validity of their stated findings.

The authors state in their intro that will be looking for positive correlations, and in the statistical analyses section they do mention correlated variables, but they do not present the value of this correlation or others, but press on with their modeling. Some of their variables (e.g. the binary ones) do not seem particularly well suited for correlations, though non-Pearson measures like Spearman or point-biserial correlations could allow the authors to calculate and present correlations among all their variables. I would strongly urge the authors to do this. It will elucidate their results in multiple ways, and may cast light on why their p-values of interest are marginal. 0.05 is an arbitrary value, but to interpret a result that is not below the threshold, the authors need context. Correlation could give that context. So could more descriptive tables and figures.

Once the authors have reconsidered their analyses, they need to also rethink their discussion. In the meantime, one clear, positive result was the association between TL and Endurance. This is discussed relatively briefly, 4 paragraphs in. This is the authors’ strongest result; they ought to give a bit more thought to it, possibly in the context of SMR in that model.

In general, thought SMR might be associated with TRB, it is a weak association, and it seems to be not at all associated with the other two traits, TL and TER. Negative results like this are interesting! Rather than focusing on the weak positive evidence to the exclusion of the negative evidence, the authors ought to consider what it means that they found no associations where they predicted. Power? Construct validity? Ecological factors? The authors, readers, and field will benefit the most from a reasoned discussion of why expectations did not bear out.
Additional comments: The following comments are specific to particular sections of the manuscript, indicated by line number. Some are major, some are minor.
32: “Correlated”, then 33: “associated”. Why different? Are these the correct terms?
35: “Moderately” what is this word’s purpose? I don’t understand what falling “moderately on a pace-of-life syndrome continuum” means.
62 – 64: this long statement in parentheses. It needs to be unpacked, not just presented as an aside. For example, the authors ought to link lizards to ectotherms, and ideally, explain why ectotherms and endotherms metabolic rates are measured differently and set up this information for later reference. Or, move this to a more relevant location, like where the authors introduce their lizards.
66: “intuitive” – it isn’t intuitive. This needs to be setup and linked better, I didn’t find this paragraph’s logic easy to follow.
72: It was good to see the references to other models, but the authors don’t define or dig into any of these, so I while I understand the desire to acknowledge them, their mention leaves the reader wondering. If the results are discrepant, then what papers and evidence match which theories? Listing the theories and then listing some evidence unfortunately does not advance the authors’ logic.
86 – 89: In what way does the POLS hypothesis “generally propose” this? Please be more concrete. You can link this to the preceding sentence, where I also was not certain of what the “logical gap” was.
105 - 106: It is worth mentioning again, this either needs to be more explicitly exploratory or the authors need to state the hypotheses they are attempting to confirm.
121 & 123: What are the “card-boxes” or “carton boxes”? I wasn’t sure what materials these terms were referring to, so please be more specific if possible.
132: What kind of link? What is the direction of the association?
137: Why was the maximum value chosen? Is there a precedent for this? (Please cite)
149: Again, why the maximum value? The authors say that performance can be variable and do provide a reference, but I would like to see a bit more justification.
In these cases where some values are thrown out, in addition to more reasoning, I would be very interested to see correlations between measures or other descriptive data that show that the measurements are reliable and/or that the measurement chosen by the authors is empirically superior.
169: I think it should read “carried out in” ?
181: SMR – why introduce one metabolism acronym earlier, in the intro, and abandon it for this, very similar acronym later? Are they the same, or just similar? I would like to know how they are linked, if both are important enough to be given acronyms. Maybe drop the RMR acronym and just write it out?
194 – 195: “minimum” – as above, why this measurement and not the others?
198 – 200: I am intrigued by these 5 individuals. They represent 10% of the authors’ sample, and seem to have a consistent phenotype. To me, that indicates an aspect of personality that is being excluded, which is both relevant to the current analyses and interesting for future research. This should be mentioned in the discussion.
213: “links among” is too vague. The authors should say how they specified the model – what was the outcome and what were the predictors.
215: There is a test for overdispersion that I assume the authors carried out. Please give the results.
224: Where are the R-squareds? I didn’t see them mentioned anywhere else in the paper.
230: See the earlier sections for my main suggestions of how to improve the results.
239: What statistic is S? Is it named after someone? I wasn’t immediately sure what this referred to, so I think in the first instance the author should give the full name, as S is not immediately recognizable like t or df or p.
250: As mentioned, this isn’t quite what the authors found due to the issues of significance. This at least needs to be clear.
254 – 257: The bighorn sheep that Reale has studied are a great example of a high-altitude living species for which personality and POLS data have been collected. If the authors haven’t already read this work in depth, I’d suggest they take a look as there may be some relevant research for comparison and discussion in that species.
Table 1: My comments above about acronyms apply here, especially. At very least, the authors should present a caption explaining what each acronym stands for *and* what trait the measure is a proxy of. We should also know what kind of regression each model is using. Also, the authors could add the R-squareds in a bottom row as well.
Figure 1: I think the caption should explain more about what occurred, temporally, in each condition, and clarify what the ice and lamp symbols are and mean.
Figure 2: I think some text is missing from the first part of the caption: “SRM. C”
I’m not sure the plots accurately represent the models. For instance, a), the model appears to be linear, but the line is not, it has a clear curve. Why? Either the model or plot are not matched properly. Similar in c), there appears to be some curvature to the line.
There’s another, major issue in b) the association looks non-linear, which might well be why the p-value falls short. There’s a clear difference in the lower and higher SMR individuals, maybe there should be a two-piece model, or a quadratic as well as linear association, but this is an interesting twist on the data that the basic linear models completely miss. I would be very interested to hear what the authors make of this.

Finally, and distinct from the comment in the earlier section, please be consistent when using acronyms. Sometimes the authors restate acronyms (SMR, line 251), but in others, they could use an acronym they’ve established, but don’ (‘pace-of-life’, line 105).


