Uptake of MicroRNAs from Exosome-Like Nanovesicles of Edible Plant Juice by Rat Enterocytes
, Kohei Taniguchi 1,2, Yuki Kuranaga 3, Nabil Eid 4, Yosuke Inomata 1, Sang-Woong Lee 1 and Kazuhisa Uchiyama 1Round 1
Reviewer 1 Report
The paper by Yuko Ito and co-workers focusing on role of exosomes-like nanovesicles of edible plants is quite interesting. Plant-derived vesicles are receiving great attention due to their potential as nanovectors for the delivery of biologically active substances. Furthermore, their applicability to mammalian cells in vitro is very interesting.
The language is good, and the methods are clearly described. Nevertheless, I suggest a revision to refine the quality of the manuscript. It would be useful to insert some more bibliographical data to support the thesis and the results.
The introduction section should provide a statement of the background of the research and whether the objective of the article has been achieved. In the present manuscript, the authors describe well the state of the art of knowledge of the subject matter, but it needs a revision, especially as regards the criterion for choosing edible plants. In addition, the concept of epidermal cell-related juice vesicles, described in section 2.2, should be introduced, and better explained.
Author Response
Reviewer 1
The paper by Yuko Ito and co-workers focusing on role of exosomes-like nanovesicles of edible plants is quite interesting. Plant-derived vesicles are receiving great attention due to their potential as nanovectors for the delivery of biologically active substances. Furthermore, their applicability to mammalian cells in vitro is very interesting.
Answer:
Thank you for reading with interest and for your valuable comments. We agree with them.
Reviewer’s comment 1
The language is good, and the methods are clearly described. Nevertheless, I suggest a revision to refine the quality of the manuscript. It would be useful to insert some more bibliographical data to support the thesis and the results.
Answer:
Thank you for your comments. We added three reference citations [13 - 15] (line 377 – 381) to the Introduction section, lines 50 - 57.
Reviewer’s comments 2 and 3
The introduction section should provide a statement of the background of the research and whether the objective of the article has been achieved. In the present manuscript, the authors describe well the state of the art of knowledge of the subject matter, but it needs a revision, especially as regards the criterion for choosing edible plants. In addition, the concept of epidermal cell-related juice vesicles, described in section 2.2, should be introduced, and better explained.
Answer to comment 2:
Thank you for your comments. We changed the following sentence in the Introduction section (lines 66):
“These findings suggest that plant miRNAs present in edible juice can be absorbed in the GI tract, opening the possibility of using such EVs as drug delivery and other agents.”
to
“These findings suggest that plant miRNAs present in edible juice can be absorbed in the GI tract, opening the possibility of using such EVs as as a drug delivery system and for gene therapy with plant miRNAs.
Answer to comment 3:
Thank you for your comments. We added an explanation scheme to Figure 2a. We also added the text (legend of Figure 2, line 105 - 106): “Modified structure of cross sections of grapefruits and semi-thin sections of grapefruit juice vesicles. Juice vesicles are shown in segment (a)."
Reviewer 1
The paper by Yuko Ito and co-workers focusing on role of exosomes-like nanovesicles of edible plants is quite interesting. Plant-derived vesicles are receiving great attention due to their potential as nanovectors for the delivery of biologically active substances. Furthermore, their applicability to mammalian cells in vitro is very interesting.
Answer:
Thank you for reading with interest and for your valuable comments. We agree with them.
Reviewer’s comment 1
The language is good, and the methods are clearly described. Nevertheless, I suggest a revision to refine the quality of the manuscript. It would be useful to insert some more bibliographical data to support the thesis and the results.
Answer:
Thank you for your comments. We added three reference citations [13 - 15] (line 377 – 381) to the Introduction section, lines 50 - 57.
Reviewer’s comments 2 and 3
The introduction section should provide a statement of the background of the research and whether the objective of the article has been achieved. In the present manuscript, the authors describe well the state of the art of knowledge of the subject matter, but it needs a revision, especially as regards the criterion for choosing edible plants. In addition, the concept of epidermal cell-related juice vesicles, described in section 2.2, should be introduced, and better explained.
Answer to comment 2:
Thank you for your comments. We changed the following sentence in the Introduction section (lines 66):
“These findings suggest that plant miRNAs present in edible juice can be absorbed in the GI tract, opening the possibility of using such EVs as drug delivery and other agents.”
to
“These findings suggest that plant miRNAs present in edible juice can be absorbed in the GI tract, opening the possibility of using such EVs as as a drug delivery system and for gene therapy with plant miRNAs.
Answer to comment 3:
Thank you for your comments. We added an explanation scheme to Figure 2a. We also added the text (legend of Figure 2, line 105 - 106): “Modified structure of cross sections of grapefruits and semi-thin sections of grapefruit juice vesicles. Juice vesicles are shown in segment (a)."
Reviewer 1
The paper by Yuko Ito and co-workers focusing on role of exosomes-like nanovesicles of edible plants is quite interesting. Plant-derived vesicles are receiving great attention due to their potential as nanovectors for the delivery of biologically active substances. Furthermore, their applicability to mammalian cells in vitro is very interesting.
Answer:
Thank you for reading with interest and for your valuable comments. We agree with them.
Reviewer’s comment 1
The language is good, and the methods are clearly described. Nevertheless, I suggest a revision to refine the quality of the manuscript. It would be useful to insert some more bibliographical data to support the thesis and the results.
