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: TOTAL INCOMBUSTIBLE (MINERAL) CONTENT OF CHERAX QUADRICARINATUS DIFFERS BETWEEN FERAL POPULATIONS IN CENTRAL-EASTERN AUSTRALIA
Review round: 2
Reviewer: 1

Output:
Basic reporting: This is second review, thus straight to constructive comments. The study reports valid results, but sampling has been very limited in 3 our 5 cases. The Discussions section is a bit confusing and should be compacted. The data from Lake Mooregah is not convinsing, and leaving it out would not take anything out of the rest of the data sets. Even the statistical comparisons would be valid.
Experimental design: See comments below.
Validity of the findings: Some notes on the results speculation:
1. Abstract and Results section claims Lake Ainsworth crayfish to differ significantly in their mineral content from other locations (Abstract, line 34) and from Emigrant Creeck Lake and Lake Somerset (Results, line 214). This is a bit confusing and should be presented in a similar manner in both cases.
2. Line 41 in Abstract, Lake mentioned but is there a word missing (Ainsworth)?
3. I would recommend using (or listing when first mentioned) common names, in addition to scientific names (line 93 onwards).
4. The authors are discussing possible reasons for observed differencies in mineral contents of the sampled populations. First, the sampled numbers are rather small in most cases (less than 5), which would suggest a rather limited speculation for the reasons. Also, the Discussions section is built with short, even one sentence paragraphs. I would suggest the authors to compact the presentation into a short note. As an example, the text in paragraphs starting from line 278 to line 303 compacted. The speculation here evolves around low pH and calcium, and could be presented in much compacted manner.
5. Lines 319-323, this sentence is very hard to understand and needs rewording. Seems like the authors are talking about mineralisation process and environmental (water quality) dynamics in this sentence, but in a manner that does not open to me.
6. Lines 326-331, in this paragraph there are two sentences, but it is hard to understand their logical connection. Word 'population' repeated here. Rewording needed.
7. Line 354, the pathway south is speculated to be some 600 km, what about the pathway north, some 600 km, too?
8. Lines 360-361, what are the authors trying to say here? It sounds strange, that the authors are doubting their data. I agree, though, that the sample numbers are too low, but are the authors stating it here?
9. Figure 2, the data from Lake Mooregah (n=2) does not look convinsing. I still suggest that this should be omitted, especially since the other too samples with low numbers do have much smaller SD (or variation).
Additional comments: The study is of value, but the presention should be compacted to a note. Most of the sample sizes are low (less than 5), which is normally considered preliminary data.


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

Input:
Title: TOTAL INCOMBUSTIBLE (MINERAL) CONTENT OF CHERAX QUADRICARINATUS DIFFERS BETWEEN FERAL POPULATIONS IN CENTRAL-EASTERN AUSTRALIA
Review round: 2
Reviewer: 2

Output:
Basic reporting: The revised manuscript is very much improved version as most of the comments have been accepted and incorporated into the revised version.
However, my only critical comment that would stay for this revised manuscript is that the research importance of this article is still limited and unfortunately nothing can be done to improve this unless there is some more data available that has not been used.
Experimental design: Greatly improved in terms of details now.
Validity of the findings: More references have been added
Additional comments: There is a great improvement in this version. The additional details in experimental design has made it more clearer now.
I still suggest to reduce some of the discussion points that are merely based on the speculation.


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: TOTAL INCOMBUSTIBLE (MINERAL) CONTENT OF CHERAX QUADRICARINATUS DIFFERS BETWEEN FERAL POPULATIONS IN CENTRAL-EASTERN AUSTRALIA
Review round: 2
Reviewer: 3

Output:
Basic reporting: The revised introduction easily leads the reader through the background information on the species and provides compelling justification for the study. I do think the discussion could continue to benefit from some smoothing out, including reformation into paragraphs with clear topic sentences (in places such as 301 and 305, it appears single sentences are comprising a paragraph).
Experimental design: Thank you for being so thorough in explaining what you were measuring in your comments. I believe I better understand the differences in our lines of thinking now. From my understanding of your methods, after the organic carbon is burned off and the sample weighed again, the difference between dry weight and ash weight is considered to be total mineral content. I remain unconvinced that what remains is only mineral. I do not at all doubt that you have found differences in composition between these populations of crayfish, but I am not fully convinced that what remains after an hour exposure to 500 deg C is mineral content, especially mineral content that can be interpreted in the context of environmental influence on calcification.

I can imagine a few instances where this may not be the case. First, crustacean cuticle is a heterogenous material where mineral and organic material are tightly intermingled. As a paper I cited in my last review demonstrates (which was published after the protocol used here was established), sometimes an hour of ignition does not fully burn off organic carbon in sediment samples, and I am curious if that may be true for a crustacean cuticle, a material with a complex biological matrix of organic content and mineral.  I have not myself examined ashes for evidence of incomplete combustion and do not know if visual inspection may identify if there is any remaining organic material on the micro scale of the matrix.

Secondly, it is my understanding that other inorganic elements and compounds will remain after this ignition protocol, which are included in the weight measurement going into calculations of the mineral content but are not strictly minerals. These may include metals that are found in crustacean cuticles, such as aluminum and manganese, and other elements such as chlorine. If one sampling location has more bioavailable metals (lower pH waters may increase bioavailability), the differences in the weights of your samples may have arisen for this reason, not because of the available calcifying elements or pH. In short, I have not yet found any other papers that use only the data from a loss of organic carbon to discuss mineralogy.

I will be more satisfied that these methods are sufficient for producing the desired result of mineral content if one or all of the following are provided: 1) peer-reviewed literature that demonstrates that these methods have been used on biological samples to quantify mineralogy, particularly for samples that include both organic content and mineral in a heterogenous material (especially crustaceans). 2) If the ashes are still accessible, determining carbonate content for the reasons I suggested in my last review (provides more specificity for quantifying the calcium carbonate polymorphs, although you would not be able to calculate any calcium associated with hydroxyapatite) or using another technique to measure the ashes for calcium and magnesium to compare to the water sample data. I do not believe knowing the exact concentrations of the minerals in the samples are required, but this approach would assuage any doubts that your methods are only targeting the calcifying minerals. 3) Using new samples to demonstrate that another established technique of measuring mineral content and this protocol produce analogous results. 4) I’d also be amenable to another option. In short, I would like to be assured  that the waterbody-specific differences in percent weight of the remaining material is indeed mineral likely to have been used in calcification (eg calcium carbonate) and not another non-organic component of the crayfish bodies. This will better support the argument that the available waterbody data does provide insight into the differences in the composition of these crayfish.

I also do have to mention a potential sticking point with the water data. I appreciate the clear effort put into procuring and organizing it for this revision. I am concerned that these samples from Lake Ainsworth were taken 18+ years before the crayfish samples were collected, and as line 227 notes, crayfish were not present in this waterbody until more than a decade after. A lot may have changed in the water conditions in intervening years that is not captured here. Ideally, more recent calcium and magnesium measurements could be made, although it has now been a few years since samples were collected, but this would potentially provide evidence that conditions have been stable. However, if the 1996 and 1987 samples were taken from the same area of the lake, or if the lake is small enough to be homogenous , there seems to be evidence that there are large changes in lake minerals. It is promising that there are not large differences in the pH you have measured and the pH from these samples. Also, thank you for being upfront about this in the figure caption. If you are not able to get more recent data, I suggest also noting the difference in the age of samples in the text as a caveat also.

What does seem more problematic is that parts of Lake Ainsworth have (or used to have) a very high amount of calcium—the maximum values are 3-4 times greater than the maximums recorded for other waterbodies. Please justify the focus on the 1996 samples rather than the 1987 samples  which include high measurements of calcium that do not support the hypothesis presented here.
Validity of the findings: Mixed in with above
Additional comments: Thank you for your revisions and well-organized comments. Please find a few remaining line comments below.

Methods
Line 142: To clarify my last comment on the pH scale, I was asking whether pH was measured on the total, NBS, or free scale. These scales can be more than 0.1 pH units different from each other. If you are only interested in the differences in pH across sites, this might not be important, but I think it would be useful to include to add context to the absolute values in Table 2 and knowing if the data in Table 1 and Table 2 are measured on the same scale is important when comparing them. I believe NBS is most common for freshwater.

Results
Line 210: To clarify my last comment on line 194 of the first manuscript, I would appreciate seeing the means +/- SE of the crayfish mineral content here in the results (potentially in Table 1) rather than trying to read values off of Figure 2. I’m sorry for being unclear about that.

Discussion
Line 288: It might not be clear to readers how acidity and low calcium levels may be two separate issues based on the information provided in above paragraphs that focus on how calcareous mineral or calcium storage and deposition are affected. I believe it would be clearer to also mention that bicarbonate is less available at reduced pHs, thereby making calcification more difficult if bicarbonate isn’t fully supplied physiologically. Thus, if acidity and low calcium levels interact, both components of calcium carbonate may be difficult to procure.

