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: ACPT GENE IS INACTIVATED IN MAMMALIAN LINEAGES THAT LACK ENAMEL OR TEETH
Review round: 1
Reviewer: 2

Output:
Basic reporting: Generally this is fine.  The authors should get help to fix English grammar, vocabulary usage, conjugation of verbs, nouns as adjectives, etc. throughout the manuscript.  The text is not so far off but does need extensive work in this context and is pretty rough in parts in terms of grammar.  I generally do not make these sorts of changes in manuscript ad hoc review.
Experimental design: The experimental design is generally fine in terms of methods, but additional sequences should be included from various enamel-less and toothless mammals that have sequenced genomes (pangolin, sloth, and perhaps others).
Validity of the findings: 1) The authors should seek to clearly note what Sharma et al. (2018) noted about inactivation of ACPT in mammals with no teeth or enamel and distinguish this clearly from what this new study shows relative to that earlier study that originally documented the gene knockouts in mammals without teeth or enamel.  For example, extending analyses to more mysticete whale species and to pygmy sperm whale, etc. and inference of inactivation times.  Did Sharma et al. (2018) estimate inactivation times as well?  If so, how do dates and error bars on dates differ?
2) line 33.  " which is earlier than the oldest pangolin" should be " which is earlier than the oldest aardvark".
3) lines 33-36.  It is not clear in the abstract what is meant by " the oldest fossil time".  Perhaps this is a word choice issue and is ambiguous as written in the context of these sentences.  i.e., "oldest fossil time" of what?
4) Is it known exactly what the ACPT gene does in enamel formation?  Or, is this still not well studied by experimental means in past literature?  Are there hypotheses regarding what role this gene plays in tooth development?
5) Why was only one species of Xenarthran analyzed in this paper?  Why are there no pangolin sequences in the study?  These are critical excluded species that are toothless (pangolins, anteaters) or lack enamel (sloths and other aramadillos).  Are genomes for these additional species not available?  Or, is the ACPT gene completely lacking from currently available genome sequences?  If the latter, are flanking genes relative to ACPT found in these toothless or enamel-less mammals?  My understanding is that Dasypus is an armadillo with very recent degradation of enamel based on previous morphological, paleontological, and molecular work and that multiple losses of enamel occurred within the armadillo clade.  Are the authors up to date on this literature?
6) line 263.  The subtitle of this section is " Is inactivation of ACPT neutral or adaptive?" but I think this challenging question was not really answered adequately in this section.  I did not find the authors' arguments here to be compelling.  There are multiple hypothesized losses of enamel in Xenarthra, not just one in the common ancestor of this clade.  Because the authors included just one Xenarthran in their analysis and this is in an armadillo that seems to have lost enamel very recently, the text of the authors does not make sense to me.  I think much more work has to be done here to make a compelling argument (i.e., more sequences from xenarthrans need to be included from sloths, anteaters, and more armadillo genera).  I did not follow the logic of the statement, " However, estimates for ACPT, ODAM and ENAM inactivation are both older than the oldest fossil aardvark, O. minutus, which is ~19Ma (Patterson, 1975). It strongly suggested that gene loss may be the reason, not the consequence, for degeneration and / or loss of enamel, which is different from the result of Sharma et al. (Sharma et al., 2018)."  What did Sharma et al. say and how does this differ logically from the interpretation of the authors here.  I am also not understanding how the very young date for inactivation of ACPT was calculated.  What was the time range used for the ancestral branch to Mysticeti, and what was this based on?  Isn't the basal node of Mysticeti estimated from previous work to be older than 15 MY, which is the authors' estimate for inactivation on the stem lineage of Mysticeti?  Or, maybe I am missing something here.  At any rate, this should be clarified in revision.  Also, there is recent evidence that the "toothless" mysticete Eomysticetus is in a clade (Eomysticetidae) in which some close relatives have vestigial teeth at the tips of their jaws.  This region of anatomy is not, I think, well preserved enough in Eomysticetus to tell whether this genus was truly toothless?
7) line 266.  The author's definition here seems off.  " adaptive evolution (direct natural / positive selection to conserve energy and / eliminate the disadvantageous effects of morphological structure)"  What about reproductive fitness implied by different genotypes?  Their definition is unconventional even if it is as in the references given.
Additional comments: Yuan Mu et al. analyze sequence data for the ACPT gene in a phylogenetic context to characterize inferred gene inactivations over the history of Mammalia.  The authors note that they seek to analyze the "mechanisms" of gene inactivation as related to tooth loss in the abstract and in the introduction.  This is a challenge as it is difficult to assess cause and effect when so many genes have been inactivated on the same evolutionary branches where inferred losses of enamel or teeth have occurred in mammalian phylogeny.  The authors document knockouts of ACPT in baleen whales, pygmy right whale, aardvark, and armadillo which are all characterized by lack of enamel or degraded enamel.  This work follows a previous paper that first noted some of these inactivations (Sharma et al., 2018) that was recently published using broad genomic screens.  So, as the authors note, it was previously known that inactivations of ACPT in mammals without enamel were present, but the authors provide extensive further detail on these events within the context of phylogenetic trees from many more mammalian species (116 species) that were extracted from published genomes and estimate inactivation times using dN/dS ratios on branches where loss of enamel is inferred.  The paper is pretty simple and straightforward and represents a contribution to the field, but the new insights presented are perhaps not great given previous work on this gene by Sharma et al. (2018).  An important issue is point #6 below.  Why were pangolin and anteater sequences not included in this study, as well as sloth sequences?  These are critical species without teeth or enamel that have published genomes?  In general the authors methods seem sound, and the results and discussion are simply stated and not overinterpreted.

1) The authors should seek to clearly note what Sharma et al. (2018) noted about inactivation of ACPT in mammals with no teeth or enamel and distinguish this clearly from what this new study shows relative to that earlier study that originally documented the gene knockouts in mammals without teeth or enamel.  For example, extending analyses to more mysticete whale species and to pygmy sperm whale, etc. and inference of inactivation times.  Did Sharma et al. (2018) estimate inactivation times as well?  If so, how do dates and error bars on dates differ?
2) The authors should get help to fix English grammar, vocabulary usage, conjugation of verbs, nouns as adjectives, etc. throughout the manuscript.  The text is not so far off but does need extensive work in this context and is pretty rough in parts in terms of grammar.  I generally do not make these sorts of changes in manuscript ad hoc review.
3) line 33.  " which is earlier than the oldest pangolin" should be " which is earlier than the oldest aardvark".
4) lines 33-36.  It is not clear in the abstract what is meant by " the oldest fossil time".  Perhaps this is a word choice issue and is ambiguous as written in the context of these sentences.  i.e., "oldest fossil time" of what?
5) Is it known exactly what the ACPT gene does in enamel formation?  Or, is this still not well studied by experimental means in past literature?  Are there hypotheses regarding what role this gene plays in tooth development?
6) Why was only one species of Xenarthran analyzed in this paper?  Why are there no pangolin sequences in the study?  These are critical excluded species that are toothless (pangolins, anteaters) or lack enamel (sloths and other aramadillos).  Are genomes for these additional species not available?  Or, is the ACPT gene completely lacking from currently available genome sequences?  If the latter, are flanking genes relative to ACPT found in these toothless or enamel-less mammals?  My understanding is that Dasypus is an armadillo with very recent degradation of enamel based on previous morphological, paleontological, and molecular work and that multiple losses of enamel occurred within the armadillo clade.  Are the authors up to date on this literature?
7) line 263.  The subtitle of this section is " Is inactivation of ACPT neutral or adaptive?" but I think this challenging question was not really answered adequately in this section.  I did not find the authors' arguments here to be compelling.  There are multiple hypothesized losses of enamel in Xenarthra, not just one in the common ancestor of this clade.  Because the authors included just one Xenarthran in their analysis and this is in an armadillo that seems to have lost enamel very recently, the text of the authors does not make sense to me.  I think much more work has to be done here to make a compelling argument (i.e., more sequences from xenarthrans need to be included from sloths, anteaters, and more armadillo genera).  I did not follow the logic of the statement, " However, estimates for ACPT, ODAM and ENAM inactivation are both older than the oldest fossil aardvark, O. minutus, which is ~19Ma (Patterson, 1975). It strongly suggested that gene loss may be the reason, not the consequence, for degeneration and / or loss of enamel, which is different from the result of Sharma et al. (Sharma et al., 2018)."  What did Sharma et al. say and how does this differ logically from the interpretation of the authors here.  I am also not understanding how the very young date for inactivation of ACPT was calculated.  What was the time range used for the ancestral branch to Mysticeti, and what was this based on?  Isn't the basal node of Mysticeti estimated from previous work to be older than 15 MY, which is the authors' estimate for inactivation on the stem lineage of Mysticeti?  Or, maybe I am missing something here.  At any rate, this should be clarified in revision.  Also, there is recent evidence that the "toothless" mysticete Eomysticetus is in a clade (Eomysticetidae) in which some close relatives have vestigial teeth at the tips of their jaws.  This region of anatomy is not, I think, well preserved enough in Eomysticetus to tell whether this genus was truly toothless?
8) line 266.  The author's definition here seems off.  " adaptive evolution (direct natural / positive selection to conserve energy and / eliminate the disadvantageous effects of morphological structure)"  What about reproductive fitness implied by different genotypes?  Their definition is unconventional even if it is as in the references given.