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: COVARIATIONS BETWEEN PERSONALITY BEHAVIORS AND METABOLIC/PERFORMANCE TRAITS IN AN ASIAN AGAMID LIZARD (PHRYNOCEPHALUS VLANGALII)
Review round: 1
Reviewer: 3

Output:
Basic reporting: The introduction works relatively well. Seems like sufficient field background/context is provided. However, the statement on line 91-93 that “few studies have directly measured all associations among personality, performance and metabolic capacity and analyze potential causal relations” is supported with 3 references (Owerkowicz & Baudinette 2008; Merritt et al. 2013; Binder et al. 2016) that did not measure “all” associations.

Figures are showing predictions instead of raw data, which is undesirable. It would be more relevant to the content of the article to show partial residuals instead of predicted values. In the end, it is not at all clear what the scatterplot figures are showing because they are not the raw data. Perhaps it’s partial residual plots, but the goodness of fit looks way too good to match the results in the table. One of the models at least on the figure appears to be the incorrect function (shape) to fit those data.

Other comments about figures: Resolution is too low. SMR acronym is not defined in the figure caption. Figure 2 states 95% standard errors, but it must be an error and should be 95% confidence intervals.

The structure of the article conforms to an acceptable format of ‘standard sections’. All appropriate raw data has been made available in accordance with our Data Sharing policy.
Lines 61-62: It makes no sense to say that animals gain and expend energy “at higher resting metabolic rates”
Experimental design: Methods are described with sufficient information to be reproducible by another investigator, except that the authors did not provide equations used for calculating SMR.

It is hard to understand how the authors analysed the data exactly. Moreover, using residuals in a second-step analysis is not recommended. Best to include body mass as a covariate in the analyses.

One of the major flaws is the design of the novel environment test. There were two refuges (lines 159-160). It is unclear if the refuges in the novel environment acted as such or as a goal of exploration? Two individuals could go strait to the refuge for different reasons: one for seeking a refuge and the other to explore it. So it is impossible to interpret this variable.

Line 139: “The subjects were warmed with an infrared heater before measuring endurance and sprint speed in order to insure the lizards were active and to standardize body temperature”, but the temperature varied between 22 and 35°C, so this is not very standardised.

Line 183-184: unclear what is meant by "within the animal’s range of activity".
Validity of the findings: Negative/inconclusive results are not treated as such. The authors “present the estimates of main effects from the full models regardless of their significance because the parameters directly relate to our hypothesis.” (lines 224-225). While this is OK, it is not OK to say that “time to enter refuge (TER) was positively associated with SMR” when the P-value of that relationship 0.86 (line 236).

The main limitation of the study is that there are no repeated measures. Repeated measures are the gold standard in the field of personality, because they allow partitioning the variance into among- and within-individual variance. It has been clearly stated that behavioural syndromes are defined at the among-individual correlations. The same applies for the POLS. Therefore, in the current study it must be assumed that the phenotypic correlations faithfully reflect the among-individual correlations.

There are many speculative comments that are not identified as such. The first is that the “The associated pattern may be related to their micro-/macro habitat” (line 253), which is based on an un-replicated two-species comparison (to be avoided at all cost). The second is the presence of a trade-off between foraging and mating, which would explain the presence of a negative correlation between risk-taking and bite force when the resources are scarce (line 279). This is a far stretch. Third, the reasoning behind the statement that motivation is a “reasonable explanation for the link between endurance and exploration” (line 290) is implicit and not fully explained. It is also not the most parsimonious explanation (perhaps high-endurance individuals are simply capable of keep moving for longer, such that their time in locomotion was higher during the novel environment test).

In the abstract, there are two sentences (lines 23-28) on how the POLS may vary depending on fitness outcomes, and therefore we need to compare patterns across populations. This is entirely misleading because the current study does not contain data on fitness or on multiple populations, such that readers will be immensely deceived when they learn they this is a study based on a single population.

Were sprint speed, endurance and bite force correlated with each other? At the moment these correlations are not presented. The best way to analyse these data would be best to use a multivariate model with all 7 traits included as response variables, and testing all possible correlations among these while conditioning on body mass. Such a model can be run using R packages like MCMCglmm and brms.
Additional comments: This manuscript is based on an interesting dataset containing behavioural, metabolic, and performance measurements. It is challenging to measure all of these traits within a given study, so the authors must be praised. However, the absence of repeated measures is a major flaw in the design. Moreover, the presence of refuges in the novel environment test is atypical and complicates the interpretation of the behaviour measured. Finally, some of the non-significant relationships are discussed as if they were significant.


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: COVARIATIONS BETWEEN PERSONALITY BEHAVIORS AND METABOLIC/PERFORMANCE TRAITS IN AN ASIAN AGAMID LIZARD (PHRYNOCEPHALUS VLANGALII)
Review round: 2
Reviewer: 1

Output:
Basic reporting: English needs to be checked again by a native speaker to correct small mistakes and improve the flow of the manuscript.

Literature cited could be broader, especially general theoretical papers from the field. Some of the basic personality/POLS papers are missing in the introduction (e.g. the Réale 2010 basic POLS paper). This point was already made in the previous review.
Experimental design: The research question is sufficiently defined, but could be made more explicit in what are the specific hypothesis and predictions of the study. So far, it comes out in between the lines.

Also, the research question is not well justified by the literature. It seems to be a revised hypothesis fitted to the findings. The statements that "Activity/exploration or risk-taking behaviors are more likely to impact resource
acquisition in organism foraging on clumped and ephemeral food sources", and "Vigilance or freezing behavior is instead more likely to covary with the pace
 of life when organisms rely on food items requiring long bouts of handling " are not supported. The only paper that is cited throughout (Dronne 1999) doesn't seem to have anything to do with the topic.