Line 309: I believe this introduction of cuticle thickness needs more context or should be left out. Yes, these papers also studied crayfish living in low pH environments, but since the response variable studied is different, I do not think they are very complementary. Cuticle thickness may not be correlated with mineral content, as mineral density might differ, and there may be different mechanisms behind more dense or reduced construction of Bouligand layers than impregnation of mineral. Without further explanation, readers may be thrown off by a somewhat abrupt injection of discussion of structure rather than composition.


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: TOTAL INCOMBUSTIBLE (MINERAL) CONTENT OF CHERAX QUADRICARINATUS DIFFERS BETWEEN FERAL POPULATIONS IN CENTRAL-EASTERN AUSTRALIA
Review round: 3
Reviewer: 1

Output:
Basic reporting: Addressed with previous reviews
Experimental design: Comments below
Validity of the findings: Comments below
Additional comments: Dear authors,

Thank you for the revision and working to clarify some points. Here are my short thoughts:

With this revision, we agree on the results provided from this methodology, and I am glad we have reached this understanding. Yet, this now means I don’t follow the conclusions from these data.  Because we both agree that the method applied does not provide quantification of the constituents of the material left over from combustion, I do not understand how the discussion may speculate extensively about how the conditions of the lakes influence a specific part of the mineral content measured. Authors, I do understand that your goal was to measure “the amount (quantified by DWT) of incombustible material in the crayfish.” Yet, the discussion does not stop here—I would be satisfied if it did, simply pointing out these differences. Just your crayfish data and the assembled water quality, separately, do add valuable information to the literature, as you assert in your last response. However, potential calcium differences in these crayfish feature heavily in the discussion—about five paragraphs’ worth—although was not studied by these methodologies, as we agree.

I’m not sure how else to bridge this gap, so please allow me to present how I see this situation, in another context. Researchers set out to study five forests and record the total abundance of furry animals (total mineral content) in each, and they find some forests have fewer than others. Discussing the results, they argue that the number of squirrels in some forests are reduced, probably because there are fewer oak trees. This is the breakdown I see; if the researchers wanted to discuss squirrels, that is the data that should have been collected (this was my sticking point on the last two reviews—why calcium content wasn’t measured when that was the component of the incombustible material that was of interest). Perhaps there are the same number of squirrels in the forests, and the differences in the number of furry animals is instead due to the number of rats (metals, for example); there’s no way to know that from just the data on furry animals alone. Or, more troubling, the differences in furry animals are due to a few animals; there are fewer squirrels in one forest and fewer rats in another—this is something else the methodology cannot parse apart. Changes in total mineral content do not provide information on how calcium differs in the body of these crayfish, as other constituents may be driving the differences in Lake Ainsworth crayfish.
I do see great value in publishing the basic data (in a reduced manuscript with less speculative discussion, as reviewers 1 and 2 have brought up), as you argued in the latest response, and I appreciate the work you as authors have put in; the changes I have seen over these few revisions have led to a stronger, refined piece of writing.  However, I remain unconvinced about the how differences in total mineral content arise.


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

Input:
Title: IDENTIFICATION OF SPECIES AND MATERIA MEDICA WITHIN SAUSSUREA SUBG. AMPHILAENA BASED ON DNA BARCODES
Review round: 1
Reviewer: 1

Output:
Basic reporting: The authors should revise the English grammar of the manuscript.
Experimental design: Appropriate.
Validity of the findings: Appropriate and accurate.
Additional comments: Saussurea is an important genus in Asteraceae with high species diversification in high mountains in northern hemisphere. The authors made a detailed and thorough barcoding analyses on an interesting group of subg. Amphilaena, which is traditional and very popular as medicine uses. All the commonly used markers have been sequenced for barcoding analyses and species identification with comprehensive sampling including both populations and individuals. Data analyses and explanations are enough and excellent. This study is fell well within the scope of the journal, it is a good paper to be accepted.
A few minor issues are given below:
1.	Line 116: a having more than two populations ?
2.	Line 119: to my knowledge, matK is included in trnK and only matK is commonly used for barcoding. Since only a small fragment is only sequenced in barcoding analyses, trnK here is only a different short fragment from matK. Please clarify the use of trnK and its difference from matK.
3.	Line 131: our sampled ?
4.	Line 197: an the individual ?
5.	Line 484 Table 2: what’s meaning for numbers after voucher numbers in the bracket.


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

Input:
Title: IDENTIFICATION OF SPECIES AND MATERIA MEDICA WITHIN SAUSSUREA SUBG. AMPHILAENA BASED ON DNA BARCODES
Review round: 1
Reviewer: 2

Output:
Basic reporting: The authors collected extensive samples from Saussurea in the field and they sequenced five DNA barcode candidates in chloroplasts (matK, trnH-psbA, trnK, and rbcL) and the nuclear ITS. This manuscript is well written, I suggested that this work should be accepted after minor revision.
Experimental design: I suggest authors should utilize incongruence length difference and partition homogeneity test when you use more than one barcode markers to discriminate species.
Validity of the findings: If possible, authors should collect some commercial herb in market to test their identity according to Pharmacopoeia of People's Republic of China.
Additional comments: No additional comments


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

Input:
Title: IDENTIFICATION OF SPECIES AND MATERIA MEDICA WITHIN SAUSSUREA SUBG. AMPHILAENA BASED ON DNA BARCODES
Review round: 1
Reviewer: 3

Output:
Basic reporting: Chen et al. have done a good job to test five candidate barcode in delimitation of medical plants in Saussurea subg. Amphilaena, the results are clear and the conclusions are umambiguous, and their English are mostly satisfied. They are familiar with taxonomy and references related to Saussurea, and their organization of the article are professional.
Experimental design: The experimental design of this work is sufficient to address key questions the authors want to ask.
Validity of the findings: The data that the authors presented in this manuscript is robust. Usually barcoding researches sample 3 individuals per species, and in this study they sampled ca. 8 individuals per species, which is more than enough. The author well addressed their key questions by running a series of analyses and the key conclusions they made are objective, i.e. well supported by their data and results.
Additional comments: Chen et al. have done a good job to test five candidate barcode in delimitation of medical plants in Saussurea subg. Amphilaena, the results are clear and the conclusions are umambiguous, and their English are mostly satisfied. They are familiar with taxonomy and references related to Saussurea, and their organization of the article are professional. The data that the authors presented in this manuscript is robust. Usually barcoding researches sample 3 individuals per species, and in this study they sampled ca. 8 individuals per species, which is more than enough. The author well addressed their key questions by running a series of analyses and the key conclusions they made are objective, i.e. well supported by their data and results. However, I have some comments which may be helpful to improve the manuscript.
(1) At the end of the Introduction as well as in the paragraph before Conclusion, the authors discussed about possible substitute of closely related medical plants. I think on this point, the author should be cautious. It would be a good strategy to put forward potential substitute species, yet it is highly necessary to warn the readers that extraction and validation of medical components should be done before such substitution be made. The other way is that, your DNA barcoding method could be use to refrain substitution of well-known medical species with species that is common yet less effective medically.
(2) In L197-191, the author claimed that lower gene flow of rbcL compare to ITS and higher mutation rate of trnK compare to rbcL is the reason why combination of ITS+rbcL perform better than ITS+trnK. However, my opinion is that, maybe weaker incongruence between ITS and rbcL than between ITS and trnK is the most important issue, i.e. ITS signal is much stronger than rbcL, and hence rbcL work as a complementary part, but both ITS and trnK signals are strong and thus lead to a conflict between barcodes. This can be tested by conducting Partition homogeneity testing that is performed in PAUP*. Just do the test for ITS vs rbcL and ITS vs trnK would be sufficient.

Minor concerns:
L69: change 'with' to 'show'?
L70: distantly related 'according to molecular phylogeny'?
L86: is is necessary to put 'S. subg. Amphilaena' after 'useful'?
L90: second-class 'protected plants in China'?
L91: 'of' may be replaced with a coma
L93: What do you mean? Do you mean identifying substitutes for medical usages is urgently needed? Or do you mean identifing fake medical materials (cheap substitute expensive) is urgently needed?
L111: I think nearly all plants you involved have been reported to have medical value, how can you find new plants? I suppose that you should have to involve previously not-recorded-as-medical plants to mine new medical plant species.
L148: I think you should tell PCR success rate and sequencing success rate separately. The current presentation is wrong and misleading. The PCR and sequencing success rate should be the product that multiply the PCR success rate and the sequencing success rate.
L175: I do not agree with this. The PCR successful rate is significantly higher than trnK and trnH-psbA, yet more or less close to rbcL and matK.
L187-188: please clarify;
L190: do you mean 'higher intraspecific differentiation between populations'?
L204-206: please clarify;
L227-228: Please rephrase to improve clarity;
L231: is 'potential' better than 'possible'?
L238-240: I think you need more evidence here. Or else, warn the authors that you are infer and assume. Plus, this is slightly contradict with the next paragraph where you said closely related species share the same or similar metabolimes and bioactivities.
L251-254: I do not think this is a good ending of the paragraph. A better ending would be suppose that these complex you proposed could be further checked with their secondary metabolimes and bioactivities, so as to make sure some plants can substitute the other, if cultivation is not possible.
In addition, I would like to recommend the author to add a figure to show some beautiful photos of some beautiful species of this section in Saussaurea. I am sure that will attract attention from many non-taxonomist readers!