Answer:
Thank you for your comments. We added three reference citations [13 - 15] (line 377 – 381) to the Introduction section, lines 50 - 57.
Reviewer’s comments 2 and 3
The introduction section should provide a statement of the background of the research and whether the objective of the article has been achieved. In the present manuscript, the authors describe well the state of the art of knowledge of the subject matter, but it needs a revision, especially as regards the criterion for choosing edible plants. In addition, the concept of epidermal cell-related juice vesicles, described in section 2.2, should be introduced, and better explained.
Answer to comment 2:
Thank you for your comments. We changed the following sentence in the Introduction section (lines 66):
“These findings suggest that plant miRNAs present in edible juice can be absorbed in the GI tract, opening the possibility of using such EVs as drug delivery and other agents.”
to
“These findings suggest that plant miRNAs present in edible juice can be absorbed in the GI tract, opening the possibility of using such EVs as as a drug delivery system and for gene therapy with plant miRNAs.
Answer to comment 3:
Thank you for your comments. We added an explanation scheme to Figure 2a. We also added the text (legend of Figure 2, line 105 - 106): “Modified structure of cross sections of grapefruits and semi-thin sections of grapefruit juice vesicles. Juice vesicles are shown in segment (a)."
Reviewer 1
The paper by Yuko Ito and co-workers focusing on role of exosomes-like nanovesicles of edible plants is quite interesting. Plant-derived vesicles are receiving great attention due to their potential as nanovectors for the delivery of biologically active substances. Furthermore, their applicability to mammalian cells in vitro is very interesting.
Answer:
Thank you for reading with interest and for your valuable comments. We agree with them.
Reviewer’s comment 1
The language is good, and the methods are clearly described. Nevertheless, I suggest a revision to refine the quality of the manuscript. It would be useful to insert some more bibliographical data to support the thesis and the results.
Answer:
Thank you for your comments. We added three reference citations [13 - 15] (line 377 – 381) to the Introduction section, lines 50 - 57.
Reviewer’s comments 2 and 3
The introduction section should provide a statement of the background of the research and whether the objective of the article has been achieved. In the present manuscript, the authors describe well the state of the art of knowledge of the subject matter, but it needs a revision, especially as regards the criterion for choosing edible plants. In addition, the concept of epidermal cell-related juice vesicles, described in section 2.2, should be introduced, and better explained.
Answer to comment 2:
Thank you for your comments. We changed the following sentence in the Introduction section (lines 66):
“These findings suggest that plant miRNAs present in edible juice can be absorbed in the GI tract, opening the possibility of using such EVs as drug delivery and other agents.”
to
“These findings suggest that plant miRNAs present in edible juice can be absorbed in the GI tract, opening the possibility of using such EVs as as a drug delivery system and for gene therapy with plant miRNAs.
Answer to comment 3:
Thank you for your comments. We added an explanation scheme to Figure 2a. We also added the text (legend of Figure 2, line 105 - 106): “Modified structure of cross sections of grapefruits and semi-thin sections of grapefruit juice vesicles. Juice vesicles are shown in segment (a)."
Reviewer 1
The paper by Yuko Ito and co-workers focusing on role of exosomes-like nanovesicles of edible plants is quite interesting. Plant-derived vesicles are receiving great attention due to their potential as nanovectors for the delivery of biologically active substances. Furthermore, their applicability to mammalian cells in vitro is very interesting.
Answer:
Thank you for reading with interest and for your valuable comments. We agree with them.
Reviewer’s comment 1
The language is good, and the methods are clearly described. Nevertheless, I suggest a revision to refine the quality of the manuscript. It would be useful to insert some more bibliographical data to support the thesis and the results.
Answer:
Thank you for your comments. We added three reference citations [13 - 15] (line 377 – 381) to the Introduction section, lines 50 - 57.
Reviewer’s comments 2 and 3
The introduction section should provide a statement of the background of the research and whether the objective of the article has been achieved. In the present manuscript, the authors describe well the state of the art of knowledge of the subject matter, but it needs a revision, especially as regards the criterion for choosing edible plants. In addition, the concept of epidermal cell-related juice vesicles, described in section 2.2, should be introduced, and better explained.
Answer to comment 2:
Thank you for your comments. We changed the following sentence in the Introduction section (lines 66):
“These findings suggest that plant miRNAs present in edible juice can be absorbed in the GI tract, opening the possibility of using such EVs as drug delivery and other agents.”
to
“These findings suggest that plant miRNAs present in edible juice can be absorbed in the GI tract, opening the possibility of using such EVs as as a drug delivery system and for gene therapy with plant miRNAs.
Answer to comment 3:
Thank you for your comments. We added an explanation scheme to Figure 2a. We also added the text (legend of Figure 2, line 105 - 106): “Modified structure of cross sections of grapefruits and semi-thin sections of grapefruit juice vesicles. Juice vesicles are shown in segment (a)."
Reviewer 1
The paper by Yuko Ito and co-workers focusing on role of exosomes-like nanovesicles of edible plants is quite interesting. Plant-derived vesicles are receiving great attention due to their potential as nanovectors for the delivery of biologically active substances. Furthermore, their applicability to mammalian cells in vitro is very interesting.
Answer:
Thank you for reading with interest and for your valuable comments. We agree with them.
Reviewer’s comment 1
The language is good, and the methods are clearly described. Nevertheless, I suggest a revision to refine the quality of the manuscript. It would be useful to insert some more bibliographical data to support the thesis and the results.