Once again, as stated in the previous review, it's a little confusing in which framework the study fits. It's not really testing the POLS as there are no life history trade offs measured. I do agree with the general point that POLS predictions should be more specific to the species natural history and ecological context, but little can be said in this respect if there is e.g. no comparison between populations. I think it should simply state that the goal is testing the predicted relationship between some traits involved in the POLS, and keep the rest for discussion.

The section on metabolism seems just attached on at the end of the introduction, but the rationale for including metabolism in the framework should be clarified before talking about the specific study species.
Validity of the findings: The discussion specific to the experiment and he study species is satisfactory, but to me is still missing a link to the broader implications of the findings.

The authors should also acknowledge throughout the lack of repeated measurements and that phenotypic correlations are used in place of among individual correlations.
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: COVARIATIONS BETWEEN PERSONALITY BEHAVIORS AND METABOLIC/PERFORMANCE TRAITS IN AN ASIAN AGAMID LIZARD (PHRYNOCEPHALUS VLANGALII)
Review round: 2
Reviewer: 2

Output:
Basic reporting: -English is satisfactory at this point, but could certainly be improved, and doing so would improve the manuscript overall.
- Literature coverage is good.
- Structure across sections is good, though there are a few issues within sections (particularly the intro) where some improvements should be made.
- Results and discussion follow from data and literature.
Experimental design: No additional comments on this revision, but see general comments for some related issues.
Validity of the findings: - Authors' take on their findings is more measured and comprehensive than in previous revision.
- Data collection is as good as before
- Statistics have been updated, so that they are clearer.
- Conclusions are reasonable to be drawn from these data, and speculation is minimal.
Additional comments: Line 28: "this pattern." - is there an "is" missing at the end of the sentence?

Line 67: "acquisition in organism" - I think this should read "organisms".

Lines 86 to 88: This is a long sentence and the double parenthetical expression doesn't help. It should be rephrased and/or broken up to make the ideas clearer.

Lines 73 to 96: There is a broader, related issue in the last two intro paragraphs. The final paragraph introduces SMR (twice, oddly enough), and that is about it. The previous paragraph (starting line 73) should be the final intro paragraph, because this is where the authors introduce the specifics of their study species and state their hypotheses. The authors should start by swapping these two paragraphs, and then carefully consider:
- the logical flow of the manuscript after making this change
- the order of and extent to which they introduce their key variables. Some variable details should obviously stay in the methods, but we need some information in the intro, but it needs to be logical, not just a string of descriptions of what metabolic rate or risk-taking are.

Lines 149 to 156: I appreciate the authors attempts to clarify their variables, but there are still some issues, particularly with Exploration. Exploration Intensity should not be used as the name for two different variables - adding a I or II does not in actuality make it much easier for the reader to keep track. Moreover, the authors have written "The time to enter refuges was taken as measurement of quickness to explore a novel environment, and time in locomotion was taken as a measurement of exploration intensity" which is inconsistent with the previous sentences, but makes more sense.
I suggest that the authors either call these two variables what they are "time in locomotion" and "time to enter refuge", or let  "time in locomotion"  be "Exploration Intensity" and "time to enter refuge" be "Quickness". But the authors should choose and remain consistent.

Lines 193 & 198: In the revised manuscript, as far as I can tell these are the first times TER and RMR are used, respectively. They are not properly defined or introduced. When the authors revise and consider variable names again, they need to keep track of all acronyms that are used, when they are first used, and where they are defined. When they are first used and where they are defined should naturally be the same.

Line 206: "produce" should be "product".

Line 211 to 222 - Results: Reporting needs to be more consistent. When the authors mention a correlation, whether it is significant or not, they should give the r or rho for the correlation coefficient and the p-value.

Lines 249 to 250: Be consistent about tense here (and all throughout- keep an eye on this). The 'was' on line 249 means the 'are' on 250 should be 'were'.

Line 253: What do the authors mean by "in general"? Please be more specific, and I would also suggest using an example or two.

Line 261: This is too short. What defines these 5 individuals? This does not need to be so much longer, but the reader will likely have forgotten about these individuals, so the authors should concisely tell us what the phenotype is.

Table 1, line 2: The authors used Pearson and Spearman correlations, so "(r)" should really be "(r or rho)" - preferably using the symbol for rho.


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: COVARIATIONS BETWEEN PERSONALITY BEHAVIORS AND METABOLIC/PERFORMANCE TRAITS IN AN ASIAN AGAMID LIZARD (PHRYNOCEPHALUS VLANGALII)
Review round: 2
Reviewer: 3

Output:
Basic reporting: Basic reporting has been improved from the previous version
Experimental design: Methods better detailed than in previous version
Validity of the findings: In the revised manuscript, the aim is now to test the hypothesis that the temporal and spatial distribution of resources drives the relationship between behaviour and the pace of life. This is a very interesting idea that would deserve more development and empirical attention. However, the current study deals with a single population. Testing such a hypothesis would require replicated sets of populations feeding on spatio-temporally varying resources. This way, we could see if the correlations are different in the precise way predicted by the hypothesis. Since the study is based on a single population, it is not possible to test the hypothesis put forward.
Additional comments: Line 86: opposite: SMR for ectotherms and BMR for endotherms

Line 212: “We found exploration intensity I was negatively correlated with exploration intensity”

Line 239-242: The absence of correlation with SMR should be noted here.

Line 242:243: this is an interesting idea. The cited paper, however does not contain the word "metab" or "scope" or "oxyg*", so perhaps it cannot be used to support the statement.

Line 261: not clear. What are “the 5 individuals potentially indicating a consistent phenotype” exactly?