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

Input:
Title: IDENTIFICATION OF SPECIES AND MATERIA MEDICA WITHIN SAUSSUREA SUBG. AMPHILAENA BASED ON DNA BARCODES
Review round: 2
Reviewer: 1

Output:
Basic reporting: Clearly.
Experimental design: Reasonable.
Validity of the findings: Accurate.
Additional comments: I have checked the manuscript again, and I found that the authors have carefully revised the manuscript according to my previously suggestions and comments. So I recommend it acceptance in its current form.


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

Input:
Title: IDENTIFICATION OF SPECIES AND MATERIA MEDICA WITHIN SAUSSUREA SUBG. AMPHILAENA BASED ON DNA BARCODES
Review round: 2
Reviewer: 2

Output:
Basic reporting: I think the authors have added these analysis i mentioned.

This manuscript should be accepted.
Experimental design: no
Validity of the findings: no
Additional comments: no


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

Input:
Title: IDENTIFICATION OF SPECIES AND MATERIA MEDICA WITHIN SAUSSUREA SUBG. AMPHILAENA BASED ON DNA BARCODES
Review round: 2
Reviewer: 3

Output:
Basic reporting: N/A
Experimental design: N/A
Validity of the findings: N/A
Additional comments: I am happy that the authors addressed all concerns that I raised in last round of review carefully and in a point-by-point manner. I think the manuscript reads stronger than last version, and I believe this work will attract interests from many readers working on medical plants, Asteraceae, DNA barcoding and high altitude flora, among others.


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: A GELATIN/COLLAGEN/POLYCAPROLACTONE SCAFFOLD FOR SKIN REGENERATION
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: A Review of:

Title: A gelatin/collagen/polycaprolactone scaffold for skin regeneration
This study investigated the effect of composition and formulation of gelatin/collagen/PCL polymeric materials on in vivo efficacy to enhance skin wound healing, especially with the aid of stem cell incorporation. Although a series of material characterizations was evaluated for various compositions, material’s functionality to facilitate in vitro cellular proliferation and in vivo skin tissue regeneration does not seem to be significant. Specifically, in order to conclude that this biocomposite material sufficiently support full thickness in vivo skin regeneration, authors should have included the experimental control group such as sham. All other comments listed below.
1.	Please revise any typos, grammatical errors, and abbreviations.
2.	Provide how statistical analysis was performed in the method section.
3.	Figure 1 (Protein release): It might be better to provide a degradation profile of biocomposites in the in vitro condition with the release of gelatin and collagen from the materials. Total release amount of gelatin/collagen, evaluated by conventional BCA assay, should be correlated with that of mass loss and physiological degeradation of biocomposites over time.
4.	Figure 2 (Surface morphology of fabricated biocomposites): It would be more informative to provide any discussion about a surface coverage (as seen in Fig 2) of the fabricated scaffold materials and any possible inhibition of cellular infiltration. In general, the topography/ surface morphology and correated overall porosity might influence cell and tissue infiltration in an in vivo environment.
5.	Figure 3 (Cellular proliferation on the top of resulting scaffolds): Please remark any statistical difference between formulations in the figure. Moreover, please elaborate discussion about how G/C/P formulation affect the in vitro cellular proliferation.
6.	 Figure 5 (in vivo functionality): Again, it is hard to conclude that G/C/P scaffold could facilitate skin would healing, unless the sham control is provided. Please also remark statistical difference in Figure 2 (Was the only significance observed on Day 21?).


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: A GELATIN/COLLAGEN/POLYCAPROLACTONE SCAFFOLD FOR SKIN REGENERATION
Review round: 1
Reviewer: 2

Output:
Basic reporting: Clear english- yes
Literature - insufficient
Structure -  acceptable, I find it better to discuss results as they are presented and not in a separate section but authors followed PeeJ standard
Figures - Not all are professional (Fig 4)
Raw data - Not all is supplied
Self contained- yes, but with limitations
Experimental design: Yes
Yes-
Yes-
Yes-
Validity of the findings: Data is not robust
Conclusions are not well supported
Additional comments: Development of more effective wound healing procedures and substitutes is a clinical need, but it is my opinion that with this work the authors did not achieve a significant advancement and conclusions are not well supported by the data.

L29- Bioplastics – definitely not a commonly used adjective to characterize polymers like gelatin, collagen and polycaprolactone. Not even the authors use it on a regular basis, the final appearance of bioplastics is on line 72, when the paper has 424. Moreover, the work contextualization, first 10 lines of the introduction, in totally off, the world use of petroleum-based plastics has nothing to do with a biomedical application of gelatin, collagen and polycaprolactone.

L111- “both biocomposites” – did the authors mean “both types of biocomposite”? , because 6 different composites were produced that can be grouped in two sets

L111- Please confirm that type B gelatin is of bovine skin origin

L155- Please check that the cross-head speed was 50 mm/min. This is a very high speed for a mechanical test, why did the authors choose it and the consequences must be discussed.

L157 – 3 measurements is insufficient given the typical variability of test results given the fluctuations in sample production and preparation.

Table 1- The way authors choose to report solution composition is in no way reader-friendly. For instance, 0.0004g/0.2ml should be reported as 2 mg/ml. This is totally different in terms of readability. Also, final percentage of each polymer in the biocomposites should be given as this simplifies the interpretation of results like protein release.

Fig1- Protein release is very fast, therefore the usefulness of having a gelatin/collagen component in the scaffold should be discussed. Also, why, given the “burst release” of the protein component the authors did not opt to stabilize this component by crosslinking it.

Fig 1, Line 243 and others- Since protein release exceeds 100% in the case of GCLPL, this means that the calibration curve may be significantly offset. Given this uncertainty, it is incorrect to state release values up to the tenth percent.

Fig 1- the authors do not give any explanation on why for some scaffolds protein loss is total whereas for others it is not. The authors simply describe their factual results without attempting an explanation. Is protein loss correlated with gelatin content? Is collagen more resistant to dissolution? Is collagen lost by day 3, or does it also dissolve? Why should the GCLPL scaffold lose all its G/C content and the GCHPL retain about 30%?

L247 – Why should scaffolds that are very similar behave so different in terms of protein loss? Why should they plateau by day 3 at different release values? Does plateau in reality mean total release?

L256– The original DSC/TGA spectra should be supplied as supplementary information.

L259- I am not sure about the explanation given for the different crystallinity values. Crystallinity seems to increase with PCL content, which leads me to ask the authors if the melting enthalpy, which the software calculates given the sample mass, was corrected for the G/C component.

L263- The 1% difference is irrelevant, smaller than the experimental uncertainty, which is more than just a standard deviation.

L265- J/g

L278- Why didn’t the authors determine mechanical properties for all scaffolds?

L278- “tensile strength” should be compressive strength, “elongation” should be strain and elastic or compressive modulus would be better than Young’s modulus.

Table 3- Uncertainties should be expressed with one digit only (not including zeroes to the left).

L284 and Table 2- Pore size cannot be determined from SEM images, period. The definition of pore in such structures is by no means straightforward. Measurement of a “pore” area is highly prone to bias. If techniques like mercury intrusion or bubble point porosimetry had been used, operator bias would have been removed and everyone knows about limitations and approximations with these methods. Here, we know nothing about the measurements because authors did not supply annotated SEM images.

L294 – Authors do not report how many times these experiments were performed and with how many replicas each time. Given the overall behavior of cell populations with time, I do not see any significantly different cell behavior on all samples tested. Differences reported are within the experimental uncertainty given the variability of these experiments.

Fig 4 has no quality. We only see blurred images impossible to interpret. Moreover, controls without primary antibody and DAPI counterstaining should have been performed.

L318 – Given that only the scaffold GCLPH was tested for its mechanical properties, how can it be selected in detriment of others based on its “proper mechanical properties” (why are they proper?)

L223- In the experimental methods the authors state that: “The wounds were divided into the following two groups: blank GCLPH biocomposite and ASCs-seeded GCLPH biocomposites”. Fig 5 reports results for these two groups, but then Fig 6 contains data for two additional groups.

L 326- “wound contraction was evidently observed only in the GCLPH group at 16 and 21 days” – Wound contraction happens in both groups and appears to be higher for the ASCs + GCLPH group.

Fig 6 – Is “Area of wound “ the area of the wound that has not been re-epithelialized? If it is, how did the authors evaluate this area?

L327 – “Gomori’s trichrome staining was performed in the ASC-seeded GCLPH, open wound, ASCs-only, and non-seeded GCLPH groups” – The open wound and ASCs-only groups were not reported until this sentence!

L338 – The novelty of this skin substitute is very limited. The relevant literature, where very similar scaffolds were studied, is not comprehensively discussed.

L348 – It is not true that gelatin has no drawbacks. Fast dissolution is one.

L365 – PCL is degraded by enzymes like collagenase, MMPs and so on?

L420 – There was no optimization, simply the choice of a scaffold with the lowest collagen content.