Answer:
Thank you for your comments. We added three reference citations [13 - 15] (line 377 – 381) to the Introduction section, lines 50 - 57.
Reviewer’s comments 2 and 3
The introduction section should provide a statement of the background of the research and whether the objective of the article has been achieved. In the present manuscript, the authors describe well the state of the art of knowledge of the subject matter, but it needs a revision, especially as regards the criterion for choosing edible plants. In addition, the concept of epidermal cell-related juice vesicles, described in section 2.2, should be introduced, and better explained.
Answer to comment 2:
Thank you for your comments. We changed the following sentence in the Introduction section (lines 66):
“These findings suggest that plant miRNAs present in edible juice can be absorbed in the GI tract, opening the possibility of using such EVs as drug delivery and other agents.”
to
“These findings suggest that plant miRNAs present in edible juice can be absorbed in the GI tract, opening the possibility of using such EVs as as a drug delivery system and for gene therapy with plant miRNAs.
Answer to comment 3:
Thank you for your comments. We added an explanation scheme to Figure 2a. We also added the text (legend of Figure 2, line 105 - 106): “Modified structure of cross sections of grapefruits and semi-thin sections of grapefruit juice vesicles. Juice vesicles are shown in segment (a)."
Reviewer 1
The paper by Yuko Ito and co-workers focusing on role of exosomes-like nanovesicles of edible plants is quite interesting. Plant-derived vesicles are receiving great attention due to their potential as nanovectors for the delivery of biologically active substances. Furthermore, their applicability to mammalian cells in vitro is very interesting.
Answer:
Thank you for reading with interest and for your valuable comments. We agree with them.
Reviewer’s comment 1
The language is good, and the methods are clearly described. Nevertheless, I suggest a revision to refine the quality of the manuscript. It would be useful to insert some more bibliographical data to support the thesis and the results.
Answer:
Thank you for your comments. We added three reference citations [13 - 15] (line 377 – 381) to the Introduction section, lines 50 - 57.
Reviewer’s comments 2 and 3
The introduction section should provide a statement of the background of the research and whether the objective of the article has been achieved. In the present manuscript, the authors describe well the state of the art of knowledge of the subject matter, but it needs a revision, especially as regards the criterion for choosing edible plants. In addition, the concept of epidermal cell-related juice vesicles, described in section 2.2, should be introduced, and better explained.
Answer to comment 2:
Thank you for your comments. We changed the following sentence in the Introduction section (lines 66):
“These findings suggest that plant miRNAs present in edible juice can be absorbed in the GI tract, opening the possibility of using such EVs as drug delivery and other agents.”
to
“These findings suggest that plant miRNAs present in edible juice can be absorbed in the GI tract, opening the possibility of using such EVs as as a drug delivery system and for gene therapy with plant miRNAs.
Answer to comment 3:
Thank you for your comments. We added an explanation scheme to Figure 2a. We also added the text (legend of Figure 2, line 105 - 106): “Modified structure of cross sections of grapefruits and semi-thin sections of grapefruit juice vesicles. Juice vesicles are shown in segment (a)."
Reviewer 1
The paper by Yuko Ito and co-workers focusing on role of exosomes-like nanovesicles of edible plants is quite interesting. Plant-derived vesicles are receiving great attention due to their potential as nanovectors for the delivery of biologically active substances. Furthermore, their applicability to mammalian cells in vitro is very interesting.
Answer:
Thank you for reading with interest and for your valuable comments. We agree with them.
Reviewer’s comment 1
The language is good, and the methods are clearly described. Nevertheless, I suggest a revision to refine the quality of the manuscript. It would be useful to insert some more bibliographical data to support the thesis and the results.
Answer:
Thank you for your comments. We added three reference citations [13 - 15] (line 377 – 381) to the Introduction section, lines 50 - 57.
Reviewer’s comments 2 and 3
The introduction section should provide a statement of the background of the research and whether the objective of the article has been achieved. In the present manuscript, the authors describe well the state of the art of knowledge of the subject matter, but it needs a revision, especially as regards the criterion for choosing edible plants. In addition, the concept of epidermal cell-related juice vesicles, described in section 2.2, should be introduced, and better explained.
Answer to comment 2:
Thank you for your comments. We changed the following sentence in the Introduction section (lines 66):
“These findings suggest that plant miRNAs present in edible juice can be absorbed in the GI tract, opening the possibility of using such EVs as drug delivery and other agents.”
to
“These findings suggest that plant miRNAs present in edible juice can be absorbed in the GI tract, opening the possibility of using such EVs as as a drug delivery system and for gene therapy with plant miRNAs.
Answer to comment 3:
Thank you for your comments. We added an explanation scheme to Figure 2a. We also added the text (legend of Figure 2, line 105 - 106): “Modified structure of cross sections of grapefruits and semi-thin sections of grapefruit juice vesicles. Juice vesicles are shown in segment (a)."
Reviewer 1
The paper by Yuko Ito and co-workers focusing on role of exosomes-like nanovesicles of edible plants is quite interesting. Plant-derived vesicles are receiving great attention due to their potential as nanovectors for the delivery of biologically active substances. Furthermore, their applicability to mammalian cells in vitro is very interesting.
Answer:
Thank you for reading with interest and for your valuable comments. We agree with them.
Reviewer’s comment 1
The language is good, and the methods are clearly described. Nevertheless, I suggest a revision to refine the quality of the manuscript. It would be useful to insert some more bibliographical data to support the thesis and the results.