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: PHYLOGENETIC AND RECOMBINATION ANALYSES OF TWO DEFORMED WING VIRUS STRAINS FROM DIFFERENT HONEYBEE SPECIES IN CHINA
Review round: 1
Reviewer: 1

Output:
Basic reporting: It meets standards of English, article structure.
Experimental design: The research is original. It appears to be rigorous, but more details are required in the description of the methods, including phylogenetic and recombination analyses.
Validity of the findings: Some recombination analyses in this study are insufficient to assess the robustness of the results, although the descriptions of the methods of recombination analyses can be amended by improving the text. Therefore, it needs substantial additional work, including some distinguishing biological property for the potential recombinants.
Additional comments: In this study, Fei et al. conduct phylogenetic and recombination analyses to investigate the evolutionary relationships of the deformed wing virus. I value the efforts that the authors have put into this study, but it is not suitable for publication at the present format. By accessing the results presented in this paper, I don't think in silico recombination analysis is sufficient to justify recombination events. In Figure 3, no putative recombination breakpoints were observed in the China1-2017 or China2-2018 strains from SimPlot analyses. Simultaneously, the recombination events were detected in the two strains by RDP packages without high confidence (at least supported by four but not three different algorithms with an associated P value of < 1.0E-6) in Table 4. To avoid false identification, the settings of key parameters in RDP packages should have to be modified according to the size of the sequence dataset before identifying them as recombinants. In addition, the authors would have to show that these chains have some distinguishing biological property. This does not appear to be the case.

Some minor comments are listed as follows:

ABSTRACT
Line 33	Please replace ‘Provinces’ with ‘Cities’

Line 33-34	‘MF770715’and ‘MH165180’ should be ‘accession number MF770715’ and ‘accession number MH165180’, respectively

Line 49-50	‘similarity’ should be ‘identity’

Line 157, 169 and 180
Please replace the terms ‘GenBank accession no’ with ‘accession number’ throughout the manuscript.

Line187-190
It is now generally accepted that model-based phylogenetic algorithms, such maximum likelihood (ML) or Bayesian inference (BI) produce more reliable results than distance-based (i.e. neighbor joining, NJ) and parsimony (maximum parsimony, MP) methods. Therefore, it would be preferred to reconstruct the phylogenetic tree using either BI or ML algorithms. Notably, which substitution model and what criterion providing the best fit to the data in this study should be mentioned.

Line191-212
For recombination analyses, the potential recombinants and parental strains were firstly identified using seven methods implemented the RDP v4 packages, and then further analysed using Simplot or Bootscan implemented Simplot v3.5. In addition, multiple different approaches were recommended to investigate and confirm the recombination events. For example, phylogenetic networks could be employed when reticulate events such as recombination. Besides, GARD implemented in the HyPhy packages could be used to further confirm the tentative recombinants.

Line 220
Please replace ‘homology’ with ‘identity’ thorough the manuscript. In addition, it is unclear that the best sequence threshold criterion for this genus species demarcation. This should be mentioned in the text.


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

Input:
Title: PHYLOGENETIC AND RECOMBINATION ANALYSES OF TWO DEFORMED WING VIRUS STRAINS FROM DIFFERENT HONEYBEE SPECIES IN CHINA
Review round: 1
Reviewer: 2

Output:
Basic reporting: Please see my 'General comments for the author'.
Experimental design: Please see my 'General comments for the author'.
Validity of the findings: Please see my 'General comments for the author'.
Additional comments: In this manuscript, the authors report complete genome sequences and phylogenetic analyses of two deformed wing virus (DWV) strains, China1-2017 and China2-2018, which were isolated from Apis mellifera and Apis cerana colonies kept in China, respectively. Although I agree that most of the experimental designs and data presented in this manuscript sound solid, I think that the authors need to add some discussions about the results obtained in this study in the Discussion section. I have some major and minor comments, as follows:

Major comments:
1)	Although the authors state in their Abstract that ‘The sequences were compared with…sequences of two closely related viruses, Kakugo virus (KGV) and Varroa destructor virus-1 (VDV-1).’, these results are completely missed in their Results and Discussion sections. These results need to be included in Tables 2-4, and Figs. 2-5 and discussed in their Discussion section.
2)	My impression is that most of the current Discussion section is a mere repetition of information already described in the Introduction and Results sections, and does not contain necessary discussion based on their findings. For example, the authors need to discuss possible reasons why the results of phylogenetic tree of the ORF and VP1 groups better explained the geographic distribution, while the results of the 3C;RdRp-coding region better explained the genotype and diversity of DWVs.
3)	The authors also need to discuss whether DWV strains are infectious to both Apis mellifera and Apis cerana in their Discussion section, based on their results of recombination analysis.
4)	Please explain why it is so far thought that ‘Generally, DWV is only prevalent in A. mellifera and V. destructor, and not common in A. cerana’ and discuss why different results were obtained in this study.

Minor points:
1)	Ls. 220-221; Although the authors state ‘The nucleotide sequence homology…BLAST alignment (Fig.1)’, Fig.1 shows the results of gel electrophoresis but not the results of sequence homology analysis.
2)	Ls. 225-228; ‘each of which contains a single…’ is correct?
3)	L. 292; ‘52%’ is correct?
4)	L. 300; (Table 3) is correctly (Table 4)?
5)	Table 1; DWV10 ‘6294-9274’ seems incorrect.
6)	Table 3 Note; Table 3 contains no ‘shaded frames’.
7)	Table 4 Note; What does ‘2’ that precedes ‘NS: not significant’ mean?
8)	Table 4; Please explain the reason why the results of event number 1 and 6 are shown with bold letters.
9)	Fig. 1; What does ‘C=Negative control’ mean?
10)	Fig. 4; Please explain the meanings of green, red and blue triangles in the legend.
11)	Fig. 5; Please consider to avoid overlapping of vertical lines and some words colored in red. Also consider to show the meanings of red, blue and black lines in the figure legend but not in an inlet (they are too small).