L423- “rapid closure and re-epithelialization of skin wounds were observed in nude mice treated with ASC-seeded GCLPH biocomposites” - an important phenomenon with clinical implications is wound contraction and scarring, which were not discussed.

L424- No advances were demonstrated in comparison with currently used treatment methods.


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: A GELATIN/COLLAGEN/POLYCAPROLACTONE SCAFFOLD FOR SKIN REGENERATION
Review round: 2
Reviewer: 1

Output:
Basic reporting: Statistical analysis is missing in so figures and relevant discussions are necessary.
Experimental design: According to #1 (Basic reporting), it is necessary to report any significance in formulations to conclude the therapeutic efficacy of the present materials.
Validity of the findings: N/A
Additional comments: N/A


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: A GELATIN/COLLAGEN/POLYCAPROLACTONE SCAFFOLD FOR SKIN REGENERATION
Review round: 2
Reviewer: 2

Output:
Basic reporting: Clear english- ok
Literature - acceptable
Structure - ok
Figures - Fig 4 is still difficult to observe
Raw data - ok
Self contained- not really: authors want to lower collagen content in scaffolds they studied previously but replaced collagen by gelatin that was not cross linked so it dissolved pretty fast and authors ended up studying several PCL scaffolds.
Experimental design: Yes
Yes- : research question is relevant but methods used do not lead to a convincing result.
Yes- : I have no reason to have doubts about technical and ethical standards, it's experimental planning that, to me, was not the best
Yes
Validity of the findings: Data is not robust, statistically weak
Conclusions are still not well supported
Additional comments: In spite of the corrections and improvements made by the authors, this new version still requires attention to quite a few issues detailed below:

L30 – The last mention of “bioplastics” still standing

L31 – The methods description became more confusing than before. My suggestion: “G/C/P biocomposites were fabricated by impregnation of lyophilized gelatin/collagen (GC) mats with PCL solutions, followed by solvent evaporation.
Two different GC:PCL ratios (1:8 and 1:20) were used.”

L40 – remove “Besides,”

L42 – “than those”

L45 – “As expected” may be an indication of some bias result analysis, not the best way to start a description of the main results achieved.

L48 – The scaffolds the authors developed are not just a “Wound dressing”, they are aimed at assisting/promoting skin regeneration.

L256 - Protein release from the G/C/P: the results are still presented without convincing explanations (those put forward by authors do not explain all results) and in a difficult to read manner. Quantitative results should be presented in a table with composite composition (percentage of each polymer in the scaffold) alongside.

- Also, the authors didn’t answer my question: “Why should the GCLPL scaffold lose all its G/C content and the GCHPL retain about 30%?”.

L258 - The authors argue that differences in protein loss between GCLPL (higher loss) and GCLPH (smaller loss) are due to the higher PCL content of GCLPH that encapsulates G and C. But then, why is it that GCHPL that has a smaller PCL content also has a smaller protein loss that the GCHPH?

L262 – “The cumulative release (w/w) at 24 hours was 100% and 82.6% for two biocomposites respectively. The released rate reached a plateau for both biocomposites after 3 days. “ – If the cumulative release reaches 100% after 24h, then the plateau is reached after 24 h, not after 3 days… Also, instead of “release rate”, “cumulative release” should be used.

L284 – The authors didn’t directly answer my question, if the melting enthalpy, which the software calculates given the sample mass, was corrected for the G/C component. However, the values they now present are higher than those in the preceding version, which suggests this correction was made. Is this the reason why values are different?

L284 – A crystallinity of 84.7% for PCL is something I don’t recall ever seeing reported. Also, I don’t understand to which scaffold this value pertains as the text reads “the 1:20 collagen:PCL biocomposite displayed the highest crystallinity of 84.7%, followed by 58.7% for 1:8 collagen:PCL biocomposites”, are these Dai et al (2004) scaffolds or are these the authors present scaffolds? Moreover, these numbers are not on table 2 that still contains the same numbers as the previous version.

L285 – In the previous version, the crystallinity of PCL films prepared by solvent casting was characterised as “notably low at 54.2 ± 5.4%” but now it is “unrealistically high at 54.2±5.4%”… what happened?

L291 – In my previous report I questioned the authors “Why didn’t the authors determine mechanical properties for all scaffolds?”. The answer, “We measured the G/C/P composites” does not really answer my question because the authors produced 4 G/C/P composites and only one, which was later used in the in vivo tests, was tested here, denoting a bias towards this scaffold.

L307 – Given the production method used and the fact that most, if not all, protein content was lost (I wonder what the advantage of incorporating one was), cells are attaching to a PCL surface. Therefore, no significant differences should show up in day 1 populations and the differences reported may be simply due to the difficulty in detaching and collecting all cells for subsequent counting using trypsin. What surprises me most is that fibroblasts, which are easy cells to culture and attach, seem to have had a great difficulty in attaching to TCP, an unusual behavior.

Fig 4 – The images I have access to are the same as in the previous submission, blurred and with some of the arrows pointing to almost dark areas. Also, without staining controls lacking primary antibody and without DAPI counterstaining there is no way to know if the arrows are pointing at cells, dirt or some other feature. I wonder how authors could measure the areas reported, what their criteria was.

L604 – “Immunohistochemical”

L328 – “involucrin expression of keratinocyte on GCLPH was only slighter lower than that of GCHPL – From Fig 4C it is about two thirds, which, in my opinion, does not qualify as “only slighter lower”. In fact, from fig 4A, the images pertaining to GCLPH exhibit the faintest staining.

L334 – Wounds heal by a combination of contraction, which is clearly seen in Fig 5, synthesis of a new dermis and a new epidermis, also called re-epithelialization. Wound contraction is easy to observe but re-epithelialization is not and at least from the figures the reader cannot distinguish between open wound (without epidermis) and closed wound (with epidermis). The authors did not answer my question: how did they evaluate re-epithelialization?

L334 – Why is it that in Fig 5A one cannot visually confirm the presence of the scaffold? Wounds receiving the scaffold and control look alike. The scaffold should impart a whitish appearance to the wound.

L343 – “We could observe that the biocomposite (GCLPH only) became incorporated into the regenerated skin” – How? What do figures show that can be unambiguously identified as the biocomposite? What is identified in Fig 6F as biocomposite is simply a scab.


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: A GELATIN/COLLAGEN/POLYCAPROLACTONE SCAFFOLD FOR SKIN REGENERATION
Review round: 3
Reviewer: 1

Output:
Basic reporting: It is hard to understand which comparisons were performed in Figure 3 (in vitro proliferation of cells). Even though authors stated "∗: indicates P<0.05 relative to control" (i.e., TCP) in the Figure caption 3, the statistical difference is indicated in the control group itself. It seems that any significance is observed in the control group as compared with other formulations. Please revise them all.

In addition, more importantly, the statistical analysis should be also performed in the Figure 5 (in vivo wound closure). In order to emphasize the therapeutic efficacy of the present platform, any significant difference in wound closing area should be indicated as compared with the untreated wound control (purple line), at each given time point.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: no comment


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

Input:
Title: A GELATIN/COLLAGEN/POLYCAPROLACTONE SCAFFOLD FOR SKIN REGENERATION
Review round: 3
Reviewer: 2

Output:
Basic reporting: All OK
Experimental design: All OK
Validity of the findings: All OK
Additional comments: The manuscript was improved and may be published after a few minor issues are solved:

- L187 “immunohistochemical”

- L280 – I still don’t understand why the crystallinity of PCL films is “unreallistically high at 54%” when the crystallinity of 1:20 collagen:PCL composites is higher, at 63%

- L321 – alpha-tubulin dimerizes with beta-tubulin to form microtubules, a major component of fibroblast cytoskeleton, but not “the main component”

- L326 – The GCLPH composite was chosen for its collagen content, the lowest of the 4 PGC scaffolds studied, not because it “showed the nearest tensile strength to 1:20 C/P biocomposites”, this is irrelevant.

- L603 and 665 – What are “positive cells”?

- L631 – The SD should be presented with only one, or at the most two, digits (not counting zeros to the left of the first non-zero digit)

- L653 – The meaning of the second set of columns (4, 5 and 6) of figure 4 is not stated, nor what is Anti-GFP, nor why there are repeated images in Fig 4.B

-L661 – Fig 6F has an arrow pointing to what is supposedly a piece of the biocomposite but that looks like debris from a scab.


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: TIME ARROW IN PUBLISHED CLINICAL STUDIES/TRIALS INDEXED IN MEDLINE: A SYSTEMATIC ANALYSIS OF RETROSPECTIVE VS. PROSPECTIVE STUDY DESIGN, FROM 1960 TO 2017
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1.1. Clear and unambiguous, professional English used throughout.
The article would require considerabe editing to get to the clarity and simplicity of expression needed.

1.2 Literature references, sufficient field background/context provided.
More complete review of the existing literature would be helpful. There is some existing literature on distribution of research in narrower topic areas that could be interesting to discuss, here are some exampes
Falagas ME, Papastamataki PA, Bliziotis IA. A bibliometric analysis of research productivity in Parasitology by different world regions during a 9-year period (1995–2003). BMC Infect Dis. BioMed Central; 2006 Dec 17;6(1):56.