Answer:
Thank you for your comments. We added three reference citations [13 - 15] (line 377 – 381) to the Introduction section, lines 50 - 57.
Reviewer’s comments 2 and 3
The introduction section should provide a statement of the background of the research and whether the objective of the article has been achieved. In the present manuscript, the authors describe well the state of the art of knowledge of the subject matter, but it needs a revision, especially as regards the criterion for choosing edible plants. In addition, the concept of epidermal cell-related juice vesicles, described in section 2.2, should be introduced, and better explained.
Answer to comment 2:
Thank you for your comments. We changed the following sentence in the Introduction section (lines 66):
“These findings suggest that plant miRNAs present in edible juice can be absorbed in the GI tract, opening the possibility of using such EVs as drug delivery and other agents.”
to
“These findings suggest that plant miRNAs present in edible juice can be absorbed in the GI tract, opening the possibility of using such EVs as as a drug delivery system and for gene therapy with plant miRNAs.
Answer to comment 3:
Thank you for your comments. We added an explanation scheme to Figure 2a. We also added the text (legend of Figure 2, line 105 - 106): “Modified structure of cross sections of grapefruits and semi-thin sections of grapefruit juice vesicles. Juice vesicles are shown in segment (a)."
Reviewer 1
The paper by Yuko Ito and co-workers focusing on role of exosomes-like nanovesicles of edible plants is quite interesting. Plant-derived vesicles are receiving great attention due to their potential as nanovectors for the delivery of biologically active substances. Furthermore, their applicability to mammalian cells in vitro is very interesting.
Answer:
Thank you for reading with interest and for your valuable comments. We agree with them.
Reviewer’s comment 1
The language is good, and the methods are clearly described. Nevertheless, I suggest a revision to refine the quality of the manuscript. It would be useful to insert some more bibliographical data to support the thesis and the results.
Answer:
Thank you for your comments. We added three reference citations [13 - 15] (line 377 – 381) to the Introduction section, lines 50 - 57.
Reviewer’s comments 2 and 3
The introduction section should provide a statement of the background of the research and whether the objective of the article has been achieved. In the present manuscript, the authors describe well the state of the art of knowledge of the subject matter, but it needs a revision, especially as regards the criterion for choosing edible plants. In addition, the concept of epidermal cell-related juice vesicles, described in section 2.2, should be introduced, and better explained.
Answer to comment 2:
Thank you for your comments. We changed the following sentence in the Introduction section (lines 66):
“These findings suggest that plant miRNAs present in edible juice can be absorbed in the GI tract, opening the possibility of using such EVs as drug delivery and other agents.”
to
“These findings suggest that plant miRNAs present in edible juice can be absorbed in the GI tract, opening the possibility of using such EVs as as a drug delivery system and for gene therapy with plant miRNAs.
Answer to comment 3:
Thank you for your comments. We added an explanation scheme to Figure 2a. We also added the text (legend of Figure 2, line 105 - 106): “Modified structure of cross sections of grapefruits and semi-thin sections of grapefruit juice vesicles. Juice vesicles are shown in segment (a)."
Reviewer 1
The paper by Yuko Ito and co-workers focusing on role of exosomes-like nanovesicles of edible plants is quite interesting. Plant-derived vesicles are receiving great attention due to their potential as nanovectors for the delivery of biologically active substances. Furthermore, their applicability to mammalian cells in vitro is very interesting.
Answer:
Thank you for reading with interest and for your valuable comments. We agree with them.
Reviewer’s comment 1
The language is good, and the methods are clearly described. Nevertheless, I suggest a revision to refine the quality of the manuscript. It would be useful to insert some more bibliographical data to support the thesis and the results.
Answer:
Thank you for your comments. We added three reference citations [13 - 15] (line 377 – 381) to the Introduction section, lines 50 - 57.
Reviewer’s comments 2 and 3
The introduction section should provide a statement of the background of the research and whether the objective of the article has been achieved. In the present manuscript, the authors describe well the state of the art of knowledge of the subject matter, but it needs a revision, especially as regards the criterion for choosing edible plants. In addition, the concept of epidermal cell-related juice vesicles, described in section 2.2, should be introduced, and better explained.
Answer to comment 2:
Thank you for your comments. We changed the following sentence in the Introduction section (lines 66):
“These findings suggest that plant miRNAs present in edible juice can be absorbed in the GI tract, opening the possibility of using such EVs as drug delivery and other agents.”
to
“These findings suggest that plant miRNAs present in edible juice can be absorbed in the GI tract, opening the possibility of using such EVs as as a drug delivery system and for gene therapy with plant miRNAs.
Answer to comment 3:
Thank you for your comments. We added an explanation scheme to Figure 2a. We also added the text (legend of Figure 2, line 105 - 106): “Modified structure of cross sections of grapefruits and semi-thin sections of grapefruit juice vesicles. Juice vesicles are shown in segment (a)."
Reviewer 1
The paper by Yuko Ito and co-workers focusing on role of exosomes-like nanovesicles of edible plants is quite interesting. Plant-derived vesicles are receiving great attention due to their potential as nanovectors for the delivery of biologically active substances. Furthermore, their applicability to mammalian cells in vitro is very interesting.
Answer:
Thank you for reading with interest and for your valuable comments. We agree with them.
Reviewer’s comment 1
The language is good, and the methods are clearly described. Nevertheless, I suggest a revision to refine the quality of the manuscript. It would be useful to insert some more bibliographical data to support the thesis and the results.