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: PHYLOGENETIC AND RECOMBINATION ANALYSES OF TWO DEFORMED WING VIRUS STRAINS FROM DIFFERENT HONEYBEE SPECIES IN CHINA
Review round: 2
Reviewer: 1

Output:
Basic reporting: It meets standards of article structure, but the language needs some work. This can be improved with extensive language editing.
Experimental design: The research is original. It seems to be rigorous, but more details are clarified in the description of the methods.
Validity of the findings: Some statistical tests are not described in sufficient detail to assess the robustness of some of the results.
Additional comments: – This is a revised version of the paper by Fei et al. on Phylogenetic and recombination analyses of DWV in China. I think the authors made some efforts to improve the manuscript that now looks much better. I am not a native English speaker, but I think that language should be checked to correct several typos and/or sentences which are not clear to me.
After going through the authors' responses, I have the following comments:

METHODS
– Line 192 and 203-208 Please delete the relevant contents of similarity analyses to avoid confusion
– Line 199 replace "substitutionmodel" by " substitution model"
In addition, I think it would be worth specifying the criterion used to select the substitution model for phylogenetic reconstruction.

RESULTS
– The authors mention that some potential recombination signals were detected by RDP package. In fact, Table 4 shows only a significant recombination signal was observed in the China2-2018 (event 3) strain according to the threshold used in RDP4, as described in the method section. However, the authors found no evidence of recombination in the data set using Simplot with reference strains, because no significant recombination breakpoints are shown in Figure 3. The results of Simplot analysed do not support the results of recombination analysis using the RDP4 software. The conclusion on recombination analyses is not only confusing but also overstated based on the results mentioned above. Although the authors answered to my comment on this contradiction by descripting that “it's not appropriate to put the SimPlot analysis and the recombination analysis together”. Unfortunately, this is not a convincing explanation on this contradiction. Thus, I am basically submitting the same review than I previously did. To avoid unnecessary confusion, in my opinion, the relevant contents about the Simplot analyses would be removed from the manuscript.
– In addition, the authors should only mention the recombination events for which the detection of potential recombinants are significant whereas should not mention any non-significant results, which are meaningless.

– Line 241 replace "isloates" by " isolates"
– Line 307 replace "alorithms" by " algorithms"
– Line 308 replace "potenial" by " potential"
– Line 313 replace "recombina" by " recombination"
– Line 315 replace "evnet 5" by " event 5"

DISCUSSION
Briefly, for quantity of provided data, contents of discussion are unsatisfactory.

– Line 323 replace "In Addition" by "In addition"
– Line 323 replace "different different" by "different"
– Line 382, 385, 386 replace "honeybee clonies" by "honeybee colonies"?
– Line 382 replace "ofter" by after?
– Line 391 replace "recombinated phenomenon" by "recombination phenomenon"

Table 3
– This table is not particularly informative, it would fit better as supplementary.

Figure 2 figure legend
replace "Komura-2-parameter" by "Kimura-2-parameter "?


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: PHYLOGENETIC AND RECOMBINATION ANALYSES OF TWO DEFORMED WING VIRUS STRAINS FROM DIFFERENT HONEYBEE SPECIES IN CHINA
Review round: 3
Reviewer: 1

Output:
Basic reporting: It meets standards of article structure, but there are still some typos in the revised manuscript although it has been proof-edited by FSeditor.
Experimental design: It appears to be rigorous, but minor details need to be clarified in the M&M section.
Validity of the findings: It is technically sound, leading to the conclusions listed in the manuscript.
Additional comments: The revised version of the manuscript by Fei et al. has improved compared with the previous version, but a few points might require some attention, especially in the revised manuscript with track changes (please see the attached).

Some minor comments and the typos that should be checked carefully are listed as follows:

-Abstract/Line 62 'fromin'? What does this mean?

-Line 68 'decreaded' change to 'decreased'

-Line 93 '…areatrophy of the wings' change to'…are atrophy of the wings'

-Line 98 'hasbeen distributed' …'change to 'has been distributed …'

-Line 127 'usingrecombination' change to 'using recombination'

-Lin 204-208	Which the nucleotide models and what criterion were selected for each dataset (ORF, VP1 and 3C+RdRp) to perform maximum likelihood (ML) analyses, respectively? The GTR+G+I and Kimura-2-parameter models were mentioned in the manuscript. Do the authors mean that the GTR+G+I model was used for three datasets? If that’s the case, please rephrased this sentence. Here are for your reference. “Then, phylogenetic trees of ORF, VP1 and 3C+RdRp were reconstructed by the maximum likelihood method using MEGA5. The best-fitting nucleotide substitution model (GTR+I+G) was used for the all alignment datasets, which was determined by the lowest BIC score in MEGA 5.”

-Line 208	Please remove the citation of “Kurmar et al, 2016” since it was not referenced by the authors in the manuscript.

-Line 246 'isloates' change to 'isolates'

-Line 389 'related to to' change to 'related to'

-Line 390 'wasdiscovered' change to 'was discovered'

-Line 449-450	Please remove the reference of 'Kumar S, Stecher G, Tamura K. 2016.MEGA5: Molecular Evolutionary Genetics Analysis Version 7.0 for BiggerDatasets. Mol Biol Evol.' because the citation is wrong. In fact, this is a MEGA 7 citation, not MEGA5.

-Figure 2 Legend please remove 'and the Kimural-2-parameter method'


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: PHYLOGENETIC AND RECOMBINATION ANALYSES OF TWO DEFORMED WING VIRUS STRAINS FROM DIFFERENT HONEYBEE SPECIES IN CHINA
Review round: 3
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The authors have made much efforts to appropriately revise their manuscript, and I think the revised version has drastically been improved when compared with the previous one.

The results reported in this manuscript are novel and important.