Falagas ME, Karavasiou AI, Bliziotis IA. A bibliometric analysis of global trends of research productivity in tropical medicine. Acta Trop. Elsevier; 2006 Oct 1;99(2–3):155–9.
Michalopoulos A, Falagas ME. A Bibliometric Analysis of Global Research Production in Respiratory Medicine. Chest. Elsevier; 2005 Dec 1;128(6):3993–8.

1.3 Professional article structure, figs, tables. Raw data shared.
The artice is well organized and adhers to the IMRAD structure.
Figures need to be reconsidered. Results that are simple enough to be displayed in pie charts should usually just be presented numerically in the text or, when there are many categories, in a table. I do not think the pie charts are needed. The line graph should not be shade.

In Figure 1, top panel, you have graphed 10 year intervals until the final, which is 5 years. This erroneously looks like production has declined.

1.4 Self-contained with relevant results to hypotheses.
Good
Experimental design: 2.1 Original primary research within Aims and Scope of the journal.
No major issues

2.2 Rigorous investigation performed to a high technical & ethical standard.
The reporting of the search makes it difficult to assess but 1. You must report your search strategy in greater detail. I cannot replicate it or determine if it is of good quality.  The following should be reported:
The date you searched
The interface or platform used to search MEDLINE (this could be Ovid, Ebscohost, PubMed, to name a few of the possibilities)
A transcript of the search

Both retrospective studies and prospective studies exist as MeSH terms and each has narrower terms representing specific study designs.  The limit to humans is reasonable. I do not know of any article type limit for "original study".  Please clarify this.
.

2.3 Methods described with sufficient detail & information to replicate.
There are quite a few points here for the authors to address.
I attempted to replicate your search, although I had to make some assumptions. My results are below. I would expect to see a transcript like this in the appendix.

Ovid MEDLINE, searched April 13, 2018.
Searches
1	exp Retrospective Studies/	681063
2	exp Prospective Studies/	469502
3	limit 2 to (yr="1960 - 2015" and journal article)	411859
4	limit 1 to (yr="1960 - 2015" and journal article)	579375
5	1 or 2	1131628

These proportions work out a little differently than from your  search; 58% retrospective and 42% prospective.

Please describe your process of randomly sampling studies.

Why was it necessary to have the full text and what do you mean "the topic was assessable by the availability of a full text? Does this mean a free full text was linked in PubMed, or that the journal was available in your institutional library or that you requested articles through interlibrary loan.  This could seriously bias your findings. Wikipedia describes this in an entry for "FUTON" bias (https://en.wikipedia.org/wiki/FUTON_bias)

How did you determine country of origin? What is the country where the research was performed or the country listed for the corresponding author? How did you classify studies that either recruited patients from multiple countries or had authors from multiple countries (although this would only be a factor if there were a mix of high and low income countries represented).

You classify the research into categores – Aesthetics, ophamlogy, pneumology etc. What is the typology you are using and how did you determine, from the article, where is should be classified?  You may be describing this in lines 110-111, but much more detail would be needed.

Please provide a source for the GDP figures used to classify countries as high or low income. Do I understand correctly that all of the countries in the world were classified and 25% fell in the high income range and 75% in the low income range?

I am not confident about which results pertain to the period 1960-2015 and which results pertain to 2004-2015,
Validity of the findings: Without more information on the methods, it is difficut to assess the accurracy of the results from MEDLINE however, the numbers presented seem reasonable.

An issue that needs to be considered is whether research conducted by investigators living and working in  low income countries is published in MEDLINE indexed journals?  See, for example Salager-Meyer F. Scientific publishing in developing countries: Challenges for the future. J English Acad Purp. Pergamon; 2008 Apr 1;7(2):121–32.   One hypothesis about the rise of "predatory publishers" is that they provide access
3.1 Data is robust, statistically sound, & controlled
The data file made available with the manuscript would require additional annotation or would need to be organized differently before being interpretable to another investigator.
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: TIME ARROW IN PUBLISHED CLINICAL STUDIES/TRIALS INDEXED IN MEDLINE: A SYSTEMATIC ANALYSIS OF RETROSPECTIVE VS. PROSPECTIVE STUDY DESIGN, FROM 1960 TO 2017
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1)  The introduction and background do not fully describe the field being reviewed. No research question is given.  From the abstract, an objective is alluded to, but not fully explained:  line 45-46 we aimed this study to assess if there is a prevalence of retrospective vs prospective design in the clinical studies/trials by querying MedLine.  Considering all research as either retrospective or prospective without considering quality seems simplistic when there are many observational designs that may be appropriate to the research question.

2) Authors do not describe a clear need for the review.  The intended audience (i.e. which field) is not described.  The  relevance to readers of PeerJ in both the immediate field and any associated fields is not clear.

3) The English language needs improvement to ensure that the audience can clearly understand your text.  Some examples where the language could be improved include:

Lines 39-43 Clarity at the outset is very important to orient the reader.  These sentences are too long and complicated:  "Clinical studies/trial are considered by the scientific community the most appropriate instrument to identify causes of disease and/or to set up/verify possible strategies to prevent or care them. In such approach the attention to the time-line of the variables under study might be focused on a single time point or to follow time, backward or forward, in the so called, respectively, retrospective and prospective design."

Lines 74-76 Very unclear.  Consider adding some definition of the concepts “time”, “rhythm of changes”, “chronobiology”.  Also the sentence is too long.
 "We generally consider time as a quantity that in clinical studies/trials plays the role of  independent variable having, often, a real value on an axis of a graph; but time is just a way to give order to changes and, in biology, time is essentially rhythm of changes, with short and long cycles that are controlled by specific allocated resources that are the matter of chronobiology."

Line 150-151  Very unclear – are the authors likening publication and research trends to human biology?  I do not understand.
"Biological aging is, invariably, accompanied by the experience of disease, a domain that seems to be ruled by chance but when clinical researchers study diseases, generally, to preserve human life and avoid unnecessary suffering, what they actually do of the time variable, namely, how the variables are followed, or sampled, in time, backward or forward?"

Line 180 this phrase is not correct English:   "Clinical studies/trial the time variable is fundamental in the design the study;"

4a) Citations:
Some citations given seem relevant but not all assertions by the authors are backed up by citations for example:

81-83 "Usually the observation of the variable(s) under study is fixed on singular time points or is followed backward or forward in the so called, respectively, retrospective and prospective design of the study."
I think a better overview of observational studies should be found and cited.

4b)  This assertion is incorrect, and based on a 1959 citation:
Line 86-87 - sources of errors due to bias and confounding, that are indeed, more common if compared with prospective studies."

Research methods have evolved since 1959 and rather than labelling a type of research design “biased” it is more appropriate to discuss how well the research design suits the research question.   A case-control, or other retrospective study design may be very appropriate, for example, in the study of rare diseases. “For rare diseases or the diseases that have a long latency period between an exposure and outcome, case control studies are often the only feasible choice.” [See:  https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3326852/]

5) The structure conforms to PeerJ standards with the appropriate headings given.

6) Figures are well labelled and described.

7) Raw data has been supplied.
Experimental design: 8) Research question is not provided.  An identified knowledge gap is not described, nor how this research fills such a gap.

9) Methods are not described with sufficient detail and information to replicate.
The full, original search strategy should be given, including which platform was used to search MEDLINE.  The reader also needs to see how the limits of “Human” and years were applied in greater detail.

10) It is unclear how authors could tell whether the studies were actually retrospective or prospective with a yield of over 1 million records.

11) There is no discussion of how technology may affect the quality or bias of retrospective or prospective studies.  Very large datasets are available to researchers, and the types of large registry studies we see today would not have been possible then.  I find the inclusion and comparison of pre-computer research and research designs inappropriate, and perhaps this should have been addressed in the limitations.  Indeed, the very reason the authors chose this time interval was because (line 90-92) “starting from 1960 to 2015, an interval that was considered representative since, before 1960, very few studies are available.”  They were are not available because they have not been added to a very large modern database.
Validity of the findings: no comments
Additional comments: I hope the authors consider adding more people to their team.  I think the work would benefit from enlisting a professional librarian with experience and advanced skills in searching the MEDLINE database, and clearly reporting the search to a current standard (see: https://www.cadth.ca/resources/finding-evidence/press).

The manuscript would benefit from the involvement of someone with strong English writing skills to the team who can clearly articulate their work for the audience.

Some other minor comments/revisions:

Line 76-79 – the example given about  “chronobiology” and illnesses of “chronodisruption” is unclear.  Was the study limited to these types of studies? Please explain the relevance.

Line 85 – spelling error:  cheeper should be cheaper

Throughout: MedLine  should be written as MEDLINE

Line 195-196 – Please remove this line:  “Secondly, a paper describing an off-label use does not necessarily support a possible future clinical use” – unless it can somehow be shown to be relevant to the objectives of the study.