Answer:
Thank you for your comments. We added three reference citations [13 - 15] (line 377 – 381) to the Introduction section, lines 50 - 57.
Reviewer’s comments 2 and 3
The introduction section should provide a statement of the background of the research and whether the objective of the article has been achieved. In the present manuscript, the authors describe well the state of the art of knowledge of the subject matter, but it needs a revision, especially as regards the criterion for choosing edible plants. In addition, the concept of epidermal cell-related juice vesicles, described in section 2.2, should be introduced, and better explained.
Answer to comment 2:
Thank you for your comments. We changed the following sentence in the Introduction section (lines 66):
“These findings suggest that plant miRNAs present in edible juice can be absorbed in the GI tract, opening the possibility of using such EVs as drug delivery and other agents.”
to
“These findings suggest that plant miRNAs present in edible juice can be absorbed in the GI tract, opening the possibility of using such EVs as as a drug delivery system and for gene therapy with plant miRNAs.
Answer to comment 3:
Thank you for your comments. We added an explanation scheme to Figure 2a. We also added the text (legend of Figure 2, line 105 - 106): “Modified structure of cross sections of grapefruits and semi-thin sections of grapefruit juice vesicles. Juice vesicles are shown in segment (a)."
Reviewer 1
The paper by Yuko Ito and co-workers focusing on role of exosomes-like nanovesicles of edible plants is quite interesting. Plant-derived vesicles are receiving great attention due to their potential as nanovectors for the delivery of biologically active substances. Furthermore, their applicability to mammalian cells in vitro is very interesting.
Answer:
Thank you for reading with interest and for your valuable comments. We agree with them.
Reviewer’s comment 1
The language is good, and the methods are clearly described. Nevertheless, I suggest a revision to refine the quality of the manuscript. It would be useful to insert some more bibliographical data to support the thesis and the results.
Answer:
Thank you for your comments. We added three reference citations [13 - 15] (line 377 – 381) to the Introduction section, lines 50 - 57.
Reviewer’s comments 2 and 3
The introduction section should provide a statement of the background of the research and whether the objective of the article has been achieved. In the present manuscript, the authors describe well the state of the art of knowledge of the subject matter, but it needs a revision, especially as regards the criterion for choosing edible plants. In addition, the concept of epidermal cell-related juice vesicles, described in section 2.2, should be introduced, and better explained.
Answer to comment 2:
Thank you for your comments. We changed the following sentence in the Introduction section (lines 66):
“These findings suggest that plant miRNAs present in edible juice can be absorbed in the GI tract, opening the possibility of using such EVs as drug delivery and other agents.”
to
“These findings suggest that plant miRNAs present in edible juice can be absorbed in the GI tract, opening the possibility of using such EVs as as a drug delivery system and for gene therapy with plant miRNAs.
Answer to comment 3:
Thank you for your comments. We added an explanation scheme to Figure 2a. We also added the text (legend of Figure 2, line 105 - 106): “Modified structure of cross sections of grapefruits and semi-thin sections of grapefruit juice vesicles. Juice vesicles are shown in segment (a)."
Reviewer 1
The paper by Yuko Ito and co-workers focusing on role of exosomes-like nanovesicles of edible plants is quite interesting. Plant-derived vesicles are receiving great attention due to their potential as nanovectors for the delivery of biologically active substances. Furthermore, their applicability to mammalian cells in vitro is very interesting.
Answer:
Thank you for reading with interest and for your valuable comments. We agree with them.
Reviewer’s comment 1
The language is good, and the methods are clearly described. Nevertheless, I suggest a revision to refine the quality of the manuscript. It would be useful to insert some more bibliographical data to support the thesis and the results.
Answer:
Thank you for your comments. We added three reference citations [13 - 15] (line 377 – 381) to the Introduction section, lines 50 - 57.
Reviewer’s comments 2 and 3
The introduction section should provide a statement of the background of the research and whether the objective of the article has been achieved. In the present manuscript, the authors describe well the state of the art of knowledge of the subject matter, but it needs a revision, especially as regards the criterion for choosing edible plants. In addition, the concept of epidermal cell-related juice vesicles, described in section 2.2, should be introduced, and better explained.
Answer to comment 2:
Thank you for your comments. We changed the following sentence in the Introduction section (lines 66):
“These findings suggest that plant miRNAs present in edible juice can be absorbed in the GI tract, opening the possibility of using such EVs as drug delivery and other agents.”
to
“These findings suggest that plant miRNAs present in edible juice can be absorbed in the GI tract, opening the possibility of using such EVs as as a drug delivery system and for gene therapy with plant miRNAs.
Answer to comment 3:
Thank you for your comments. We added an explanation scheme to Figure 2a. We also added the text (legend of Figure 2, line 105 - 106): “Modified structure of cross sections of grapefruits and semi-thin sections of grapefruit juice vesicles. Juice vesicles are shown in segment (a)."
Reviewer 1
The paper by Yuko Ito and co-workers focusing on role of exosomes-like nanovesicles of edible plants is quite interesting. Plant-derived vesicles are receiving great attention due to their potential as nanovectors for the delivery of biologically active substances. Furthermore, their applicability to mammalian cells in vitro is very interesting.
Answer:
Thank you for reading with interest and for your valuable comments. We agree with them.
Reviewer’s comment 1
The language is good, and the methods are clearly described. Nevertheless, I suggest a revision to refine the quality of the manuscript. It would be useful to insert some more bibliographical data to support the thesis and the results.