I am satisfied with all of the authors responses to my and other Reviewers' comments and have no more concerns to this 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: SCREENING OF IMMUNOSUPPRESSIVE FACTORS FOR BIOMARKERS OF BREAST CANCER MALIGNANCY PHENOTYPES AND SUBTYPE-SPECIFIC TARGETED THERAPY
Review round: 1
Reviewer: 1

Output:
Basic reporting: Good
Experimental design: Fine
Validity of the findings: Fine
Additional comments: The manuscript entitled “Screening of immunosuppressive factors for biomarkers of breast cancer malignant phenotypes and subtype-specific targeted therapy” is a well written manuscript. In this manuscript authors have tried to screen and validate immunosuppressive factors in luminal- and basal-like breast cancer cell lines and tissue samples associated with malignant phenotypes. I have following comments to improve the quality of this manuscript:

1. What would be the survival curve (overall survival ) if combination of the screened Immunosuppressive factors are plotted together for CXCL12 and CXCL5 (both Low vs high) or CXCL12 and IDO1, , CXCL5 and PTGS2 & VEGFA and MIF.
2. In your analysis, luminal-like breast cancer tissue contains both luminal A and B?
3. I would suggest quantifying staining (IHC) of MIF and VEGFA in Fig. 7 and plotting it side by side.

Minor changes:
1. Lane 43. The expression monitoring arrays raw data for were downloaded from Gene Expression... For must be removed.
2. Lane 237: stromal cells can be induced to synthesize and/or secret immunosuppressive factors to evade…. It should be secrete.


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: SCREENING OF IMMUNOSUPPRESSIVE FACTORS FOR BIOMARKERS OF BREAST CANCER MALIGNANCY PHENOTYPES AND SUBTYPE-SPECIFIC TARGETED THERAPY
Review round: 1
Reviewer: 2

Output:
Basic reporting: 'Screening of immunosuppressive factors for biomarkers of breast cancer malignant phenotypes and subtype-specific targeted therapy' by Liu et al., is mostly clearly written with relevant literature references. The author can make better use of figure legends to describe data.
Experimental design: Experiments are well designed, however authors should work on interpretation and presentation style.
Validity of the findings: It is an interesting manuscript, but I think authors have not done a great job of explaining their results and giving it larger clinical perspective. I have few comments which could be helpful to revise the current manuscript.
1) Figure-1: is not adequately discussed/explained in text/legends, it is not clear how authors determine basal-like (blue) and luminal-like (red) groups in tumor samples (GSE1561). PCA plot (1A)  also shows four green color triangles (what is the identity of this group?) while hierarchal clustering shows many more unidentified patients (assuming they are green too). Authors should annotate all the patients (and cell lines) in Red, Green and Blue color to main the consistency between both panels.
2) Figure-2: It is not clear why there are more module in cell line data while less module in tumor data. Statement 'For cell lines, module lightyellow, lightgreen, darkmagenta etc.' is ambiguous. Which modules define the basal and luminal like behaviour should be stated clearly in text or figure legend.
3) Figure-3: It appears that author took lead from figure-2 where 'module black in tissue samples were relatively uniform in immune-related events' and investigated immune genes in cell line and tumor data. Since overlap with immune-related and IO gene is very modest author should apply statistical test to what is probability of finding such association by simple chance event. Author may like consider test genes associated with un-related models to test the significance of association with immune genes.
4) Figure-4: Author may like to validate some of the GOBO observations by qPCR analysis in cell lines. In text data should be discussed as described in each panel by specifying observations made in each panel (such as Fig.4A, etc).
5) Figure-5: Panel names are missing from figures, authors can should guide reader via panels, such as 'UALCAN analysis showed that only CXCL12 (Fig. 5B) has lower expression level in basal-like..'
6) Figure-6: Authors claim that 'Analysis of overall survival showed that higher CXCL12, CXCL5, IDO1 and PTGS2 mRNA expression levels were correlated with a comparatively higher survival rate (p<0.01),' However, Figure 6 (Since there are no panel names, I assume-A) CXCL5 P value is 0.14 which is not <0.01 please correct this.
7) Figure-7: Authors should also use markers to identify which cells are expressing VEGFA and MIF.
8) Figure-8: Is CD274 and IL-8 are also highly expressed in Basal-like tumors? Also does higher CD274 and IL-8 expression is associated with poor prognosis?
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: SCREENING OF IMMUNOSUPPRESSIVE FACTORS FOR BIOMARKERS OF BREAST CANCER MALIGNANCY PHENOTYPES AND SUBTYPE-SPECIFIC TARGETED THERAPY
Review round: 2
Reviewer: 1

Output:
Basic reporting: Good
Experimental design: Good
Validity of the findings: N/A
Additional comments: Authors have incorporated all the suggested changes.


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: SCREENING OF IMMUNOSUPPRESSIVE FACTORS FOR BIOMARKERS OF BREAST CANCER MALIGNANCY PHENOTYPES AND SUBTYPE-SPECIFIC TARGETED THERAPY
Review round: 2
Reviewer: 2

Output:
Basic reporting: Pass
Experimental design: Pass
Validity of the findings: Pass
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: AUTOMATIC GENERATION OF OBJECTIVE FOOTPRINT OUTLINES
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript is unambigous and well structured and well written; literature is properly cited, and some additions should be considered only after my comments.
Experimental design: The research questions are well defined, and the code seems solid. However, the test is based only on a reduced sample size (6 footprints) and more data should be included to valdiate the quality of the approach and of the algorithms. It would be also interesting to see how the algorithm deals with steep and low/gentle walls and if the "steepest slope" approach applies properly to different kind of tracks.

The method is properly described, but need a larger sample to be validated. It also need more clear ojectives (e.g., outline comparisons) the results lacks important information for other studies (e.g., biomechanical, ichnotaxonomical)
I have added some link to published 3D material in the pdf and I'm happy to share unpublished data if you need more.
Validity of the findings: The algorithm seems solid, but need more test to be validated and to highlight the limits of the "steepest slope" approach.
Additional comments: Having tryied different objective apporach to the study of tracks I am a bit skeptical it would ever be possible to achieve a fully objective study of footprints. In any case, I think the manuscript is an important contribution to the discipline and is accetable for publication after major revisions.