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: ACPT GENE IS INACTIVATED IN MAMMALIAN LINEAGES THAT LACK ENAMEL OR TEETH
Review round: 2
Reviewer: 1

Output:
Basic reporting: This is fine.  English grammar and word usage could still use some help.
Experimental design: no comment
Validity of the findings: This is generally fine.  I think there is still some confusion on stem baleen whales (Mysticeti).  Some have teeth, and even eomysticetids that have been described in the past as toothless show some evidence of having teeth at the tips of their jaws in at least one species (see Boessenecker papers from 2015).  This affects some of the discussion section on whales, and it would be best to get this right.
Additional comments: I think the authors have generally dealt with the comments from my initial review in an adequate way.


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: PREBIOTIC SUPPLEMENTATION EFFECT ON ESCHERICHIA COLI AND  SALMONELLA SPECIES ASSOCIATED WITH EXPERIMENTALLY INDUCED INTESTINAL COCCIDIOSIS IN RABBITS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Add some detail about coccidiosis their prevalence and than link with bacterias and antibiotic resistant.

Please add uptodate reference (is the major cause of antibiotic resistance (WHO 2000)

can you add composition of prebiotic supplements available in field and their effects and possible role in antibiotic resistant
Experimental design: Why you only choosed male rabbit?

Please add composition of diet it is must to know because diet is potential risk of coccidiosis

Please add name of antibiotics which was used in 12 antibiotic discs and give reference
Validity of the findings: Study is some how interesting and results are looking good, but it need some details before making final decision
Additional comments: Please response above mention question and try to add latest reference


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

Input:
Title: PREBIOTIC SUPPLEMENTATION EFFECT ON ESCHERICHIA COLI AND  SALMONELLA SPECIES ASSOCIATED WITH EXPERIMENTALLY INDUCED INTESTINAL COCCIDIOSIS IN RABBITS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript entitled “Prebiotic supplementation effect on Escherichia coli and Salmonella species associated with experimentally induced intestinal coccidiosis in rabbits” describes the effect of prebiotic supplementation on the presence of Escherichia coli and Salmonella species in rabbits experimentally infected with intestinal coccidiosis was investigated. I found the topic of this study interesting in the Egyptian context and fitting in the scope of the journal. The study is well executed and conducted and the manuscript is well written.
Experimental design: The study is well executed and conducted.
Validity of the findings: The findings are an added value to the scientific community.
Additional comments: Well-done!


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: PREBIOTIC SUPPLEMENTATION EFFECT ON ESCHERICHIA COLI AND  SALMONELLA SPECIES ASSOCIATED WITH EXPERIMENTALLY INDUCED INTESTINAL COCCIDIOSIS IN RABBITS
Review round: 1
Reviewer: 3

Output:
Basic reporting: no comment
Experimental design: The prepatent period for E. magna, E. flavescens and E. intestinalis is much longer than 4 days, and in turn the oocyst shedding peaks are around 8-10 days PI. The most severe period for the coccidiosis, mainly with secondary bacterial infection, usually appears since the shedding of oocysts. For such situation, Trials with more animals and sampling at more time points after 8 day PI would be valuable for the evaluation.
Validity of the findings: no comment
Additional comments: the oocyst output in Table 1 could not fully demonstrate the prophylactic effect of the product, as the oocyst shedding peaks are around 8-10 days PI.
the number of E.coli and Salmonella subtypes detected in this study reveals the severity of the bacterial infection, but not all of it, the infection intensity would be in consideration.


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: PREBIOTIC SUPPLEMENTATION EFFECT ON ESCHERICHIA COLI AND  SALMONELLA SPECIES ASSOCIATED WITH EXPERIMENTALLY INDUCED INTESTINAL COCCIDIOSIS IN RABBITS
Review round: 2
Reviewer: 1

Output:
Basic reporting: yes
Experimental design: yes it is written well
Validity of the findings: yes
Additional comments: yes


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: EXOGENOUS MELATONIN IMPROVES GROWTH IN HULLESS BARLEY SEEDLINGS UNDER COLD STRESS BY INFLUENCING THE EXPRESSION RHYTHMS OF CIRCADIAN CLOCK GENES
Review round: 1
Reviewer: 1

Output:
Basic reporting: The authors investigated the possible mechanism by which melatonin pretreatment can promote the growth of hulless barleyunder cold stress conditions by studying the effect of melatonin on the rhythm of the circadian clock system and some physiological indicators. The work appears to have been carefully done. But there are still some revision needed.
Experimental design: (1) Please explain why you use the 1μM melatonin, why do not use melatonin at other concentration to compare the effect of melatonin on the growth of plants?
(2) Please detected the concentration of melatonin after treatment.
Validity of the findings: Topic is interesting and fit for the aims and scope for Peer J, and it’s had practical production value.
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: EXOGENOUS MELATONIN IMPROVES GROWTH IN HULLESS BARLEY SEEDLINGS UNDER COLD STRESS BY INFLUENCING THE EXPRESSION RHYTHMS OF CIRCADIAN CLOCK GENES
Review round: 1
Reviewer: 2

Output:
Basic reporting: The English is good. The structure of the papers is correct, but the data interpretation is misleading.
Experimental design: The methods are appropriate.
Validity of the findings: The data are sound, but the conclusions do not reflect some of the data.
Additional comments: The paper discusses melatonin mechanisms promoting growth of hulless barley under cold stress were investigated. The application of 1 μM melatonin alleviates the growth inhibition of hulless barley seedlings caused by cold stress. Exogenous melatonin also restores circadian rhythmic oscillation of circadian clock genes, such as HvCCA1 and HvTOC1 and reduces accumulation of key physiological indicators under cold stress, including malondialdehyde (MDA) and soluble sugars.

The introduction is detailed and informative and provides good background to present study. However, the results are complex and the interpretation of the circadian results needs more careful consideration. At all temperatures, melatonin has different effects on the various circadian clock associated genes. Both the control and the melatonin data at 5 deg C (Fig. 6) are quite variable and do not show clear periodic trends. Even at 15 deg C the melatonin data seem less periodic than the control. So, the title of the paper does not correspond to the results. The data trends of chlorophyll, carotenoids, MDA, soluble sugars and proline contents at different temperatures and with melatonin are interesting.

It is interesting that the dry weight of plants at 5 deg C is greater than that at 25 and 15 deg C (Fig. 2). Authors, please, comment.

Minor changes: Line 48: “temperature gradients” implies that the temperatures were changed at the time of the experiment.

Lines 196, 236, 250: “section 2.1, section 2.6” There are no numbered sections in the MS.

Fig. 2 Parts C – F would be more informative presented in the same way as part B


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: EXOGENOUS MELATONIN IMPROVES GROWTH IN HULLESS BARLEY SEEDLINGS UNDER COLD STRESS BY INFLUENCING THE EXPRESSION RHYTHMS OF CIRCADIAN CLOCK GENES
Review round: 1
Reviewer: 3

Output:
Basic reporting: The English language could be improved to inrease the quality of this manuscript.
I feel that some parts of this paper shall be reorganized, for instance, if I understand correct, the Materials and growth condition part of the Methods and Materials seems overlap with the description of 'Effect of exogenous melatonin on the germination rate of hulless barley seeds' and other places. In the abstract, the authors repeated a statement of the general study purpose 'In this study, we investigated the possible mechanism...' in the results part, which seems better to start directly' the results showed/revealed....In the end of introduction, the authors wrote that 'The protective effects of exogenous melatonin against ... were also detected' which seems a result.
This study used 1 μM melatonin for treatment, however, higher concentration may inhibit the plant growth while a low concentration of melatonin may promote the growth, as found our in a related study (Chen et al., Journal of Plant Physiology 166: 324-328) . the authors may want to discuss the implication of that finding for their results.
Experimental design: The study used three temperature treatments of 25 ℃(Normal), 15 ℃(Low temperature) or 5 ℃(Cold) and each was incubated in each of three growth chambers, however, sometimes the authors mentioned room temperature of 25 ℃.
I understand that there is no repeat of treatment for temperature due to the actual experimental condition/facilities; the authors may want to mention their limited findings due to limited experimental design.
Validity of the findings: This study had provided evidences for the melatonin in alleviating the cool stress response in hulless barley and proved the response by gene expression evidences.
The authors may want to provide reason(s) why they apply only a concentration of 1 μM for Melatonin treatment, if it is by chance or by other purpose?
Additional comments: Not sure why legends of each figure always repeat the first sentence, I guess it could be the format request by the journal? The legend for Figure 7 seems unfinished.