Answer:
Thank you for your comments. We added three reference citations [13 - 15] (line 377 – 381) to the Introduction section, lines 50 - 57.
Reviewer’s comments 2 and 3
The introduction section should provide a statement of the background of the research and whether the objective of the article has been achieved. In the present manuscript, the authors describe well the state of the art of knowledge of the subject matter, but it needs a revision, especially as regards the criterion for choosing edible plants. In addition, the concept of epidermal cell-related juice vesicles, described in section 2.2, should be introduced, and better explained.
Answer to comment 2:
Thank you for your comments. We changed the following sentence in the Introduction section (lines 66):
“These findings suggest that plant miRNAs present in edible juice can be absorbed in the GI tract, opening the possibility of using such EVs as drug delivery and other agents.”
to
“These findings suggest that plant miRNAs present in edible juice can be absorbed in the GI tract, opening the possibility of using such EVs as as a drug delivery system and for gene therapy with plant miRNAs.
Answer to comment 3:
Thank you for your comments. We added an explanation scheme to Figure 2a. We also added the text (legend of Figure 2, line 105 - 106): “Modified structure of cross sections of grapefruits and semi-thin sections of grapefruit juice vesicles. Juice vesicles are shown in segment (a)."
Author Response File: 
Author Response.docx
Reviewer 2 Report
In this manuscript, the authors focused on plant juice-derived exosome like nanoparticles, which are taken up by enterocyte. Although Plant-derived exosomes are attracting attention as carriers for drug delivery, their biological significance is still poorly understood. So, what the authors are arguing in this paper is very important. However, the data in this manuscript are poor and there is little novelty in this paper. There are several concerns which authors should address before they publish this paper. #1. In figure 3a-c, the uptake of EVs labeled with PKH into rat IEC6 cells was shown using fluorescence microscopy. Firstly, the figures were rough. It should be changed to something more clear. Furthermore, the authors described that EVs were localized in the cytoplasm, especially in the perinuclear area of IEC6 cells; however, the nucleus in these figure is difficult to recognize. Co-staining of EVs with the nucleus is necessary. #2. In addition to #1, in figure 3a, how many hours after the addition of EVs is this photograph taken? The morphology of the cytoplasm looks very different compared to Fig. 3b at 1 hour after EV addition. The authors should explain this difference between figure 3a and 3b. #3. The RT-PCR results showed that the hand-squeezed juice contained more miRNAs in its EVs than the commercial juice. Conversely, in Fig. 1d, the number of EVs in commercial grapefruit juice was about four times the number of EVs in hand-squeezed paprika juice. If the authors claimed that these miRNAs are ubiquitous, they should explain why there is such a discrepancy between the number of EVs and the amount of miRNAs in those EVs. Please add the number of EVs of hand-squeezed grapefruit juice to Figure 1d. #4. From the result of table 2, EVs derived from hand-squeezed grapefruit juice were taken up by IEC6 cells. Have you checked to see if EVs taken from commercial juices are taken up by IEC6 cells? Please add data to see if there was any difference in the uptake capability. #5. Although the authors discussed the limitation of this manuscript, it should be confirmed the characterization of the collected EVs by western blotting. International Society of Extracellular Vesicles has published a position paper: Minimal information for studies of extracellular vesicles 2018 (MISEV2018). It is stated that one should always show that the isolated EVs contain EV specific protein. Refs. 8 and 19 in the references are papers on Sunflowers and Arabidopsis. Therefore, it is very informative to show expression of these protein levels in plant juice-derived EVs. #6. Generally speaking, if EVs are co-cultured with cells, a certain amount will be taken up by endocytosis or phagocytosis. If the authors assume that enterocytes can take up EVs because of their ability to absorb nutrients, they need to confirm that EVs accumulate specifically in enterocytes in animal experiments with oral administration. #7. Please add the scale size in figure legends 1f-h.
Author Response
Reviewer 2
In this manuscript, the authors focused on plant juice-derived exosome like nanoparticles, which are taken up by enterocyte. Although Plant-derived exosomes are attracting attention as carriers for drug delivery, their biological significance is still poorly understood. So, what the authors are arguing in this paper is very important. However, the data in this manuscript are poor and there is little novelty in this paper. There are several concerns which authors should address before they publish this paper.
Answer:
We appreciate the valuable comments of the reviewer and agree with them. Here we address the comments one by one:
Reviewer’s comments 1 and 2
Firstly, the figures were rough. It should be changed to something more clear. Furthermore, the authors described that EVs were localized in the cytoplasm, especially in the perinuclear area of IEC6 cells; however, the nucleus in these figure is difficult to recognize. Co-staining of EVs with the nucleus is necessary.
#1. In figure 3a-c, the uptake of EVs labeled with PKH into rat IEC6 cells was shown using fluorescence microscopy.
#2. In addition to #1, in figure 3a, how many hours after the addition of EVs is this photograph taken? The morphology of the cytoplasm looks very different compared to Fig. 3b at 1 hour after EV addition. The authors should explain this difference between figure 3a and 3b.
Answer:
Thank you for your valuable comments. IEC6 cells are shown with an overlay, differential interference contrast (DIC), and fluorescence from PKH67.
We indicate the nuclei with black arrows in Figs.3a ~ 3c, and an enlarged view was added to Fig. 3c. We also added the following sentence to the figure legend (lines 156 -157): “ Black arrows: nuclei of IEC cells are shown with differential interference contrast (DIC). The inset in c shows an enlarged boxed area.”