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

Input:
Title: TIME ARROW IN PUBLISHED CLINICAL STUDIES/TRIALS INDEXED IN MEDLINE: A SYSTEMATIC ANALYSIS OF RETROSPECTIVE VS. PROSPECTIVE STUDY DESIGN, FROM 1960 TO 2017
Review round: 2
Reviewer: 1

Output:
Basic reporting: The introduction needs to include the research question or problem, in one simple sentence.  Also please clearly state the aims of the paper.   (Explain in one aim per sentence).

The first paragraph contains excessive background information which is difficult (impossible?) to understand without knowing its relevance to the research question.  Lines 90-98 are actually methods, and they do not belong in the "Introduction and Aims"

Clarity of the written English could be improved throughout the paper.
Experimental design: The research question is not well defined.  See comment above.  The reader should understand the research problem in the first paragraph.

The search methods are still not reported to a current expected standard.
At the minimum, the authors should provide the exact search string that was used to search Pubmed, including boolean operators and date limits.
This can be provided either by cutting and pasting, or screen shot either in the body of the paper, or an appendix.  If more than one search was done, all the search strings should be reported.  Without this information, there is not sufficient detail & information to replicate.
Validity of the findings: no comments
Additional comments: I thank the authors for the improvements to this manuscript, and for their letter explaining the actions taken to address my comments.

I strongly urge the authors to add more people to their team.  The paper needs to be edited by someone with very strong English writing skills.  A professional librarian could ensure that the MEDLINE (actually Pubmed)  search is executed and reported accurately.


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: TIME ARROW IN PUBLISHED CLINICAL STUDIES/TRIALS INDEXED IN MEDLINE: A SYSTEMATIC ANALYSIS OF RETROSPECTIVE VS. PROSPECTIVE STUDY DESIGN, FROM 1960 TO 2017
Review round: 3
Reviewer: 1

Output:
Basic reporting: Thank you for the opportunity to review this manuscript in its revised form. I agree that the authors have robustly assessed alternative search strategies and selected one that balances simplicity and accuracy. This is a sound approach for a bibliometric study such as this.
The English usage and style in this version is much easier to read and understand. I will leave the decision as to whether further refinement is needed to the journal editors.
There are a few remaining issues, I think the authors will be easily able to address all of them.
Finally, may I say that this paper would have been much stronger if the authors had examined the prior literature on research capacity and productivity across the spectrum of low to high resource countries.
Experimental design: Issue 1. Reporting of the search strategy. You need to say what you did, exactly, so others can replicate your search.
Please change the reporting in methods, line 111-113, reporting this almost exactly as you did in the reply to reviewers document:
We searched PubMed in September 2017 as follows;
(retrospective study AND ( "1960/01/01"[PDat] : "2017/12/31"[PDat] )) sorted by Best Match
(prospective study AND ( "1960/01/01"[PDat] : "2017/12/31"[PDat] )), sorted by Best Match

When I paste the string
(retrospective study AND ( "1960/01/01"[PDat] : "2017/12/31"[PDat] )) into PubMed today, October 18, 2018, I retrieve 744,511 records, pretty close to your reported 787,938.  When I enter what you have in methods “retrospective study” using best match sort and keeping the publication dates the same above, I get 115,502 records, which is vastly different.
Issue 2 – Survey findings

Lines 177 – 182. Now you are citing the actual survey instrument in SurveyMonkey. In earlier version of the manuscript, you cited this preprint in ResearchGate: Ciulla MM. The arrow of time in clinical studies. Retrospective vs prospective: a survey on the propensity of researchers in clinical study’s design. ResearchGate (2016). doi:10.13140/RG.2.1.1725.1443

The citation of the preprint is necessary unless you want to include the methods and results in this manuscript. Either way, a couple of points are; 1. what is the response rate of the set you were able to survey? 2. What were the reasons investigators chose a prospective design? The pre-print seems to show results only for those who chose a retrospective design. 3. You state in line 181-182 “Furthermore, the surveyed researchers believe that the prospective design is more expensive, statistically accurate and clinical predictable.” The survey question reads “statistically appropriate” so the wording in the manuscript should also say “appropriate”.

Issue 3

In the  Discussion, line 213  you state “previously ascertained through a comparison also with other databases such as EMBASE”
  – introducing Embase here is too late, assuming you mean the exploratory work you did in Embase and described in the reply to reviewers document. You should report this work in the methods section, lines 114-116, pretty much the way you described it in the reply to reviewers.
Validity of the findings: An assumption that the authors seem to be making throughout the paper is that all articles indexed in PubMed are clinical studies. Many won’t be, they may be animal studies, studies of health systems, even methodological research on retrospective or prospective study designs. A note in Limitations section could just indicate you are using this term very broadly and there will be some imprecision in your results.

Another limitation to mention is that only the address of the corresponding author was considered (and this is a limitation of MEDLINE) whereas other research databases such as Web of Science provide all authors, allowing consideration of international collaborations (see for example Huffman MD, Baldridge A, Bloomfield GS, Colantonio LD, Prabhakaran P, Ajay VS,Suh S, Lewison G, Prabhakaran D. Global cardiovascular research output, citations, and collaborations: a time-trend, bibliometric analysis (1999-2008).PLoS One. 2013 Dec 31;8(12):e83440. )
Additional comments: Key word selection: I would add “bibliometric study” to represent the methods used.

Line 92 – delete the word appropriately

Line 116 – I am not convinced at all that it prevents classification problems.  I recommend deleting this phrase. As well citation 8 (Volpato) is not a great source for this, as the best match was deployed in 2017 and Volpato does not test anything like it. I would defer to the National Library of Medicine documentation or an article that studies Best Match directly such as Fiorini N, Canese K, Starchenko G, Kireev E, Kim W, Miller V, Osipov M,Kholodov M, Ismagilov R, Mohan S, Ostell J, Lu Z. Best Match: New relevance search for PubMed. PLoS Biol. 2018 Aug 28;16(8):e2005343. doi:10.1371/journal.pbio.2005343. eCollection 2018 Aug. PubMed PMID: 30153250; PubMed Central PMCID: PMC6112631.

Line 165. Reference 8 (Volpato) is cited here as well. Likely, 9 is the intended reference (Ciulla)


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: TIME ARROW IN PUBLISHED CLINICAL STUDIES/TRIALS INDEXED IN MEDLINE: A SYSTEMATIC ANALYSIS OF RETROSPECTIVE VS. PROSPECTIVE STUDY DESIGN, FROM 1960 TO 2017
Review round: 4
Reviewer: 1

Output:
Basic reporting: In terms of the overall structure of the document, the quality of the English writing is now ranges from good to excellent.  I did notice a little type in line 222 "MEDLINE is t is constantly".

I think the limitations section (line 212 – 224) belongs in the discussion section rather than the conclusion.
Experimental design: First, the authors (or their advisors) do not seem aware that PubMed Best Match is a ranking algorithm. It has a very small effect on which articles are retrieved, but has a significant effect on the order they are presented it. Thus it doesn't address bias and it doesn't exclude animal studies.  This is pretty easy to test empirically  and I suggest you repeat your searches comparing counts for best match and the same search sorted by date. Then limit each to human, than to animal and repeat the sorts. I think you will quickly understand what is happening.  Therefore, line 117-118 needs to be altered from "we relied on the free text search by using the "best match", a function available on PubMed that prevents incorrect classification problems by including MESH." to a simple statement that they searched using the Best Match feature, without making any claims about what that feature does.

Likewise, under the heading "limitations" in lines 218 where the authors state "Secondly, by using the keywords without the quotes, inevitably, produces some noise, nonetheless, by using the recently updated “best match” function for PubMed that includes MESH, we prevented most incorrect classification problems." This is simply false and the authors need to accept that their methods have some limitations.  May I suggest something along these lines:

"Secondly, bibliometric methods examine a body of work at a macro level and our results must be interpreted in light of this.  Using a simple search has the benefits of being readily understood and easily replicated but inevitably produces some imprecision or noise. In our study 93% of prospective studies and  95% of retrospective studies were indexed as human, some were index as animal studies, some were indexed as both human and animal studies, and some were indexed as neither. As well, about 8% of prospective studies were also retrieved by the search for retrospective studies and are included in both analyses. "

Some data;
		Total	Animal	%	Human 	%	Both Human and Animal %	Neither	  %
prospective	652002	21520	3.3	607159	93	10414			1.6	33737	5
retrospective	789806	18207	2.3	746473	95	7746			0.9	32872	4

both pro & retro	53429
% of prospective 	8.1%
Validity of the findings: The authors may have misunderstood my suggestion that using the address of only the first author may overlook international collaborations between researchers in high and low GDP countries. This leaves the slightly awkward final sentence (line 223) "Finally we considered only the address of the corresponding author for the attribution papers to the country of origin trusting in the authorship general criteria." I was not implying at all that authors were misrepresenting their contribution.  I would prefer that this paper acknowledge that is not able to capture collaborations, but either way I would prefer to see this sentence deleted.
Additional comments: The changes you need are small but vital to the credibility of your paper.