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: EXOGENOUS MELATONIN IMPROVES GROWTH IN HULLESS BARLEY SEEDLINGS UNDER COLD STRESS BY INFLUENCING THE EXPRESSION RHYTHMS OF CIRCADIAN CLOCK GENES
Review round: 1
Reviewer: 4

Output:
Basic reporting: Line 56 is missing a space between "barley" and "under".
Line 182, "and" after "water" should be changed to "or".
Line 187. It should be the effect of melatonin treatment on plant vegetative growth.
Line 196. Which title is section2.1, the expression here is not very clear
Line 236. "section 2.1" the same problem as above
Line 250. "section 2.6"the same problem as above
Line482-493. There should be a picture of the physiological indicators of untreated barley at different temperatures, which can reflect the original excellent resistance of hulless barley and the powerful regulation ability of melatonin.
The suppose  in the discussion is wonderful and can explain the experimental conclusions to a certain extent, but there is a lack of experimental evidence. If it can be further proved, it must be an excellent article.
Experimental design: In the content of lines 165-167, what is the meaning of continuous light for a week?
Wang et al. (Plant Cell & Environment. 2020; 43: 637– 648 ) studied the influence of light and temperature on soybean circadian rhythm, they used a 14d 12/12 day-night ratio to Entrainment  the soybean materials, and there was no 7 days‘ continuous light before that.

Line 317-318. The seeds were soaked with MT 12 hours, germinate 2 days, continuous light for 7 days, Entrainment for 2 days, and then 3 days of continuous light for sampling to determine circadian gene expression. To what extent can these explain the results are the effect of melatonin on the biological clock genes?
Suggestions: 1. Construct different melatonin overexpression and interference hulless barley strains, and obtain melatonin differential strains for relevant experiments to illustrate the effect of melatonin on the circadian rhythm.
2. After Entrainment, spray melatonin to determine the biological clock-related genes.
Validity of the findings: no comment
Additional comments: Rhythm genes are mainly regulated by light, then by temperature. After these, it is melatonin that comes into play.
This article combines temperature and melatonin, which is a good starting point. But it feels that the combination is not very well, the experimental design is not ideal. In addition, the fluorescence quantification is only related data, and there is no direct causal 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: EXOGENOUS MELATONIN IMPROVES GROWTH IN HULLESS BARLEY SEEDLINGS UNDER COLD STRESS BY INFLUENCING THE EXPRESSION RHYTHMS OF CIRCADIAN CLOCK GENES
Review round: 2
Reviewer: 1

Output:
Basic reporting: The author addressed all questions, and I agree to accept the MS.
Experimental design: No question.
Validity of the findings: No question.
Additional comments: No question.


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: EXOGENOUS MELATONIN IMPROVES GROWTH IN HULLESS BARLEY SEEDLINGS UNDER COLD STRESS BY INFLUENCING THE EXPRESSION RHYTHMS OF CIRCADIAN CLOCK GENES
Review round: 2
Reviewer: 2

Output:
Basic reporting: The English needs some attention:

For instance:

Line 48: “there is…” change to “there are…”
Line 153: “to instigate” change to “to investigate”
Line 418: “In some studies, It also shows…” change to “ In some studies, the data also show…”
Experimental design: The experimental design is satisfactory.
Validity of the findings: The findings are interesting and novel.
Additional comments: The authors improved the MS along my suggestions.

I still think that the title is misleading, as exogenous melatonin seems to have different effects on different clock genes at different temperatures. I suggest more general title, such as:

Exogenous melatonin improves growth in hulless barley seedlings under cold stress by influencing the expression rhythms of circadian clock genes.


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: PSEUDOMONAS CHLORORAPHIS PA23 METABOLITES PROTECT AGAINST PROTOZOAN GRAZING BY THE PREDATOR ACANTHAMOEBA CASTELLANII
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: This manuscript describes the toxicity of pyrrolnitrin (PRN), phenazine (PHZ) and hydrogen cyanide (HCN) produced by Pseudomonas chlororaphis on the amoeba, Acanthamoeba castellanii. The results show that all 3 secondary metabolites inhibit and/or repel amoeba in vitro and that the expression of secondary metabolites increases in the presence of amoeba trophozoites but not supernatants from amoeba cultures.

The manuscript is well-written and clear and there are sufficient references in the background. All the figures are necessary and well presented. While the inhibition of predators by secondary metabolites have been shown previously, the bacterial strain used here has not been explored previously with respect to predation inhibition.

For the co-culture assays, it states that extracellular bacteria were counted (line 107-108) but what about intracellular bacterial numbers. In some cases bacteria can multiply inside the amoeba and you would not capture this by only plating extracellular cells. Have you considered lysing the amoeba and doing total bacterial counts?

What was the setup for the motility experiments? Were the amoeba added to the swim plate and if so how?

Specific comments
Line 130 – To maintain consistency the heading should be sentence case (e.g. Chemotaxis assay)
Lines 13 and 307 and – there should be a space between the number and unit (e.g. 72 h)


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: PSEUDOMONAS CHLORORAPHIS PA23 METABOLITES PROTECT AGAINST PROTOZOAN GRAZING BY THE PREDATOR ACANTHAMOEBA CASTELLANII
Review round: 1
Reviewer: 2

Output:
Basic reporting: no coment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Very interesting story and very excellent English. I learned a lot from it. However, just minor faults :1) in background introduction, I think the regulation of antibiotics production might not be necessary for this study. So I suggested this part is deleted (Line 40-46); 2) most references are listed with the full name of journal, but some are not (Line 425, 457, and 466).


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: PSEUDOMONAS CHLORORAPHIS PA23 METABOLITES PROTECT AGAINST PROTOZOAN GRAZING BY THE PREDATOR ACANTHAMOEBA CASTELLANII
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript is well written and care has been taken to explain key concepts to the reader. Background to the study provides clear context and concisely conveys the current level of understanding in the field and the knowledge gap to be addressed. All figures are of a high quality and accompanied by detailed legends. All raw data has been supplied and is in an understandable format.
Experimental design: The study aims to determine if PA23 metabolites facilitate survival in the presence of a protozoan predator. It also aims to determine how these metabolites potentially protect the bacterium which produces them. As such, the study has a clear and well-defined research question.
Experiments are well conceived and well controlled. I commend the authors for the use of appropriate controls throughout their study. The number of replicates and experimental repeats performed allow for robust statistical analysis of the data.
Methods are detailed and could be easily reproduced by another researcher. For some experiments, the number of replicates are missing (see specific comments below). The methods employed are appropriate.
Validity of the findings: Statistical analyses have been applied where appropriate. The significance level of all analyses have been stated and provided on all figures. However, I am not 100% convinced regarding the appropriateness of a 2-way ANOVA for some of the analyses.  Results are well described and easy to follow. Conclusions drawn are justified based on a solid analysis of the results. Potential anomalies are well explained. For example, potentially unexpected results for the PHZ- which can be explained by elevated levels of PRN produced by this strain.
Additional comments: Overall, I commend the authors for an interesting study investigating the mechanisms by which bacteria protect themselves from potential predators. I only have a few minor comments which are listed below:
Line 76, the abbreviated version of Escherichia coli (E. coli) is used prior to the full name being provided.
Line 212, “Conversely, trophozoite numbers were significantly elevated when grown on the PRN- and HCN- strains (Fig. 1)”. I find this description confusing; it implies that trophozoite numbers increased compared to Day 0. A more specific statement such as “trophozoite numbers were significantly higher when grown on PRN-  and HCN- strains compared to those grown on the PHZ- strain at day 15.
Figure 2: I would find a line chart easier to view with regards to changes of the viability for each mutant over time. Is there a specific reason the authors chose to represent these data with a histogram?
In the methods section, a number of experiments are missing details on the number of replicates and repeats performed. These experiments are the “P. chlororaphis PA23 – Ac co-culture assays” “Chemotaxis assays “ and  “ analysis of transcriptional fusions”


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: ESTIMATING THE AVERAGE DAILY RAINFALL IN THAILAND USING CONFIDENCE INTERVALS FOR THE COMMON MEAN OF SEVERAL DELTA-LOGNORMAL DISTRIBUTIONS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The text is written clearly and in good English. These authors say what they want to do.
Experimental design: not applicablöe here.
Validity of the findings: I think all findings make sense and are plausible.
Additional comments: Dealing with continuous distributions with a spike at zero is a non-standard problem and it honours the authors to seriously model this situation and the delta-lognormal model is a reasonable approach to cope with the issue.
Here the focus is on estiamting a common mean when data arise from several distributions.
I would like to ask the authors a few questions if I may.
a) In equation (3) the authors define the common mean as a weighted mean where the weights depend on the sample estimate of the respective subpopulation. This is a bit unusal as the population parameter should be defined independently of the sample which is used. A simple solution qwould be define theta identical for all subpopulations.
b) would it then not make sense to define the estiamte as a weighted mean as you intend in (3)?
c) A critical person might argue it would be simpler to pool all the data and treat it as a single population which would be a lot easier. So, I think you need to arge a bit here that your approach is more beneficial. I beleive it is quite similar to meta-analysis.
d) I am not clear with eq. (6) and the arguments leading. The last line in (6) says it is infact unbiased (which I am not clear from the previous line), but why is then there a limitation to obtain an unbiased estimate?

MINOR:
a) I would use a different name for Algorithm 6 as it is not a method that is compared.
b) Presentation of results: it is not clear from tables or grpahs whow the winner is. I would invest a bit more work to illuminate this.
c) application: you do this for a very selectied data set. But if this is done routinely per day or per week, which method should be used taking into acocunt that it must be practicable?
d) I think you need to take this work much more forward. Suppose you have a number of covariates such as time and location how would you take this forward? A way to go could be a two-component model where you model the zeros according to a logistic model and the positive observations with a linear model using a log-transformation on the dependent variable. very much similar to the Lambert model in zero-inflation modelling. But this cleary is for another paper to come.