We think that the IEC6 cells in Fig. 3a had no phagosome containing PKH67-labeled EVs, and the number of phagosomes was increased in 1h-cells. We added an explanation to the text to the effect that control experiment showed no phagosome containing PKH67-labeled EVs (Fig. 3a) (line 139). We also added "0 h" to Figure 3a and the legend (line 154).
Reviewer’s comments 3
#3. The RT-PCR results showed that the hand-squeezed juice contained more miRNAs in its EVs than the commercial juice. Conversely, in Fig. 1d, the number of EVs in commercial grapefruit juice was about four times the number of EVs in hand-squeezed paprika juice. If the authors claimed that these miRNAs are ubiquitous, they should explain why there is such a discrepancy between the number of EVs and the amount of miRNAs in those EVs. Please add the number of EVs of hand-squeezed grapefruit juice to Figure 1d.
Answer:
The concentration of SJ-grapefruits juice was 339.6 x 108 /mL. It was lower than that of commercial grapefruits juice (please find an attached file). We think EVs of commercial grapefruits juice and SJ-paprika juice contained pigment granules. Hence, we used filter paper to remove pigment granules through squeezing and pre-filtration. We do not think that every EV contained miRNAs, and we consider that the number of EVs did not correspond to the volume containing miRNA.
We added these points to the Discussion section as follows (line 205 - 207):
“Although the concentration of grapefruits SJ (339.6 x 108 /ml) was lower than that of grapefruits J (Fig. 1d), expression of miRNAs for grapefruits SJ were higher than that of grapefruits J (Table 1).”
Reviewer’s comments 4
#4. From the result of table 2, EVs derived from hand-squeezed grapefruit juice were taken up by IEC6 cells. Have you checked to see if EVs taken from commercial juices are taken up by IEC6 cells? Please add data to see if there was any difference in the uptake capability.
Answer:
Thank you for your comments. We did not check the differences in uptake capability. We conducted the uptaking experiments using IEC6 cells for grapefruits SJ because their expression of miRNAs was higher than that of grapefruits J, as shown in Table 1. We will evaluate uptake capability and its effects on enterocytes in our subsequent work. Thank you for the suggestion.
Reviewer’s comments 5
#5. Although the authors discussed the limitation of this manuscript, it should be confirmed the characterization of the collected EVs by western blotting. International Society of Extracellular Vesicles has published a position paper: Minimal information for studies of extracellular vesicles 2018 (MISEV2018). It is stated that one should always show that the isolated EVs contain EV specific protein. Refs. 8 and 19 in the references are papers on Sunflowers and Arabidopsis. Therefore, it is very informative to show expression of these protein levels in plant juice-derived EVs.
Answer:
Thank you for your comments. We tried to perform western blots using the plant EV markers TET-8 and Rab 11 as previously described (8). However, because of animal antigens, we did not observe any signal. We think that antibodies suitable for plant EVs are not commercially available. We are preparing antibodies against some plant EVs, and we will show the results of grapefruit EVs in a future publication.
We modified the text regarding this point in the Discussion section as follows (lines 248 - 252):
“First, we did not use any specific plant EV marker, such as Rab11a and TET8,9 that have been suggested in previous studies [8,22], nor detected the expression of proteins. We mainly detected plant EVs based on morphology. Further experiments are needed to more easily observe plant EVs.”
Reviewer’s comments 6
#6. Generally speaking, if EVs are co-cultured with cells, a certain amount will be taken up by endocytosis or phagocytosis. If the authors assume that enterocytes can take up EVs because of their ability to absorb nutrients, they need to confirm that EVs accumulate specifically in enterocytes in animal experiments with oral administration.
Answer:
Thank you for your comments. Of course, your opinion is valid. The subject of this project was to perform in vitro experiments before the animal experiments. We are now designing in vitro experiments as the next project. We now mention this point in the Discussion section as follows (lines 228 - 230):
“Further studies, especially using in vivo systems, are needed to demonstrate the fate of EVs incorporated into enterocytes.”
Reviewer’s comments 7
#7. Please add the scale size in figure legends 1f-h.
Answer:
Thank you for your comments. As suggested, we added scale bars to Figure 1f-1h and in the legend as follows (line 91):
“Scale bars: f, 667 nm; g, 600 nm; h, 750 nm.”
Reviewer 2
In this manuscript, the authors focused on plant juice-derived exosome like nanoparticles, which are taken up by enterocyte. Although Plant-derived exosomes are attracting attention as carriers for drug delivery, their biological significance is still poorly understood. So, what the authors are arguing in this paper is very important. However, the data in this manuscript are poor and there is little novelty in this paper. There are several concerns which authors should address before they publish this paper.
Answer:
We appreciate the valuable comments of the reviewer and agree with them. Here we address the comments one by one:
Reviewer’s comments 1 and 2
Firstly, the figures were rough. It should be changed to something more clear. Furthermore, the authors described that EVs were localized in the cytoplasm, especially in the perinuclear area of IEC6 cells; however, the nucleus in these figure is difficult to recognize. Co-staining of EVs with the nucleus is necessary.
#1. In figure 3a-c, the uptake of EVs labeled with PKH into rat IEC6 cells was shown using fluorescence microscopy.
#2. In addition to #1, in figure 3a, how many hours after the addition of EVs is this photograph taken? The morphology of the cytoplasm looks very different compared to Fig. 3b at 1 hour after EV addition. The authors should explain this difference between figure 3a and 3b.
