Identification of Proteins Responsible for High Activity of Cysteine Proteinase Inhibitor in the Blood of Nile Tilapia Oreochromis niloticus
Round 1
Reviewer 1 Report
General comments
This publication presents the identification of cysteine proteinase inhibitor in the blood of Nile tilapia. The publication topic and results are interesting and publish-worthy. The topic of the paper is essential for the meat/fish industry and researchers.
The study was planned and executed correctly. The results have been compiled and described in an accessible form. The results are given the discussion correctly and include the most important literature. The methodology, description of results, and chromatography graphs need to be corrected. All figures and tables are of good graphic quality.
Specific comments
L66: please give papain product number in sigma shop
L75: this sentence more fit to Discussion section
L76: don’t use “We”. Better will be: Papain inhibitory activity as indication of the presence of CPI in the sample was measured based on the previous methods [7, 22-24] with some modifications.
L78: what was tissue to buffer ratio?
L80: please give centrifuging parameters
L85: please give concentration of Fast Garnet GBC, sodium nitrite and mersalyl-Brij
2.3. Papain inhibitory activity in different tissues – please give how many repetitions you did for this assay
2.4.1. Isolation of CPIs from tilapia serum – please give the volume of fractions in each of chromatography method. Give name of FPLC equipment?
In all figures, please format X and Y axis - add Tick marks (major type).
Fig 2. – please give abc letters for statistical significant difference
Fig 3. Please mark on figures where is moment of (a) samples uptake, (b) buffers changes
Fig 3. What means CPI activity in 90th fraction?
L180: give a number of this fractions
L176: this sentence is the same as sentence in L180.
Fig. 4: please add on figure calibration marks of column, add marks of ovalbumin 45kDa, bovine serum albumin 66 kDa and aldorase 158 kDa
Fig. 5: Here are separated 2 figures, please combine to one or add legend for fig. 5A and 5B
L187-188: the 54 and 100 kDa proteins there are not in fraction number 20 on fig. 6
L186-188: please add sentence about mass of protein in line 2 on fig. 6
L206: “two major CPIs” in its regards to CPI inhibitor-activity or to CPI protein-concentration?
Table 1: “QVVAG was found both in tilapia fetuin-B and kininogen-1 sequences.” I can not see QVVAG sentence in 27 peptides
Why are selected peptide sequences underlined in Table 1?
L238: should be Summary
Author Response
Dear reviewer 1
Thank you very much for your kind corrections on our manuscript (fishes-1780014). We are happy with most changes you suggested, and most of them have been incorporated into the revised manuscript.
Response to Specific comments
L66: please give papain product number in sigma shop
- it was added to "Materials and Methods" as suggested
L75: this sentence more fit to Discussion section
- this sentence was removed
L76: don’t use “We”. Better will be: Papain inhibitory activity as indication of the presence of CPI in the sample was measured based on the previous methods [7, 22-24] with some modifications.
- Replaced the whole sentence as you suggested
L78: what was tissue to buffer ratio?
- the ratio was added to "Materials and Methods" as suggested
L80: please give centrifuging parameters
- centrifuging parameters were added to "Materials and Methods" as suggested
L85: please give concentration of Fast Garnet GBC, sodium nitrite and mersalyl-Brij
- final concentrations of them in the mixture was added to "Materials and Methods" as suggested
2.3. Papain inhibitory activity in different tissues – please give how many repetitions you did for this assay
- it was done three times: this info was added to "Materials and Methods" as suggested
2.4.1. Isolation of CPIs from tilapia serum – please give the volume of fractions in each of chromatography method. Give name of FPLC equipment?
- volume of each fraction was 0.5 ml and AKTA FPLC syatem was used. this infomation was added to "Materials and Methods" as suggested
In all figures, please format X and Y axis - add Tick marks (major type).
- tick marks are added to all figures as suggested
Fig 2. – please give abc letters for statistical significant difference
- statistical difference was marked in this figure as suggested
Fig 3. Please mark on figures where is moment of (a) samples uptake, (b) buffers changes
- fractions were collected and analyzed for CPI activity after the injection of elution buffer. we have marked the timing of elution buffer in this figure as suggested
Fig 3. What means CPI activity in 90th fraction?
- this is most difficult for us to explain. we haven't paid much attention to the unexpected CPI activity in the fraction (actually it is 86th) because abs of the fraction was 0.008. it is possibly a mistake but we couldn't just get rid of it.