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: ESTIMATING THE AVERAGE DAILY RAINFALL IN THAILAND USING CONFIDENCE INTERVALS FOR THE COMMON MEAN OF SEVERAL DELTA-LOGNORMAL DISTRIBUTIONS
Review round: 1
Reviewer: 2

Output:
Basic reporting: n/a
Experimental design: n/a
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: ESTIMATING THE AVERAGE DAILY RAINFALL IN THAILAND USING CONFIDENCE INTERVALS FOR THE COMMON MEAN OF SEVERAL DELTA-LOGNORMAL DISTRIBUTIONS
Review round: 1
Reviewer: 3

Output:
Basic reporting: The Science looks fine but the English needs improvement. More specifically, the Introduction section needs to be edited (re-written).
Experimental design: No comment
Validity of the findings: No comment
Additional comments: Please re-write the introduction section by getting help from a native English speaker.


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: ESTIMATING THE AVERAGE DAILY RAINFALL IN THAILAND USING CONFIDENCE INTERVALS FOR THE COMMON MEAN OF SEVERAL DELTA-LOGNORMAL DISTRIBUTIONS
Review round: 1
Reviewer: 4

Output:
Basic reporting: The article is well clear. Couple  of typos were detected. For example: in abstract, there should be space in between 'in' and 'a'. A thorough overview should be given for a flawless article.
Experimental design: 'No comment'
Validity of the findings: More discussion on "Simulation study and results" could give the readers a clearer idea.
Additional comments: Overall, this is a very well written article and the problem was well stated. The problem and the conclusions drawn from the article plays a major role in the country like Thailand.
The tables and figures could be labeled properly to give clear idea about each of the cases.
I recommend this article to be accepted with very very minor corrections.


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: ESTIMATING THE AVERAGE DAILY RAINFALL IN THAILAND USING CONFIDENCE INTERVALS FOR THE COMMON MEAN OF SEVERAL DELTA-LOGNORMAL DISTRIBUTIONS
Review round: 2
Reviewer: 1

Output:
Basic reporting: ok
Experimental design: n/a
Validity of the findings: ok
Additional comments: I am happy with the revision.


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

Input:
Title: GENOME-WIDE IDENTIFICATION, EVOLUTION, AND EXPRESSION OF THE SNARE GENE FAMILY IN WHEAT RESISTANCE TO POWDERY MILDEW
Review round: 1
Reviewer: 1

Output:
Basic reporting: I had gone through the manuscript entitled 'Genome-wide identification, evolution, and expression of SNARE genes family in wheat'. The manuscript is well written using good English language. The authors also provided background information on SNAREs, still they can include the following two papers under discussion and mention a few lines:
1.Gaggar P, Kumar M, Mukhopadhyay K (2020) Genome-scale identification, in silico characterization and interaction study between wheat SNARE and NPSN gene families involved in vesicular transport. IEEE/ACM Transactions on Computational Biology and Bioinformatics. DOI: 10.1109/TCBB.2020.2981896
2. Wang X, Wang X, Deng L, Chang H, Dubcovsky J, Feng H, Han Q, Huang L, Kang Z, (2014). Wheat TaNPSN SNARE homologues are involved in vesicle-mediated resistance to stripe rust (Puccinia striiformis f. sp. tritici). Journal of experimental botany, 65,4807-4820.
Experimental design: All the experiments are properly designed. Aim of the manuscript clearly mentioned
Validity of the findings: A bit lenient method had been followed for identification of the genes from wheat genome, which is acceptable. A more stringent method would have been better, but that will reduce  the number of identified genes.
Additional comments: The following aspects should be rectified:
1. When all authors belong to the same lab of the same University, there is no need to add superscript 1 after all names
2. Line 43 and 361 the author names should be:  Snyder BA and  Nicholson RL
3. Line 64 and 73: Italicize the pathogen names Phytophthora infestans and Magnaporthe oryzae
4. Line 108: Remove one set of the two sets of parenthesis.
5. Lines 184-185: Explain why the colinear gene pairs do specifically exist on adjascent chromosomes. Has similar situations found with any other genes in wheat?
6. Lines 220-223: What are A,B and C elements, please explain. In Fig. 4 also, as referred in the text, nothing is mentioned about these elements.
7. Line 301: Capitalize t after full stop
8 Many article titles in the reference list has been copied from original article showing capitalized first letters. Please provide running titles.


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

Input:
Title: GENOME-WIDE IDENTIFICATION, EVOLUTION, AND EXPRESSION OF THE SNARE GENE FAMILY IN WHEAT RESISTANCE TO POWDERY MILDEW
Review round: 1
Reviewer: 2

Output:
Basic reporting: There are numerous grammatical mistakes, misspell words which needs to be checked thoroughly by a native English speaker, otherwise not considered for publication in PeerJ.

Line 38-39 scientific names not written in italic.
Experimental design: The title of the manuscript should include expression analysis of snare genes in wheat against powdery mildew infection to make it more sound.

The SNARE protein full form is not mentioned once in the manuscript.

In introduction part no background is given on the fungus used in the study as well as why was this fungus chosen for experimental work.

Line 88 says all plant snare proteins sequences, which plants, name them or add a supplementary file.

In section 2.5 kindly mention clearly the time points of sample collection after fungus treatment.

Which method was used for powdery mildew conidia infection on wheat is missing.
Validity of the findings: Fig. 6 font needs to be increased. As each row in the fig depicts one family of SNARE eg first row is of Qa snare then this fig  can be made more clear by separating each family so that patterns are visible among each family.

In Section 3.6  the expression results are reported in a straight manner and the inference of the expression data obtained is missing.

Why did the authors choose to do a RNA seq expression of SNARE genes which reveal only abiotic stress like drought and heat and no biotic stress like fungal infection time points   as only the later was focused in this study. The RNA seq expression profile and the Qrt-pcr results does not tally to give a conclusion on snare roles in biotic stress. Please explain.

In Fig 4, the pie charts shows 882,8%, 1647,15% and so on, this is confusing and should be written as 882 (8%) and so on.
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: GENOME-WIDE IDENTIFICATION, EVOLUTION, AND EXPRESSION OF THE SNARE GENE FAMILY IN WHEAT RESISTANCE TO POWDERY MILDEW
Review round: 1
Reviewer: 3

Output:
Basic reporting: I carefully read the text of the manuscript «Genome-wide identification, evolution, and expression of SNARE genes family in wheat.» Overall, it describes a perfect study, but the authors should correct the style and mistakes/typos in English.
Also, I think you should expand your keyword list by adding words that more closely specify the research.
Line 77-81: The last paragraph of the introduction can be expanded to indicate the article's purpose and main results more accurately.
The full form for the abbreviation SNARE must be indicated at the beginning of the introduction.
Experimental design: In the introduction part, the authors should introduce more information about the fungus used in the study and specify why they used this fungus for experimental work.
The authors should check the methodology described in "Fungus and Wheat materials": the method of infection and how long after infection did they fix the research material?
Validity of the findings: no comment
Additional comments: Line 64 and 73: The pathogen names ''Phytophthora infestans'' and ''Magnaporthe oryzae'' should be italicized.
Fig 4. Please, check the % values, you should divide all values by 100.
Fig6. It is necessary to increase the font of the axis labels.


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

Input:
Title: GENOME-WIDE IDENTIFICATION, EVOLUTION, AND EXPRESSION OF THE SNARE GENE FAMILY IN WHEAT RESISTANCE TO POWDERY MILDEW
Review round: 2
Reviewer: 1

Output:
Basic reporting: English language still needs polishing, still there are a few spelling mistakes and grammatical errors. I'm mentioning the following few:
Line 15: Phylogenetic tree
Line 99: Synaptobrevin, not synaptebrevin
Line 135:Use growth chamber instead of incubator
Line 137: method
Line 138: use collected instead of cut
Line 174: Actin gene; also provide the NCBI accession number within parenthesis
Line 211: in terms of their relative position on chromosomes
Line 374: SNAP4, P is missing
Line 382: Provide the publication year after Zhang et al., as there are two references with Zhang
Line 384: use we instead of I

All gene names should be italicized

There is a huge confusion between homologous and homoeologous at various places in the manuscript.
Experimental design: OK
Validity of the findings: Very good
Additional comments: English language still needs polishing, still there are spelling mistakes and grammatical errors.


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

Input:
Title: GENOME-WIDE IDENTIFICATION, EVOLUTION, AND EXPRESSION OF THE SNARE GENE FAMILY IN WHEAT RESISTANCE TO POWDERY MILDEW
Review round: 2
Reviewer: 2

Output:
Basic reporting: In the manuscript wherever this kind- Sun et al (Sun et al. 2017) of reference is put can be simply replaced by Sun et al. 2017.

In section 2.5 writing should be uniform for eg. at some points 24h is written whereas others are written as 48 h. Correct all as including space or no space in between. Apply this to all.

Line 153 omit extra bracket.