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

Input:
Title: GENES OF THE PIG, SUS SCROFA, RECONSTRUCTED WITH EVIDENTIALGENE
Review round: 1
Reviewer: 1

Output:
Basic reporting: Basic reporting is generally sound, however there are several sections that could do with proof checking to provide a clearer understanding of what the author means, please see general comments below. I also think some time spent checking grammar and spelling would be warranted.

One area that could do with improvement is in regards to context/aims in the introduction. E.g. line 44 please give the reasons/context for why gene set reconstruction fails. Based on this, set out how the methods applied here, inc the new automated pipeline, aims to address this.  – also you don’t say what it is you are demonstrating in the para (which is important)

While I commend the author for providing a thorough data and software citations section that provides links to all the projects/data and software used, I do feel that it does a disservice to their authors/contributors by not citing the appropriate literature, which should be rectified in the revision.
Experimental design: The method section, while presenting an ok overview of the board steps involved does lack sufficient detail to be replicable. The whole section reads rather jumbled and does not flow logically. I would like to see clear description of steps involved and citations to techniques implemented if not described here.

Line 70 – what types of modelling, what are their qualities over one another – cite works that has already shown this if available.
99 – what assembly methods and parameters – while these are listed later (128) parameter choice is not given. Maybe a summary table of assembler and parameters would help?
104-106 you say data was selected on tissue sample, methodology and others; then point to data section – yet these details are not given in that section. Please be clear about how you selected data. i.e to cover the majority of tissue types, only selecting for Illumina PE data etc etc
130 – k-mer sizes selected would also be good to should in the table suggested above.
134 – provide detail on normalization methods.
Validity of the findings: The implemented pipeline/method shows substantial improvement in terms of gene set reconstruction and will be useful to future work, which is in line with the stated conclusions. The data seems robust and sound (I like the persistent DOI’s that provide clear data and results).

However, I find the results section particularly hard to follow and glean the important information from. It reads largely as text pointing to where results data can be found (e.g.175-186 and 192-201, 248 onwards) rather than actually presenting results in a clear and interpretable manner.

161 How many subsequent runs – how much did they iteratively improve the results?
228-229 is more of a description of a dataset rather than results of its analysis.
248-253 – where are the results in this section?

I feel the discussion is missing important evidence (in terms of citations or data shown in results) to back up statements made. E.g. Lines: 301-303, 311-314). An important consideration is that the authors ‘sells’ the note as a demonstration of his pipeline. While examples of how to execute it are available online I think a more clear demonstration in this paper is warranted. Be this either as a clear step by step example with embedded code (which would be highly valuable to readers) or a clear flow diagram (and associated text) that clearly shows the computational steps of the pipeline.
Additional comments: It is unfortunate that this note reads as being largely unfinished, as I am keen to see it published because the method is exciting and valuable. I think it is easily rectifiable with further work.

Please give full species names in para 4 of Intro.
The language used can be difficult to follow, for example the following sentences could do with rewording to improve their clarity. Some examples follow, but it is not an exhaustive list: Lines: 38, 97, 106, 111, 130-135,160, 163, 190, 205, 268-271
111-115 does not fit in methods – maybe this should be moved into the discussion in a considerations section or removed entirely.


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: GENES OF THE PIG, SUS SCROFA, RECONSTRUCTED WITH EVIDENTIALGENE
Review round: 1
Reviewer: 2

Output:
Basic reporting: In this manuscript, the author described the result of his annotation pipeline “the EvidentialGene” for a pig genome. However, I could not evaluate it properly due to the lack of critical information in the current manuscript. He cited his previous works published as conference posters, which does not have a full description of his method.

For example, gene construction part is critical in this process, but he only described all steps in one paragraph (2nd paragraph of the method), and his cited poster also does not have a full explanation: how to make a score for each step? What is the minimum evidence score? What kind of evidence was used, and how were they weighted? I think this manuscript would be better to describe the Evidential Gene pipeline more technically, with a pig transcriptome as an example.

There is no figure in this manuscript, but all tables need to be revised substantially. Table 1 is hard to read the information. Table 2 needs more information to understand what “Full”, “Align”, Miss”, and “Best” mean. Table 3 also requires more explanation about the criteria for “Miss”, “Frag”, and “Short”. Also, the author mentioned four data in the manuscript, but he only reported two cases here. The more comprehensive report should be presented for each table.
Experimental design: De novo transcriptome assembly is one of the key steps in this method, but the detailed experimental procedure is lacking. For each step, the author needs to provide detailed parameters, and how to evaluate a tremendous amount of transcriptome candidates for refinement. In the current manuscript, the author only reported the number of outputs, without explaining how that outcome was produced. The author also mentioned multiple assemblers, but it is not clear which assembler is used for each data. It should be clarified.

More explanation is required why a pig is a good case to apply the EvidentialGene pipeline. Although the author described some reasons in Introduction (many data, good quality of genome, etc), the genome is published already and major genome resources such as EnsEMBL and UCSC provided comprehensive annotation, and a recent version of EnsEMBL (Sscrofa11.1) also incorporated PacBio long reads and RNA-seq data for their annotation. At least, the author needs to compare current annotation with the result of EvidentialGene, which is completely missing in this manuscript.
Validity of the findings: I agree with the author that the approach presented in this manuscript (EvidentialGene) is useful to evaluate different version of genome annotation in many organisms. However, current manuscripts need to be revised substantially, to support its contribution. Without systematic comparison with current annotation, it is hard to know the benefit of this approach. Although the author provides the comparison with RefSeq data, at least two other major annotation data (EnsEMBL, UCSC) should be analyzed.

The conclusion is also not well presented by data. The main description is the number of transcripts and their associated loci, but the more important thing is how to reduce false positive (mis-annotated genes), or false negative (missing genes). By analyzing genes not identified in this pipeline, and those only identified here, the author may argue the benefit of the EvidentialGene to reduce these errors.
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: GENES OF THE PIG, SUS SCROFA, RECONSTRUCTED WITH EVIDENTIALGENE
Review round: 1
Reviewer: 3

Output:
Basic reporting: This manuscript was well-written and relatively easy to understand. One point that I think really could be improved (for clarity) is how the author refers to the different data sets used. I think I counted 4 different methods used (full database name, submitter, pig1a or just pig 1) throughout the manuscript. Please select one method. My preference would be something easy for the reader to relate to; ie. submitting institution, or reason the data set was chose, rather than a 5 letter code followed by 4-6 integers or a random number assignment. My only other item to mention is Table 1 looked more like a slide than a table. Otherwise this was a very easy manuscript to read.
Experimental design: I think the overall design of this study was quite sound. However, the description of some methods are clearly lacking. First and foremost, the gene sets were mapped to the swine genome, but the author never states what build of the swine genome was used. This is a must! Also, I hope it was build 11.1, because if it wasn't, then the author needs to go back and re-map these loci.
Second, the author selected 4 public data sets. The description of these data sets (lines 117-126) is extremely inadequate! I suspect each data set was selected for a reason. I suspect the first data set was selected as it probably has the most reads, the last data set as it had low read depth and may contain more developmental genes and the second data set may have been selected as it was the only one with Iso-Seq data (PacBio longread), but this isn't specifically stated. Also, lines 120-121 states all data were short-read, paired end data, but the results indicate PacBio reads were used (lines 231-236) for the second data set, this needs to be corrected.
Validity of the findings: The results of the new analysis pipeline appear to fix many issues with current automated annotation pipelines. While there are a number of different methods that can be used to annotated transcripts, no single method is perfect. Thus, it is quite useful to have a variety of methods developed. Comparison among results of different methods can either validate annotation or point researchers to regions that may need additional experimentation to resolve.
If I understand correctly, the final list of transcript are for protein coding mRNA. How are non-coding transcripts identified and annotated with the current methods? Do some (or all) end up in the culled transcripts group? If they do, I think it may be interesting if the author could elaborate more on these important transcripts.
Additional comments: Overall I really enjoyed this manuscript and believe it offers a valuable method for semi-automated annotation of transcripts. I also thought the discussion on how results are affected by k-mer length selection was very enlightening. While I am sure this method has some limitations (as all methods do) it clearly is superior to the current methods used for the pig.


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: GENES OF THE PIG, SUS SCROFA, RECONSTRUCTED WITH EVIDENTIALGENE
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Firstly, thank you for the effort in revising the manuscript – especially the inclusion of additional comparisons to NCBI and ENSEMBL sets and the much needed methodological detail (inc the script archive), which improve not only rigor but increase the papers usefulness.

The paper is much improved since my last evaluation. However, I still find the language used hard to digest, but this may be personal preference of writing styles. I would request an additional careful read through before final acceptance.

Minor comments:

Line 66. “The automated pipeline of SRA2Genes is introduced and will be detailed in other papers.”

Change to say either “The automated pipeline of SRA2Genes is introduced” or “The automated pipeline of SRA2Genes is introduced and the EviGene reconstruction method will be detailed in forthcoming papers.” I don’t like “here’s this, but im not talking about it yet” – my preference would be for the first edit.