The mansucript is well written, but lacks a bit of focus on the objectives: tracing an authomatic outline of the tracks, as currently possible with your code, allows broad morphological comparisons, good to study track variability, but the lack of internal details and the misinterpretation of extramorphologies prevent any ichnotaxonomical comparison or study. Such fucus should be more clearly stated in the discussion/conclusion highlighting the limits of the method.

All my comments in the attached pdf.


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

Input:
Title: AUTOMATIC GENERATION OF OBJECTIVE FOOTPRINT OUTLINES
Review round: 1
Reviewer: 2

Output:
Basic reporting: No comment.
Experimental design: No comment on the experimental design.

I recommend that demonstrating the necessity of a new method of determining track outlines, especially given how close the algorithm outlines were to the human-interpreted outlines, will strengthen the manuscript.
Validity of the findings: I would like to see more discussion of how congruent the algorithm outlines are with the human interpreted outlines. It appears that the algorithm results actually provides support for the accuracy of human interpretations and that we can use the older track outlines (especially if that's the only specimen we have to work with) with some confidence.

This leads to my next question: exactly how is this method an improvement over the conventional human-interpreted track outlines? I wish to see presented specific scenarios in which this method would be relied on and be an improvement for interpretations in ichnology. For example, would this method allow for the analysis of intra-trackway variability more efficiently than with using human-interpreted outlines? Does this method assist with interpreting very poorly preserved footprints? Also, when conducting quantitative morphometric studies using footprint outlines, is there a significant difference between the results produced using human-interpreted outlines and the results using the algorithm presented herein?

Based on the 3D models in the supplementary information, there did not appear to be a great deal of preservational variation in the test tracks. It would be beneficial to the reader to see this method applied to tracks with a variety of preservational states. The algorithm-outlines are already demonstrated to not interpret extramorphological features: what other preservational conditions might also produce suboptimal algorithm outlines? These issues should be addressed or at least discussed in greater detail.
Additional comments: The algorithm-based track outlines do have the potential to be used in morphometric studies. However, given the similarities of the algorithm-outlines to the human-interpreted outlines, the paper should clearly demonstrate that the algorithm-based outlines will have a noticeable benefit to ichnological interpretations both for morphometric analysis and for qualitative interpretations. What preservational scenarios will the ichnologist find the algorithm-based outlines preferable to traditional outlines? More discussion is needed to justify the benefit of the algorithm-based outlines over the human-interpreted outlines.


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: AUTOMATIC GENERATION OF OBJECTIVE FOOTPRINT OUTLINES
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Dear Author,
I am pleased to see that all the suggestion were taken into account. The manuscript has greatly improved, and is now suitable for publication.

I still see some limitations for ichnotaxonomical purposes, as internal morphology is not recorded, but it is an important milestone to begin with. I'm confident that further development in this and other computational tools will lead to an integrated (traditional and computational) approach to ichnotaxonomy.

All the best


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: BIOCHEMICAL AND MOLECULAR CHARACTERIZATION OF N66 FROM THE SHELL OF PINCTADA MAZATLANICA
Review round: 1
Reviewer: 1

Output:
Basic reporting: Comments and suggestions:
1. Some grammatical, syntax and spelling errors should be corrected all along the manuscript. English language needs to be improved to be clearer.
Abstract
-The words “homologues” (this word does not exist in the English language) should be replaced by homologs
- Lane 27-  and its full-length cDNA sequenced
- Lane 29- two carbonic anhydrase domains
Introduction
- Lane 47- calcite, and nacre), are
- Lane 55- Reference should be corrected (Sanchez)
- Lane 63-  C-terminal end
- Lane 65- belong
Methods
- Lane 147- Reference last names should be corrected
- Lane 156- Escherichia coli TOP 10 cells were transformed
- Lane 158-  5 mL of the culture medium were transferred
- Lane 161- incubated. Bacterial cells
- Lane 166- Recombinant His-tagged proteins were purified
-Lane 196- As described by Laemmli (1970) and stained with silver nitrate in 10% acrylamide gels. To prepare…
- Lane 211- Native or recombinant??
- Lane 220- 10 mL of 0.2 mg protein/ mL
-Lane 232-  5 mL of 0.012 M
-Lane 234-  magnetic stirring bar
-Lane 335- were added
-Lane 247-  As a control,
-Lane 248- Each solution experiment was made in triplicates and incubated
-Lanes 280-281-  O-glycosylation and phosphorylation sites are…
-Lanes 291 – and silver stained gel were observed
-Figure 3 caption-  coomassie
-Figure 4 caption- exposing the plate to saturated…
-Lane 324- in the presence of 0.2 mg protein/ mL
Discussion
-Lane 373- its recombinant form/protein
-Lane 381- this suggests
-Lane 389- glycosylation plays
-Lane 408- its recombinant protein
Experimental design: 2. Methods
a)	Some sample data are missing. How many organisms were sampled?
Do samples come from one single adult/larvae? Male/female? Or from a pool?
b)	Authors should mention in this section how/why they suggest the native N66 protein was His-tagged to be then isolated by affinity chromatography? It is not clear how do they know these proteins had a His-residues tag exposed to be correctly attached to the Nickel column? Did they build a predictive model of the protein? If they did it, it would be really helpful to include it in the manuscript to reinforce their findings and explanations.
c)	After proteins purification, it is not clear whether authors are talking about native or recombinant proteins in the SDS-PAGE and mass spectrometry analysis, it should be clearer.
Validity of the findings: 3. Results
a) Lane 262- Data was not confirmed since no record was found at NCBI.
b) Table data are not required.
c) Figure 2 should be replaced by a multiple alignment including the same protein sequences that were drawn in figure 2.
d) Table 4 data are already included in the results section, the table is not needed.
e) Figure 3. Authors should clearly explain why two bands were observed? Are there two N66 proteins in the organism extract? Why are there two proteins from the recombinant extracts?
As I understand two proteins may occur naturally in native extracts as isoforms probably, but what about recombinants?
f) If native N66 proteins are O-glycosylated, it would be expected that their molecular masses were higher than those of recombinant proteins which are supposed to be no-glycosylated or phosphorylated… Gels proteins do not match with described results since those from the periodic acid/Schiff staining should be larger and no smaller as the figure shows.
4. Discussion
a)	What does it means that O-glycosylation and phosphorylation sites are flanking the NG domain? Why is glycosylation required for these proteins?
b)	A clear explanation is expected to be given about the two bands, and the no differences in the molecular masses of the native and recombinant forms of N66.
5. Conclusion
As part of the conclusion section, it would be desirable for authors to state clearly their main findings and future perspectives.
Additional comments: Manuscript ID: 35907  Peer J
Title: Biochemical and molecular characterization of N66 from the shell of Pinctada mazatlanica
Authors: Crisalejandra Rivera-Perez, Catalina Magallanes-Dominguez, Rosa Virginia Dominguez-Beltran, Xehu Ojeda- Ramirez, Norma Yolanda Hernandez-Saavedra