Line 186: Qa, Qb, Qc, Qb+Qc and R SNARE proteins have 48(27.7%), 187 37(21.4%), 39(22.5%), 13(7.5%) and 36(20.8%) respectively can be replaced by  Qa, Qb, Qc, Qb+Qc and R SNARE proteins comprised 48(27.7%), 187 37(21.4%), 39(22.5%), 13(7.5%) and 36(20.8%) respectively.

Line 204:The results revealed 173 SNARE genes in the chromosomal location information (Fig 2) can be changed to Fig. 2 reveals the chromosomal location of 173 SNARE genes.
Experimental design: Section  2.2 mentions that maximum number of 10 motifs were taken whereas in Fig 3 15 number of motifs are obtained. 10 should be replaced with 15.
Validity of the findings: No comment
Additional comments: No additional comments


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

Input:
Title: GENOME-WIDE IDENTIFICATION, EVOLUTION, AND EXPRESSION OF THE SNARE GENE FAMILY IN WHEAT RESISTANCE TO POWDERY MILDEW
Review round: 2
Reviewer: 3

Output:
Basic reporting: The manuscript has been revised according to the comments of the reviewers and the text and figures have been greatly improved. In addition, the authors additionally corrected grammar.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: I think you have done a good job and I think the editor should accept it for publication.


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

Input:
Title: A REVIEW OF THE CAROTID ARTERY AND FACIAL NERVE CANAL SYSTEMS IN EXTANT TURTLES
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: For the uninitiated reader, this lengthy study is a marathon through turtle diversity (both fossil and extant) and the variation of cranial innervation and vascularization patterns – with possible systematic and phylogenetic implications. Starting with Gaffney’s ground-breaking papers published in the 1970s, a tradition became established in turtle systematics to reconstruct with increasing sophistication – both technologically [CT scanning] as well as with respect to taxon sampling – the variation of cranial (‘bony’) ‘canals’ that transmit arterial systems (most prominently derivatives of the internal carotid) and cranial nerves (most prominently derivatives of the facial nerve), and the possible systematic/phylogenetic implications. One of the central issues of contention here is the pleurodire – cryptodire distinction, as well as the placement of certain ‘stem-turtles’. The whole conceptual frame of the study is evolution from bottom up – along the stem towards the crown. There is no question that the authors are very much on top of the task they set themselves. The manuscript is detailed, well informed, and well written (the occasional unavoidable typo notwithstanding). The references are up-to-date and exhaustive (for the squamate terminology that relates to cranial nerves and blood vessels [line 104] I would personally have consulted Oelrich, T.M., 1956. The Anatomy of the Head of Ctenosaura pectinata (Iguanidae). Misc. Publ. Museum of Zoology, University of Michigan, No. 94 (available online). The illustrations are excellent and informative. In that sense, the paper is publishable as is (once it has been sent through the spell-check one more time). It also conforms in style and substance to other recent analyses of turtle cranial morphology and phylogeny, as exemplified by the frequently cited paper by Evers and Benson (2019). But ever since the groundbreaking work by Gaffney (1972, 1975), there remains a big pink elephant in the room: the correlation between osteological characteristics and inferred soft anatomy relies on preciously thin ice, with only a few studies investigating a limited number of taxa providing any guidance to inferred correlations (most importantly the studies of Albrecht [1967, 1976], investigating the arterial vascularization of the turtle head based on the dissection of injected specimens). As the authors note (line 275): “As our primary source of information are skulls, we are not able to observe the cranial circulation and innervation systems directly” – but they still base far-reaching conclusions about character evolution on these latter systems. There is no ‘quick fix’ of this situation – short of a time-consuming investigation of serially sectioned and properly stained turtle heads.


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 REVIEW OF THE CAROTID ARTERY AND FACIAL NERVE CANAL SYSTEMS IN EXTANT TURTLES
Review round: 1
Reviewer: 2

Output:
Basic reporting: -	Generally the manuscript is very well written and easy to read. Some typographical/grammatical errors are indicated in the annotated PDF
-	The relevant literature is well-reviewed and relevant. How the authors’ work agrees or disagrees with that of others is well-documented
-	Structure conforms to PeerJ standard. While this is a very long descriptive piece, I don’t believe it can be substantially shortened without compromising quality.
-	Figures are relevant. See annotated PDF for comments, and please pay close attention to the following:
o	Throughout, the authors use a red-green-blue colour scheme to refer to canals and their contents. Please be aware that this colour scheme may be difficult for readers who are colour-blind; consider a friendlier colour scheme
o	Fig 19 is hard to read, and the dashed lines are almost invisible on the review copy at 100% magnification. Further, the legend does not indicate the meaning of the colours on Panel B.
-	Raw data are not supplied, but these constitute CT scans which are mostly held on public repositories (MorphoSource and Digimorph), with a few exceptions. It is not clear if those not publicly deposited are available from the authors.
Experimental design: -	This is a descriptive piece, but as such constitutes original primary research. While the work builds on that of others, it goes well beyond the most recent literature on the topic and is thus both novel and valuable.
-	The research fills a knowledge gap by substantially broadening the taxonomic coverage for our understanding a key feature of turtle morphology that is relevant for systematic and evolutionary studies, using higher quality imaging and therefore better describing key characters within this system.
-	The work is rigorous and of a high standard
-	I have suggested moving a portion of the Discussion to the Methods to improve clarity.
Validity of the findings: -	Findings are valid and statistical techniques are appropriate.
-	I would like to see clarification about how the possible contribution of allometric variation might be affecting these results, however. Allometry is not mentioned at all, but is well understood to affect turtle skull shape (e.g. Claude J. et al. 2004. Ecological Correlates and Evolutionary Divergence in the Skull of Turtles: A Geometric Morphometric Assessment, Syst Biol 53(6): 933–948, https://doi.org/10.1080/10635150490889498). I would be surprised if it isn’t in play here, and this should be confirmed or ruled out. Indeed, it may add further complexity (and evolutionary interest) to the results.
-	The Results section implies a discussion of canal development on the basis of a highly asymmetrical specimen. I looked forward to this discussion, and did not really find it. More would be useful here, especially since trouble was taken to figure this interesting specimen. Epigenetic interactions are known to structure development of cranial canals and vasculature, and it may be worth some discussion of how that might relate to this specimen and to the variability present in the patterns you have observed. See for example Jamniczky HA, Hallgrímsson B. 2011. Modularity in the skull and cranial vasculature of laboratory mice: implications for the evolution of complex phenotypes. Evol Dev. 2011;13(1):28-37. doi:10.1111/j.1525-142X.2010.00453.x. While this work is in mice and therefore tangential, the idea that much variation in these systems may not necessarily be phylogenetic is likely worth exploring here.
-	Conclusions drawn are appropriate to the data presented (but see above for clarification regarding effects of allometry on canal size).
Additional comments: Congratulations to the authors on a very large amount of work and a very nice dataset to address the continuing importance of the cranial canal networks in turtles. This is a very extensive re-examination of ideas proposed more than 40 years ago and makes some important revisions to our understanding of this system. The authors make good use of state-of-the-art 3D imaging and reconstruction tools in order to provide new insights that will be useful for the study of turtle evolution. The paper is well written and easy to read (thank you!), and contains useful figures with only minor corrections required (see below). Minor criticisms relate to the fullness of the discussion regarding canal development, where I was left disappointed after being shown the tantalizingly asymmetrical Manouria specimen; and the lack of discussion of allometric effects on canal size that may confound studies of cross-sectional area. This second point in particular should be addressed before publication.


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

Input:
Title: A REVIEW OF THE CAROTID ARTERY AND FACIAL NERVE CANAL SYSTEMS IN EXTANT TURTLES
Review round: 1
Reviewer: 3

Output:
Basic reporting: This manuscript presents a comprehensive set of reconstructions, descriptions, and comparisons of the carotid circulation and (some) cranial nerves for 66 species of turtles, supplemented by quantitative and statistical evaluations of relative arterial sizes that supply blood to the turtle head. Even though the manuscript is long, it is not unnecessarily long, being full of detailed and useful data, easy to understand, and a pleasant read for turtle specialists. I have only some very minor suggestions in the manuscript (provided in a word file with marked changes). A more noteworthy comment is regarding the description of the spaces, canals, and foramina, in which, in a number of times, the authors refer the osseous structure solely to a soft tissue organ, which hampers its identification in fossils or macerated specimens. I'm sure for most of those cases you have osteological correlates that could be used to identify those structures, so I just suggested using those in this part.
In conclusion, this is an outstanding contribution to turtle anatomists and systematists, which can also be useful for experts in other taxa, which is well-written and provides very important data. As such, I congratulate the authors and recommend it for publication after a few minor revisions.
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 REVIEW OF THE CAROTID ARTERY AND FACIAL NERVE CANAL SYSTEMS IN EXTANT TURTLES
Review round: 1
Reviewer: 4

Output:
Basic reporting: x
Experimental design: x
Validity of the findings: x
Additional comments: “A review of the carotid artery and facial nerve canal systems in extant turtles” is an impressive review of the cranial circulation in turtles, which has been a subject of extensive anatomical studies. This manuscript is a rich source of anatomical information and a welcome contribution. However, the description is impenetrably dense and frustratingly crunchy, and the discussion misses some key citations.