Line 250. “There are many more alternate isoforms in Evigene models than in the other two gene sets.” Does the author believe these are real isoforms, or potential misassemblies? Does mapping the RNA-Seq data (esp pac bio data) confirm that some of these are in fact real? This would add additional evidence that evigene produces a ‘better’ gene set. Otherwise, maybe evigene is producing a ‘better’ passing set, but the alt set has much higher prevalence of false isoforms?

Line 330: Im not happy with comments such as “This author often compares NCBI and Ensembl genes when constructing Evigene models, observing that NCBI methods now commonly surpass those of Ensembl, as is tabulated in these pig gene sets. “ . Unless the author can back them up with valid data across multiple species. Either just say that NCBIs current pig gene set is better, or back up your generalisation with evidence.


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: GENES OF THE PIG, SUS SCROFA, RECONSTRUCTED WITH EVIDENTIALGENE
Review round: 2
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The revised version has addressed all of my concerns. I have a few minor edits that need to be done.
Line 19, I think indicating improved alternate transcripts and more full-length transcripts would be better than just stating "other improvements".
Line 39 change 'pubic' to 'public'.
Line 74 insert 'and' in front of 'instrument'.
Line 97 delete the first 'are' on this line.
Line 196 are the 5177 exceptions coding-sequences or loci?
Line 395 a full citation for Zhao is required.
Table 2 does not define 'Full' and has a definition for 'Short' that is not used.


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

Input:
Title: IDENTIFICATION, EXPRESSION, AND PHYLOGENETIC ANALYSES OF TERPENOID BIOSYNTHESIS-RELATED GENES IN SECONDARY XYLEM OF LOBLOLLY PINE (PINUS TAEDA L.) BASED ON TRANSCRIPTOME ANALYSES
Review round: 1
Reviewer: 1

Output:
Basic reporting: The study presents a transcriptome analysis of secondary xylem of loblolly pine. However, the English language of the manuscript should be improved to ensure that your international audience can clearly understand.
Experimental design: I notice that the loblolly pine genome has been sequenced by Zimin et al. (2014).Why did the authors still adopt a de novo method for transcriptome analysis? Once when the gene sequences have been obtained, it is more interesting to analyze their relative expression levels in such a specific tissue.
Validity of the findings: Six key genes (i.e. DXR, DXS, HMGR, IPPI, GGPS and FPS) involved in terpenoid biosynthetic pathways were selected for multiple sequence alignments, conserved motif prediction and phylogenetic analysis. Why did the authors choose these genes? Are they completely assembled? Are there additional homologs in the genome?
Additional comments: 1.	The title of the manuscript should be changed based on the content presented.
2.	The loblolly pine genome should be mentioned in the introduction part. Besides, the reason to sequence the secondary xylem should be introduced.


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

Input:
Title: IDENTIFICATION, EXPRESSION, AND PHYLOGENETIC ANALYSES OF TERPENOID BIOSYNTHESIS-RELATED GENES IN SECONDARY XYLEM OF LOBLOLLY PINE (PINUS TAEDA L.) BASED ON TRANSCRIPTOME ANALYSES
Review round: 1
Reviewer: 2

Output:
Basic reporting: The content of the manuscript is clear. Comprehensive background have been provided. Tables and Figures are up to the mark. In some sentences grammatical errors are noticed.
Experimental design: Methods  are described in sufficient details.
Validity of the findings: Enough informative data are generated. Discussion and conclusion are well described.
Additional comments: No additional comments


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

Input:
Title: IDENTIFICATION, EXPRESSION, AND PHYLOGENETIC ANALYSES OF TERPENOID BIOSYNTHESIS-RELATED GENES IN SECONDARY XYLEM OF LOBLOLLY PINE (PINUS TAEDA L.) BASED ON TRANSCRIPTOME ANALYSES
Review round: 1
Reviewer: 3

Output:
Basic reporting: Based on transcriptome data from RNA-seq of secondary xylem of loblolly pine, the authors obtained many genes related to terpenoid biosynthesis, and performed multiple sequence alignment analysis, conserved motif prediction and phylogenetic analysis on six key genes involved in terpenoid biosynthetic pathways. Overall, the manuscript provides some useful information for studying the regulatory factors involved in the terpenoid biosynthesis of loblolly pine. However, the manuscript still has some main issues need to be addressed. Moreover, the authors should check the manuscript carefully, and many minor mistakes need be corrected.
Experimental design: no comment
Validity of the findings: A major concern is that the results lack experimental validation on the expression patterns of key genes involved in terpenoid biosynthesis in different oleoresin-yielding stages (e.g. from low to high) by qPCR. The authors only sequenced the samples from a high oleoresin-yielding stage. Thus, the qPCR validations are important for identifying the candidate key genes.
Additional comments: Title
1. The title “Genome-wide” should be deleted because no genome-wide screening was conducted within the whole genome of the loblolly pine (Pinus taeda) in the study.

Introduction

1. Line 37-38, the biotic stresses obviously include insect herbivory, fungal attacks. Thus, the sentences should be reworded.

2. The sampled tissues in the study were from secondary xylem of loblolly pine. How secondary xylem tissues are associated with location of resin canals or terpenoid biosynthesis? The authors should add the related description in the Introduction section to provide more justification for secondary xylem as the studied tissues.

Materials and methods

1. Based on the NCBI SRA accession number PRJNA482703 provided by authors, I searched and found the sampled tissues named as cambium according to BioSample information(https://www.ncbi.nlm.nih.gov/biosample/9709534), which is not consistent with the RNA-seq samples in this manuscript (secondary xylem tissues). Cambium or secondary xylem? Please explain.

2. The biological replicates of RNA-seq should be stated clearly in the Method section.

3. Lines 70-73. The authors should provide more detailed information on sampling, such as the pictures of sampled tissue, sampled location in the trunk, and methods of sampling, which can be easily understood by readers.

4. Line 90. Illumina HiSeq 2000 or 4000？The inconsistence is shown between this manuscript and NCBI sequencing information (SRA experiments) (https://www.ncbi.nlm.nih.gov/sra?linkname=bioproject_sra_all&from_uid=482703 ).

5. Please explain why not use the genome of Pinus taeda as the transcriptome reference. After all, the genome of Pinus taeda has been sequenced and reported in 2014 (Zimin et al; Neale et al) and 2017 (Zimin et al).

Zimin A, Stevens K A, Crepeau M W, et al. Sequencing and assembly of the 22-Gb loblolly pine genome. Genetics, 2014, 196(3): 875-890.

Neale D B, Wegrzyn J L, Stevens K A, et al. Decoding the massive genome of loblolly pine using haploid DNA and novel assembly strategies. Genome biology, 2014, 15(3): R59.

Zimin A V, Stevens K A, Crepeau M W, et al. An improved assembly of the loblolly pine mega-genome using long-read single-molecule sequencing. Gigascience, 2017, 6(1): 1-4.

Results
1. Lines 130-132. The scientific name of species should be italic. Please carefully check this problem throughout this manuscript.

2. Lines 133-157. The descriptions about GO, KOG and KEGG annotations need to be revised because much annotation information is not important. For example, the largest functional group in each database was extensively highlighted by authors. The authors should conclude the results on the important functional classifications close to the topic of this manuscript.

3.  Lines 158-168, line 199. To better understand the genes involved in terpenoid biosynthetic pathway, the authors should provide the schematic diagram of terpeniod biosynthetic pathway and label the corresponding key genes, such as some upstream or downstream genes (line 199).

4. Lines 199, 207,209, 211, …. If an abbreviation of gene name represents a specific gene, this gene name should be italic. Please check them throughout this manuscript.

5. Line 216. As shown in Fig. 7, the“orthologous to the Ginkgo biloba” is “orthologous to Elaeis guineensis”?

Discussion

Mirror errors, for example, line 228 “significanc”; line 236 “transript material”; line 290 “has”, and etc.

Conclusion
Lines 310-311. The authors claimed that the expression profiles analysis were performed in this study, however, there is no any expression profile results throughout this manuscript. Please revise this conclusion.

Reference
Some format errors are displayed among these references. For example, the abbreviation or full name of journals should be unified. Please check them carefully.


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

Input:
Title: IDENTIFICATION, EXPRESSION, AND PHYLOGENETIC ANALYSES OF TERPENOID BIOSYNTHESIS-RELATED GENES IN SECONDARY XYLEM OF LOBLOLLY PINE (PINUS TAEDA L.) BASED ON TRANSCRIPTOME ANALYSES
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have carefully modified and responsed my questions.
Experimental design: No additional comment.
Validity of the findings: No additional comment.
Additional comments: I suggest the title to be "Transcriptome identification, phylogenetic, and  expression analyses of terpenoid biosynthesis-related genes in secondary xylem of loblolly pine (Pinus taeda L.)".


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

Input:
Title: IDENTIFICATION, EXPRESSION, AND PHYLOGENETIC ANALYSES OF TERPENOID BIOSYNTHESIS-RELATED GENES IN SECONDARY XYLEM OF LOBLOLLY PINE (PINUS TAEDA L.) BASED ON TRANSCRIPTOME ANALYSES
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Thanks for responding to my comments. All my questions have been addressed.