The manuscript is about the shell soluble protein N66 from P. mazatlanica. As only a few similar proteins have been studied in other mollusks species, results contribute to gain original knowledge about the mechanisms that mollusk have developed to maintain their integrity and survive. Some major and minor aspects were found to improve the manuscript before it can be accepted for publication.


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

Input:
Title: BIOCHEMICAL AND MOLECULAR CHARACTERIZATION OF N66 FROM THE SHELL OF PINCTADA MAZATLANICA
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comments
Experimental design: no comments
Validity of the findings: no comments
Additional comments: This paper deals with characterization of N66 isolated from the shell of P.mazatlanica and also its recombinant protein expressed in E.coli.
  Many shell matrix proteins have been isolated from nacreous layer of bivalve and N66 is one of them. So, it is not much interesting if papers are merely devoted to similar results reported before. Although the reviewer could not understand well this paper due to poor English writing, it seems that this paper does not provide any new findings compared with those reported previously.
  Furthermore, there are some mysterious results for examples as described below.
1)	SDS-PAGE analyses show intensities of protein bands not dependent on concentrations of various fractions obtained by chromatography (Fig, S2 and S3). This raises the possibility that these are proteins contaminated during purification process.
2)	Recombinant protein from E.coli repression system should give only one protein without any isoforms because this system give no post-translation modification. However, this paper gives two protein bands in SDS-PAGE (Fig.3).
3)	No spectral data for LC/MS are given in this paper, which doubts any guarantee for N66 obtained in this paper.
4)	Other ambiguities are also found in cDNA cloning, Fig. 4 and Fig. 5.


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: BIOCHEMICAL AND MOLECULAR CHARACTERIZATION OF N66 FROM THE SHELL OF PINCTADA MAZATLANICA
Review round: 1
Reviewer: 3

Output:
Basic reporting: It is well known that post-translational modification plays a very important role during the protein interactions, as well as in the process of biomineralization. The author mentioned that a higher carbonic anhydrase and crystallization ability has been observed in in vitro prokaryotic expression of the recombinant N66, rather than the natural N66 extracted from oyster shell, indicating that the modification of the N66 protein might inhibit its own functions. Although, it‘s meaningful for us to understand how matrix proteins regulate shell formation, the overall conclusion is not well supported by the authors’ experimental findings. The experiments can be further improved.
Experimental design: The experiments can be further improved.
a. Compare carbonic anhydrase and crystallization ability of recombinant N66 with native one, after in vitro glycosylation. And also compare both activitis of the native N66 with recombinant one, after de-glycosylation.
b. Does Native N66 have other post-translational modifications? If so, can it also affect carbonic anhydrase and crystallization ability of N66?
Validity of the findings: The conclusion is not accurate.
The author made a conclusion that the lack of posttranslational modifications in the recombinant N66 modulates its activity via simply comparing the carbonic anhydrase and crystallization ability of natural N66 with recombinant N66. However, protein post-translational modifications are not only glycosylation modifications, but also other post-translational modifications such as phosphorylation.
Additional comments: no comment


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

Input:
Title: BIOCHEMICAL AND MOLECULAR CHARACTERIZATION OF N66 FROM THE SHELL OF PINCTADA MAZATLANICA
Review round: 2
Reviewer: 1

Output:
Basic reporting: I deeply reviewed the last version of the manuscript. All comments were taken by authors, and mistakes were corrected. The corrected English version is clear and fully understandable.
Experimental design: Each of the major concerns about methods and missing date were corrected. This section is now described in sufficient detail and clarity.
Validity of the findings: New and original data are part of the manuscript, perspectives were also included.  All data were validated and confirmed to be correct from the newly added explanations.
Additional comments: THE MANUSCRIPT PROVIDES BASIC KNOWLEDGE ABOUT THE SHELL SOLUBLE PROTEIN N66  FROM P.  mazatlanica.  INFORMATION IS NOW COMPLETE AND CLEAR. RESULTS WERE CORRECTLY ANALYZED AND COMPARED WITH OTHER PREVIOUS STUDIES TO  BETTER UNDERSTAND THE PARTICULARITIES OF THIS ORGANISM PROTEIN. THE MANUSCRIPT SHOULD BE NOW ACCEPTED.


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: BIOCHEMICAL AND MOLECULAR CHARACTERIZATION OF N66 FROM THE SHELL OF PINCTADA MAZATLANICA
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The mechanism on the regulatory role of matrix proteins during biomineralization in mollusks remains unclear. It's great that the auther used the modifications of matrix protein as a springboard for studying its functions. I hope the auther will dig more deeper.