The authors are busy positioning their paper as a definitive account instead of letting their work speak for itself.  They repeatedly make a claim, such as: “global comprehension was hindered by poor sampling and a lack of synthetic studies that addressed both systems (Line 16–17).” This is a false narrative. For example, Kuratani (1987) described the chondrocranial development of Caretta with extensive observation of both the carotid and facial nerve systems (J. Anat. 154, pp. 187-200). Rieppel (1990) described similar observations on Chelydra (Zool. J. Linn. Soc. 98, pp. 27-62). Their observations were picked up later by Miyashita (2012), who proposed that the spatial association between the internal carotid artery and vidian branch acts as a constraint, thus interactions among the carotid artery, facial nerve, and chondrocranium at early developmental stages control configuration of the foramina and canals (Gene Gaffney Festschrift, Springer). None of these papers was cited, even though their relevance to the authors’ discussion seems clear. This lack of attributing due credit to a long string of previous works is quite frustrating.

The authors provide quite puzzling and imprecise anatomical description by ignoring standard practice. Numerous examples caught my attention across the entire text.

-       Artery irrigates, does not “support” (Line 94–95 and throughout) or “feed” (Line 626 and elsewhere). “Supply” may be acceptable but is unclear unless specific structure is named as “supplied” (supply brain, mandible, rostrum? An artery does not exactly supply a bony element, unless discussing capillaries invading the bone.) “Cranial arterial circulation (Line 34)” is simply cranial irrigation.
-       Vein does not “pass” (Line 1033). Vein drains, as opposed to artery that irrigates.
-       Sensory component of a nerve fires from their innervation to the ganglion. So it does not “leave” or “exit” a canal closer to innervation. If purely a sensory branch, the nerve may enter the canal from a point closer to innervation toward the ganglion. However, a motor neuron has a reverse polarity (it fires toward innervation). So avoid all polarity terms in general. The authors can say it is no longer enclosed by a particular bone, but fundamentally speaking it cannot “leave” or “start”.
-       Artery or nerve does not pick itself up and “run” or “travel” (Line 112 and throughout). It is the observer’s focus that “runs” or “travels” on the structure. Artery or nerve extends.
-       Artery (Line 83 and throughout) or ganglion (Line 697 and throughout) does not “give off” branches. Artery or ganglion has branches, splits into branches, or branch into (or leads/connects to) subsidiary structures.
-       A canal itself has no inherent polarity so it does not “start” (Line 587 and elsewhere). It extends.
-       Canals do not fuse (Lines 782-783). Canal is a space, and spaces do not fuse to each other. Canals may merge, join, or become confluent.

The rest of the description is not helpful. The practice of sticking to Latin terms in the turtle literature downstream of Gaffney always puzzled me. Up to 45% of characters in some sections of the manuscript pertain to those Latin terms like “canalis caroticus interni” and frankly these sections are non-comprehensible to most who needs the information. Curiously, the authors are inconsistent. They return to Anglicized common terms for soft tissues (e.g., “mandibular artery”) and some of these osteological features in Discussion. I do not see a rationale for this. Either use accessible English terms throughout (and provide the proper Latin terms in glossary in Nomenclature and Homology), or find some formatting scheme to distinguish Latinized terms, such as italicize (muscle names are commonly italicized), bold, or underline.

In addition to the imprecision and the terminology, the description is meandering, redundant, and inaccessible. Under each family, create subheadings by each main structure discussed (just like in Nomenclature and Homology) so the readers can find the information they are looking for. In more extreme form, the authors should even consider creating character shorthand and adopt telegraphic or bullet point style.

The meandering description exposes another fundamental issue: the authors present a rhetorical/semantic argument for many of the osteological correlates. Despite the rationales of homology presented at the outset, often I was unable to tell if the absence of a particular structure is purely of semantics (e.g., canal versus foramen, or how the homology of “canals” is interpreted when a nerve and a vessel extend endosseously as a bundle), pertains to configuration of the soft tissue interacting with the bone, or arises from the loss of the tissue altogether.

The first example is in Line 424-441, and it is a recurring issue through the rest of text. Or Line 542-545 is symptomatic: what would otherwise be identified as a cavernous canal is a groove within a canal and therefore considered “absent”, even though the difference is whether this space is fully enclosed within the bone or partly open. This only muddies anatomical understanding. Another extreme example is the podocnemidid fossa: correlates are absent but structural configurations remain generally same for the soft tissues.

In these parts of the ms, the description is so bone centric (apparently canals “fuse” according to the authors, Line 782-783) that the authors seem to disregard the fact that most structures they describe are merely correlates. That is, their identity (canals, foramina, cavities) is conferred by a tissue that the bone interacts with, so applying a blanket definition of homology to all is inappropriate. The authors should ask themselves if they are describing an anatomical state or a terminological state. When a canal is absent or present, it is contingent on spatial configuration of the soft tissue(s) filling the canal. To avoid confusion, the authors should present possible arrangement(s) of the carotid and facial systems first, and then interpret their osteological correlates (=identify canals and foramina with proper names), for each family.

At the very least, distinguish semantic/terminological issue versus homology issue. Line 72-74 does not make sense, for example. The identification of a canal where a vessel and a nerve co-occur is a terminological issue, not a homology issue (=pertaining to presence/absence of a structure). It is not that “inconsistent nomenclature […] makes a deeper understanding of homology difficult (Line 90–91).” It is the authors’ expectation for homology to be perfectly translated into nomenclature, that makes a deeper understanding difficult.

Discussion is valuable, but disorganized. Construct taxonomically segregated comparative table(s) to contrast which lineages have what state.

Figures are generally excellent, but Figures 16-18 are confusing. These describe “patterns” of entirely different nature, and distinguish them by Roman numerals, alphabet, and Arabic numerals. They are confusing both in figures and text. Please come up with a better system so we don’t have to get ourselves lost which pattern is discussed. Why can’t they be differentiated as “Facial Patterns 1a-2c” ” “Hyomandibular Subpatterns 1-3”, “Vidian Subpatterns 1-5”?

For description and figures, it is often difficult to verify the characters discussed in text against illustrations in the figures. Each character described in should have reference to relevant labels and panels of the figure.

The authors should provide basic schemes of cranial circulation and facial innervation in turtles at the outset, and refer to the schematics in Lines 77 onward. This is to introduce readers unfamiliar to this problem (likely >90% of pdf downloads) to the basic scheme of the anatomical system.

Line 214-272: This method description is unhelpful and non-repeatable. There is no mention of statistical procedure or dataset (it is available with the paper?). I cannot figure out how measurements were collected (e.g., where in the canals were diameters taken?), whether the measurements were corrected for body size or ontogenetic stages, etc. Please ensure repeatability in methods description.

The order in which families are treated in the description puzzles me. The authors should provide a tree showing their general relationships.

In multiple places in the manuscript (Line 1057, 1267, elsewhere) the authors “contradict” Jamniczky and Russell (2007). But the authors provide no explanation for the discrepancies. Jamniczky and Russell (2007) also measured the same canals in different specimens using CT scan. Why are these observations at odds with each other? Are their observations/mesurements still valid? Did they misidentify structures? Are these due to differences in methods? Different sections of the canals? Different ontogenetic stages?

Line 525 and throughout: when discussing canal sizes, is it in diameter? Is it corrected for differences in body size, or allometry, or ontogeny? Is it maximum, minimum, or average diameter?

Line 547, Line 550 “infer” — what ‘infers’ each of these?

Line 603-604: I do not understand what is “trifurcation formed by the prootic, pterygoid, and basisphenoid.”

Line 629: “The canalis cariticus basisphenoidalis is formed by the basisphenoid.” This sentence is awkward. It’s like reading “the brain is housed within the braincase.”

Line 631: “separated” from each other.

Line 778-779: “while the others either get lost in the porosity of the bone…”
This should be “while the others either become unidentifiable/untraceable in the porosity of the bone…”

Line 829: “mandibular artery traverses…”
Again, an artery does not travel, run, or traverse. In this case, it extends within the cavernous canal (laterally?).

Line 1034: Lateral head vein does not pass “anteriorly.” The lateral head vein drains, and the blood flow is toward the cardinal vein (posteriorly).

Line 1039: “The canalis nervus facialis exits the prootic…”
A canal does not exit bone. A nerve may (to the direction of firing polarity; opposite between sensory and motor).

Line 1041-1042: Another issue with rhetorical/nomenclatural homology. Is canal absent or merged with the carotid canal? Go back to my original comment for this and many other examples.

Line 1075-1076: “the anterior wall of the canalis cavernosus is extremely thin and that the “foramina” are elongate slits.”
This is a good place for the authors to consider how they are mixing up nomenclatural and anatomical states.

Line 1234: “The canal does not extend along the pterygoid/basisphenoid suture, as in all turtles with an unambiguous canalis caroticus lateralis.”
As in all turtles do, or do not?

Line 1239-1240: an artery does not “split” from a canal. It splits from another artery.

Line 1257: an artery does not “join” a chondrocranial element. It penetrates a chondrocranial element.

Line 1262-1267: The majority of characters in these two sentences pertain to Latin terms, and as written these sentences make zero sense to me.

Line 1411 “all extant turtles…”
This should be “almost all extant turtles…” (the authors immediately follow this sentence with ‘exceptions’).