L180: give a number of this fractions
- numbers were added to all figures as suggested
L176: this sentence is the same as sentence in L180.
- yes, you are right. they have to be the same because the same fractions were applied to both gel filtration and ion exchange chromatography as you can see in fig 1.
Fig. 4: please add on figure calibration marks of column, add marks of ovalbumin 45kDa, bovine serum albumin 66 kDa and aldorase 158 kDa
- Thanks for this comment. we found the figure legend was a little confusing. we changed the legend and added marks of ovalbumin and bovine serum albumin as you suggested. aldorase was not marked to avoid crowding the figure
Fig. 5: Here are separated 2 figures, please combine to one or add legend for fig. 5A and 5B
- we have changed the figure to 5A and 5B as you suggested
L187-188: the 54 and 100 kDa proteins there are not in fraction number 20 on fig. 6
- Great thanks for spotting on this error. That was number 22, not 20.
L186-188: please add sentence about mass of protein in line 2 on fig. 6
- it was added as suggested
L206: “two major CPIs” in its regards to CPI inhibitor-activity or to CPI protein-concentration?
- it was based on CPI activity. we made it clearer by adding "major CPIs in terms of inhibiting activity" into the text
Table 1: “QVVAG was found both in tilapia fetuin-B and kininogen-1 sequences.” I can not see QVVAG sentence in 27 peptides
- the QVVAG is found in the partial sequence of fetuin B. However the sequence is not found in the partial but in full sequence of kininogen-1 in database. we have put accession numbers for each protein to Table1
Why are selected peptide sequences underlined in Table 1?
- the underline was removed
L238: should be Summary
- that is not Discussion but Conclusion. Discussion are writtin together with Results. we have made necessary changes to manuscript for this
Reviewer 2 Report
fishes-1780014 “Identification of proteins responsible for high activity of cysteine proteinase inhibitor in the blood of Nile tilapia”.
GENERAL COMMENT:
The subject of the work entitled “Identification of proteins responsible for high activity of cysteine proteinase inhibitor in the blood of Nile tilapia” is of current interest.
This study was conducted to screen CPI activity by examining papain inhibitory activity from different tissues of Nile tilapia Oreochromis niloticus and to identify major proteins for the activity in the blood.
The title is adequate. I suggest adding the scientific name of the fish.
In the abstract some details need to be added.
Introduction section needs to be expanded and improved.
Central argument is supported by evidence and analysis.
The methodology described by the author is accurate.
Discussion section needs to be expanded and improved.
Moderate editing of English language and style required.
This work needs some changes to be improved; for this reason, I require minor revision.
DETAILED COMMENT:
· Title
The title is adequate. I suggest adding the scientific name of the fish.
· Abstract
In the abstract the objective is clearly described. I suggest adding the total number of fish used in the study and where they came from.
· Introduction
I suggest expanding the section, it is not very exhaustive
· Materials and Methods
Add the total number of fish used in the study.
· Results
This section is detailed.
· Discussion
The discussion section is not exhaustive and need to be improved and expanded.
· Tables and figures
Tables and Figures are clear and understandable.
· References
The references are adequate.
Author Response
Dear reviewer 2
Thank you very much for your scientific comments on our manuscript (fishes-1780014). We agree to most of the points you raised, and correction has been made unless otherwise reasoned below.
DETAILED COMMENT:
- Title
The title is adequate. I suggest adding the scientific name of the fish.
- scientific name of Nile tilapia was added to the title as you suggested
- Abstract
In the abstract the objective is clearly described. I suggest adding the total number of fish used in the study and where they came from.
- fish info including number of fish, place of purchase and etc would be found in the first part of "Materials and Methods"
- Introduction
I suggest expanding the section, it is not very exhaustive
- thanks for this suggestion. we have expanded the 4th paragraph to address the lack of information on blood CPI in fish
- Materials and Methods
Add the total number of fish used in the study.
- this can be found in the first part of "Materials and Methods" (n = 5)
- Results
This section is detailed.
- Thanks. We have made some corrections to improve readability of this manuscript
- Discussion
The discussion section is not exhaustive and need to be improved and expanded.
- that is not Discussion but Conclusion. Discussion are writtin together with Results (Results and Discussion). we have made necessary changes to manuscript for this
- Tables and figures
Tables and Figures are clear and understandable.
- References
The references are adequate.
Round 2
Reviewer 1 Report
The authors corrected the manuscript according to the reviewer's suggestion. The manuscript has quality enough to press.