Line 1415-1417: It is good that the authors clarify here about “a matter of semantic preference” but the problem is that they are absolutely vague about this where this clarification matters in Description.

Line 1413, 1419: plural/singular inconsistent in multiple words.

Line 1429-1432: Is only a bony canal considered a canal? Part of the canal wall unossified and it becomes something else? The authors should remind themselves that canal is not the variation. It is a substate of the presence/absence of ossification in that particular locale, that varies.

Line 1446-1448: “A canalis caroticus lateralis is furthermore absent in chelonioids, but not a result of the absence of the palatine artery, but due to its anterior displacement.”
Again, it is nice of the authors to clarify, but this has to be explicit in description. It is vague in the original section, and therefore highly confusing.

Line 1453: what is a “trough let”?

Line 1456-1459: “Our re-interpretation is more parsimonious…”
In the absence of tree, I am not able to evaluate this claim, or what is the difference between Hermanson et al.’s interpretation and their re-interpretation.

Line 1593-1604: Informative and valuable paragraph. A perfect example where a comparative table would greatly enhance the communication.

Line 1620 onward: This is informative but meandering, long, and redundant. Enhance with a comparative table.

Line 1728: “internal carotid and palatine artery canals…”
A sudden introduction of Anglicized terms. Inconsistent.

Line 1828: “higher ‘medial’ blood flow”
“greater ‘medial’ blood flow”

Line 1846-1848: “In summary, our canal size data quantitatively confirm that variation in the stapedial artery size is countered by oppositional variation in the internal carotid size and that variation from the mean can partially be explained by the placement of the mandibular artery.”
This should be: “In summary, our canal size data quantitatively confirm that variation in the stapedial artery inversely correlates with the internal carotid size and that departure from the mean can partially be explained by the configuration of the mandibular artery.

Line 1877 “basal stem-turtles”
I don’t know what this means.

Line 1902-1904: “fossil taxa belonging to stem lineages of extant clades of Testudines”
Awkward.

Line 1911: “slight nomenclatural updates”
Slight nearly equals none. But the authors propose many changes.


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

Input:
Title: A REVIEW OF THE CAROTID ARTERY AND FACIAL NERVE CANAL SYSTEMS IN EXTANT TURTLES
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have made substantial changes in response to mine and the other reviewers' comments. These changes are adequate to respond to my concerns. While I am not in position to re-read the entire manuscript closely, I am satisfied that the changes have been made that allow publication of this version.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: No additional comment; see above.


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

Input:
Title: A REVIEW OF THE CAROTID ARTERY AND FACIAL NERVE CANAL SYSTEMS IN EXTANT TURTLES
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors satisfactorily replied to all of my comments on the original manuscript and made the pertinent modifications. I have nothing further to add and am content with the resulting manuscript and suggest its publication as it is.
Experimental design: no further comment
Validity of the findings: no further comment
Additional comments: No additional comments


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

Input:
Title: A REVIEW OF THE CAROTID ARTERY AND FACIAL NERVE CANAL SYSTEMS IN EXTANT TURTLES
Review round: 2
Reviewer: 3

Output:
Basic reporting: The authors addressed many of the comments and concerns raised. For a few items, however, they relied on rhetorical arguments instead of providing evidence.

In their response they argue their purpose is to describe "the evolutionary development of the carotid and facial nerve system" while they "in no way claim to contribute to the question as how these systems originate or interplay during ontogeny."
"Evolutionary development" means phylogenetic investigation of development (ontogeny). In that sense, once again, it is puzzling why they choose not to include the studies of turtle chondrocranial development in which these systems are described together (such as those on Caretta and Chelydra) and considered in the context of development.

They proceed to attribute this to the misunderstanding arising from the word "global" and revised this as:

"Although a significant number of studies related to these structures exists, A BROADER comprehension OF VARIATION ACROSS THE TREE HAS BEEN hindered by poor sampling and a lack of synthetic studies that addressed both systems together"
(UPPERCASE refers to their change)

Simple substitution of words accomplishes little here, and the same problem remains. Why is the structure that has generated "a significant number of studies" so "poorly sampled" at the same time? And to the point of "the lack of synthetic studies that addressed both systems together", there are studies that addressed these systems together, but these works are either not cited or relegated to the sideline along with other previous works.

Is this the "synthetic" study that the authors consider has been "lacking" in the literature, or is this a compendium of anatomical information? Or is this a follow-up to the series of works that has a deep historical root?

I repeat that arteries do not typically "supply" a bone. So the arterial blood supply for the face is correct, but "the arterial blood supply for the facial region of the skull" (Line 225) is not. Examples of this usage persist in Line 1922, 1931, 1938, 1949, 1953, 1995, .

Line 1949-1950: "these arterial reductions affect the blood supply by shifting the origin of arteries supplying the anterior skull region or the mandible to whichever artery is not reduced."
This sentence does not make sense. Please revise.

I do not intend this to be the place to fight this issue of Latinized terms in turtle literature (for example, chondrocrania are also full of them, even though they are used widely across different taxa and therefore more accessible), and the authors have clearly spoken in defence of the common practice in that field. However, if the authors wish to deliver the turtle data to a wider audience so they are also useful in a broader context, they should also be aware that these rather insular practices are not exactly helping that cause.

Line 2042: "basal-stem turtles"
"Basal" and "stem" are redundant. "Basal" is also imprecise. Why not simply stem turtles, or, to be more precise, non-XXX stem turtles?

Line 2067-2068: "fossil taxa belonging to stem lineages of the primary crown clades of Testudines"
This is as clear as mud. What are "primary crown clades of Testudines"? Why can't this be "stem cryptodires" "paracryptodires" etc.? Or simply "fossil taxa within the testudine crown"?
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: No additional comments


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

Input:
Title: COVID-19 MORTALITY IN CANCER PATIENTS: A REPORT FROM A TERTIARY CANCER CENTRE IN INDIA
Review round: 1
Reviewer: 1

Output:
Basic reporting: I think it is a well-designed and well-written paper. The aims and design are clear. The discussion is well-structured with up-to-date references. However, I think that some comments should be added about the collateral damage in the introduction and discussion and about the mechanism of increased COVID-19 contracting risk in cancer patients as we did in one of our papers (Guven DC, Aktas BY, Aksun MS, et al. COVID-19 pandemic: changes in cancer admissions. BMJ Supportive & Palliative Care. Published Online First: 14 July 2020. doi: 10.1136/bmjspcare-2020-002468). The English is generally very good but I added some suggestions and highlighted them via the track changes feature in order to further improve some sentences. It's up to the authors to use or not use these changes. The figures and tables are largely acceptable but the reporting of values should be uniform.
Experimental design: I think the study is in the scope of the journal. The study tries to answer a clinically meaningful and important question. The authors tested a significant number of patients before study inclusion. The patient numbers are adequate for conducting the analyses. The methodology is explained in detail.
Validity of the findings: Although the study hypothesis was not a novel hypothesis, it is a very important study considering the relative paucity of data from the region of the study. This study could add to the literature about the situation of the pandemic in low-resource settings like India. Conclusions are well written and are generally in-line with previous research in similar patient cohorts.
Additional comments: First of all, I want to congratulate the authors for conducting this elegant and precious work while dealing with the pandemic in a country which was significantly hit with the pandemic. I think the study is a well-constructed and well-designed study with enough. However, it could be further improved with minor changes. I think that some comments should be added about the collateral damage in the introduction and discussion and about the mechanism of increased COVID-19 contracting risk in cancer patients as we did in one of our papers. The English is generally very good but I added some suggestions and highlighted them via the track changes feature in order to further improve some sentences. It's up to the authors to use or not use these changes.


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

Input:
Title: COVID-19 MORTALITY IN CANCER PATIENTS: A REPORT FROM A TERTIARY CANCER CENTRE IN INDIA
Review round: 1
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: Overall, your manuscript is informative, but some edits/improvements need to be made to correctly convey the data:

•	Discussion:
The 6% incidence in COVID-19 in your study is based on HOSPITALIZED cancer patients and not cancer patients, in general. Therefore, your comparison with the general populations of India and Europe is relatively meaningless since one would expect a higher incidence of COVID-19 for all hospitalized patients, whether or not they have cancer. Comparison of COVID-19 incidence in your institution’s outpatient department to the national incidence would likely be more appropriate and would better quantify the effect of the presence of cancer, itself, on the higher risk of COVID-19 infection.

The same point can be made for your stated CFR of 14.5% vs the national average of 1.9% -- you are comparing hospitalized patients to non-hospitalized + hospitalized patients. The comparison gives an incorrect impression of a very pronounced difference. If this is not the case, then please better specify the patient populations being compared.

•	Conclusion:
Your conclusions reflect the exaggeration of COVID-19 incidences that I just discussed and should be appropriately modified. Comparison of your data with NON-CANCER patients hospitalized in India and elsewhere would better tell the story of how the presence of cancer impacts COVID-19 incidence.

•	Results:
Providing a comparison of the fatality rates for hospitalized cancer patients with and without COVID-19 would also provide a level field for comparison of the effect of COVID-19 on fatality on hospitalized cancer patients.

Was the difference in CFRs for those with solid vs hematological cancers statistically significant? If so, this is an important finding and worthy of more investigation (not necessarily in this report) to discover why this is the case. If it is not, I suggest deleting the sentence since it gives the false impression of a true difference – or provide a p-value for this statement.
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: COVID-19 MORTALITY IN CANCER PATIENTS: A REPORT FROM A TERTIARY CANCER CENTRE IN INDIA
Review round: 2
Reviewer: 1

Output:
Basic reporting: I think the paper is significantly improved and acceptable after this revision.
Experimental design: I think the paper is significantly improved and acceptable after this revision.
Validity of the findings: I think the paper is significantly improved and acceptable after this revision.
Additional comments: I think the paper is significantly improved and acceptable after this revision.


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

Input:
Title: GENOME-WIDE IDENTIFICATION AND ANALYSIS OF CYSTATIN FAMILY GENES IN SORGHUM (SORGHUM BICOLOR (L.) MOENCH)
Review round: 1
Reviewer: 1

Output:
Basic reporting: No
Experimental design: The present study "Molecular characterization and expression profiling of cystatin family genes reveal their high evolutionary conservation and functional divergence in sorghum (Sorghum bicolor L.)" attempt to underpin the evolutionary conservation and functional divergence of cystatin family genes in Sorghum bicolor. It is an interesting study and relevant to current focus. This study does highlight cystatin gene characterization evolutionary relatedness and their expression profiles. However, considering the interest of the global readers of this journal, authors must improve the manuscript further. Real time expression analysis can be designed in a comprehensive manner. some more studies like Protein modelling and inhibition sites finding can make it a novel work in Sorghum.
Validity of the findings: Conclusion can be written focusing mainly on the present work carried out instead of generalized Findings. Mention the group of genes fall into different localizations, exon-intron characterization and real time expression analysis, duplication events, phylogeny etc.,
Additional comments: This study does highlight cystatin gene characterization evolutionary relatedness and their expression profiles. However, considering the interest of the global readers of this journal, authors must improve the manuscript further. Real time expression analysis can be designed in a effective manner. some more studies like Protein modelling and inhibition sites finding, finding interacting partners can make it a novel work in Sorghum.


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

Input:
Title: GENOME-WIDE IDENTIFICATION AND ANALYSIS OF CYSTATIN FAMILY GENES IN SORGHUM (SORGHUM BICOLOR (L.) MOENCH)
Review round: 1
Reviewer: 2

Output:
Basic reporting: This manuscript studied the cystatin – inhibitor of cysteine protease in Sorghum. Real-time quantitative polymerase chain reaction (RT-qPCR) and some bioinformatic prediction w tools were used to support the functional diversity of SbCys genes related to specific inhibition activity in different tissues.
The introduction doe well organize paragraphs in order to emphasize the necessity of this study. Literature is well referenced and relevant on studied topic.
Experimental design: Methods was described with sufficient detail and experimental/bioinformatics tools used in this research was well defined, relevant and meaningful.
Validity of the findings: The impact and novelty of this manuscript are assessed. Overall, this manuscript was qualified for publication.
Minor comments:
Figure 1: Description in more detail may needed for protein ID
Figure 6,8: Unit for heat map scale.
Figure 7: similar scale for Figure 7A and 7B
Page 10, line 115 – 116: remove space between number and %unit (>75%)
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: GENOME-WIDE IDENTIFICATION AND ANALYSIS OF CYSTATIN FAMILY GENES IN SORGHUM (SORGHUM BICOLOR (L.) MOENCH)
Review round: 1
Reviewer: 3

Output:
Basic reporting: In the manuscript entitled “Molecular characterization and expression profiling of cystatin family genes reveal their high evolutionary conservation and functional divergence in sorghum (Sorghum bicolor L.)”, the authors predicted 18 sorghum cystatin genes (SbCys) with an HMM and analyzed their protein motifs, chromosome localization, cis-elements and expressions. These analyses for 18 SbCys genes are important for sorghum research. In the current manuscript, however, the connection of these analyses is not clear. Adding a schematic representation of putative regulatory flow for the cystatin genes would help readers to their deeper understanding.
Experimental design: Experimental design for identifying 18 SbCys genes with an HMM is clearly written but the authors need to mention the reasons why they chose this method instead of simply applying blast searches nor domain searches.
Validity of the findings: 1)	The authors describe that SbCys genes have a chromosomal preference in Results (Ll. 167-) and Discussion (Ll. 300-). In a related comparison with O. sativa (Wang W. et al. 2015 Plant Cell Rep), the chromosomal preference looked different between rice and sorghum. How do you interpret these differences? Also, the number of cystatin family genes is larger in sorghum than others (e.g. 7 genes in A. thaliana, 11 genes in O. sativa and 10 genes in N. tabacum). What is the biological and/or evolutional meaning? To check the evolutional duplication, Wang, et al. checked the Ka/Ks ratios between duplicated cystatins. I recommend the authors to refer this work.
2)	As for the promoter analysis, the authors mentioned that Skn-1 and GCN4 motifs are abundant. The motif length of Skn-1 is short. Isn’t it possible that the observed abundance is just because of its shortness? Please perform the statistical test. Also, are there any previous reports that Skn-1 or GCN4 act on cystatin genes in any other plants?
3)	Fig. 6: How is the expression profile of transcription factors, Skn-1/GCN4 ?
Additional comments: Minor comments:
L. 93 and L. 133: Please describe the parameters for HMMER and the RNA-seq applications.
Supplemental tables: Please add JGI gene numbers. Many sorghum researchers use JGI gene numbers (https://phytozome.jgi.doe.gov/pz/portal.html#!info?alias=Org_Sbicolor).
L. 117-: The explanation of microsynteny analysis is not clear enough.

Fig. 2(a): Please increase the font size
Fig. 2(b): Please remove unnecessary all “-“ columns and make the figure larger.
Fig. 6,7: Please add the explanation of each scale bar.
Fig. S1: Difficult to read the gene names.

L. 56: a-helix ->α
(L. 95: E-value ->e-value)
L. 134: P -> p
L. 138: 10-cm -> 10 cm


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

Input:
Title: GENOME-WIDE IDENTIFICATION AND ANALYSIS OF CYSTATIN FAMILY GENES IN SORGHUM (SORGHUM BICOLOR (L.) MOENCH)
Review round: 2
Reviewer: 1

Output:
Basic reporting: The author has addressed all comments and improved the manuscript in well defined manner in grammatical mistakes, references, etc.,
Experimental design: The author has addressed and included modelling of proteins, and interaction maps also. why author feels that results were unsatisfactory for Protein interaction map. Have they tried and crosschecked with other tools like Genemania and cytoscape.
Validity of the findings: All the data  of experimental findings, figures, discussion and conclusions were clearly written according to reviewer comments.
Additional comments: If the author feels unsatisfactory with protein interaction studies, they can remove it (it is not mandate, but in future, can try to predict and prove experimentally  to validate their findings).


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

Input:
Title: GENOME-WIDE IDENTIFICATION AND ANALYSIS OF CYSTATIN FAMILY GENES IN SORGHUM (SORGHUM BICOLOR (L.) MOENCH)
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The manuscript and figures were widely improved and the authors answered all my concerns. However, there are still some small mistakes. I recommend to fix the following mistakes.

Response: To identify the SbCys genes, two methods (HMM and blast searches) were used in the study. The information was shown in lines 132-147.
It is better to describe your method in “Identification of SbCys family members in Sorghum genome”, not in the other section.

Fig 2 legend: Better to remove (a) and (b) from the legend.

Fig 6: In the Wang B et al. 2018 Genome Res, they released Iso-seq data, too. Can you check the TSSs for 18 SbCys genes and add able to put Fig. 6?

Fig 10: Please carefully put “*” in the figure. Some “*” are slightly shifted. Especially Fig C at KXG22254 gene.


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

Input:
Title: GENOME-WIDE IDENTIFICATION AND ANALYSIS OF CYSTATIN FAMILY GENES IN SORGHUM (SORGHUM BICOLOR (L.) MOENCH)
Review round: 2
Reviewer: 3

Output:
Basic reporting: The manuscript shoud be gone through by a native English speaker.
Experimental design: these preliminary data could not support this big title, and authors need to do more experimental works rather than present some rough results if they really want to state the importance of SbCys in Sorghum.

The experimental design and writing logic wait to be improved.
Validity of the findings: no  comment.
Additional comments: Dear Editor,

I was very glad to be a reviewer of PeerJ. However, after reviewing this manuscript carefully that entitled ‘Molecular characterization and expression profiling of cystatin family genes reveal their high evolutionary conservation and functional divergence in Sorghum (Sorghum bicolor L.)’, I got little insightful messages from this manuscript.

In my opinion, these preliminary data could not support this big title, and authors need to do more experimental works rather than present some rough results if they really want to state the importance of SbCys in Sorghum. Nowadays, transient expression assays are easy to do and transgenic Arabidopsis is easy to be generated in any scientific institutions. In addition, the experimental design and writing logic wait to be improved.

As some details, I fully agree with former reviewers. For example, using gene ID as gene name makes it difficult to link the context. No one use ID number as their name.

Considering some efforts had been made by former reviewers, I suggest authors to supply some practical evidences proving the potential function of these SbCys genes in plant responses to drought or other abiotic stresses.