Answer:
Thank you for your valuable comments. IEC6 cells are shown with an overlay, differential interference contrast (DIC), and fluorescence from PKH67.
We indicate the nuclei with black arrows in Figs.3a ~ 3c, and an enlarged view was added to Fig. 3c. We also added the following sentence to the figure legend (lines 156 -157): “ Black arrows: nuclei of IEC cells are shown with differential interference contrast (DIC). The inset in c shows an enlarged boxed area.”
We think that the IEC6 cells in Fig. 3a had no phagosome containing PKH67-labeled EVs, and the number of phagosomes was increased in 1h-cells. We added an explanation to the text to the effect that control experiment showed no phagosome containing PKH67-labeled EVs (Fig. 3a) (line 139). We also added "0 h" to Figure 3a and the legend (line 154).
Reviewer’s comments 3
#3. The RT-PCR results showed that the hand-squeezed juice contained more miRNAs in its EVs than the commercial juice. Conversely, in Fig. 1d, the number of EVs in commercial grapefruit juice was about four times the number of EVs in hand-squeezed paprika juice. If the authors claimed that these miRNAs are ubiquitous, they should explain why there is such a discrepancy between the number of EVs and the amount of miRNAs in those EVs. Please add the number of EVs of hand-squeezed grapefruit juice to Figure 1d.
Answer:
The concentration of SJ-grapefruits juice was 339.6 x 108 /mL. It was lower than that of commercial grapefruits juice (please find an attached file). We think EVs of commercial grapefruits juice and SJ-paprika juice contained pigment granules. Hence, we used filter paper to remove pigment granules through squeezing and pre-filtration. We do not think that every EV contained miRNAs, and we consider that the number of EVs did not correspond to the volume containing miRNA.
We added these points to the Discussion section as follows (line 205 - 207):
“Although the concentration of grapefruits SJ (339.6 x 108 /ml) was lower than that of grapefruits J (Fig. 1d), expression of miRNAs for grapefruits SJ were higher than that of grapefruits J (Table 1).”
Reviewer’s comments 4
#4. From the result of table 2, EVs derived from hand-squeezed grapefruit juice were taken up by IEC6 cells. Have you checked to see if EVs taken from commercial juices are taken up by IEC6 cells? Please add data to see if there was any difference in the uptake capability.
Answer:
Thank you for your comments. We did not check the differences in uptake capability. We conducted the uptaking experiments using IEC6 cells for grapefruits SJ because their expression of miRNAs was higher than that of grapefruits J, as shown in Table 1. We will evaluate uptake capability and its effects on enterocytes in our subsequent work. Thank you for the suggestion.
Reviewer’s comments 5
#5. Although the authors discussed the limitation of this manuscript, it should be confirmed the characterization of the collected EVs by western blotting. International Society of Extracellular Vesicles has published a position paper: Minimal information for studies of extracellular vesicles 2018 (MISEV2018). It is stated that one should always show that the isolated EVs contain EV specific protein. Refs. 8 and 19 in the references are papers on Sunflowers and Arabidopsis. Therefore, it is very informative to show expression of these protein levels in plant juice-derived EVs.
Answer:
Thank you for your comments. We tried to perform western blots using the plant EV markers TET-8 and Rab 11 as previously described (8). However, because of animal antigens, we did not observe any signal. We think that antibodies suitable for plant EVs are not commercially available. We are preparing antibodies against some plant EVs, and we will show the results of grapefruit EVs in a future publication.
We modified the text regarding this point in the Discussion section as follows (lines 248 - 252):
“First, we did not use any specific plant EV marker, such as Rab11a and TET8,9 that have been suggested in previous studies [8,22], nor detected the expression of proteins. We mainly detected plant EVs based on morphology. Further experiments are needed to more easily observe plant EVs.”
Reviewer’s comments 6
#6. Generally speaking, if EVs are co-cultured with cells, a certain amount will be taken up by endocytosis or phagocytosis. If the authors assume that enterocytes can take up EVs because of their ability to absorb nutrients, they need to confirm that EVs accumulate specifically in enterocytes in animal experiments with oral administration.
Answer:
Thank you for your comments. Of course, your opinion is valid. The subject of this project was to perform in vitro experiments before the animal experiments. We are now designing in vitro experiments as the next project. We now mention this point in the Discussion section as follows (lines 228 - 230):
“Further studies, especially using in vivo systems, are needed to demonstrate the fate of EVs incorporated into enterocytes.”
Reviewer’s comments 7
#7. Please add the scale size in figure legends 1f-h.
Answer:
Thank you for your comments. As suggested, we added scale bars to Figure 1f-1h and in the legend as follows (line 91):
“Scale bars: f, 667 nm; g, 600 nm; h, 750 nm.”
Reviewer 2
In this manuscript, the authors focused on plant juice-derived exosome like nanoparticles, which are taken up by enterocyte. Although Plant-derived exosomes are attracting attention as carriers for drug delivery, their biological significance is still poorly understood. So, what the authors are arguing in this paper is very important. However, the data in this manuscript are poor and there is little novelty in this paper. There are several concerns which authors should address before they publish this paper.
Answer:
We appreciate the valuable comments of the reviewer and agree with them. Here we address the comments one by one:
Reviewer’s comments 1 and 2
Firstly, the figures were rough. It should be changed to something more clear. Furthermore, the authors described that EVs were localized in the cytoplasm, especially in the perinuclear area of IEC6 cells; however, the nucleus in these figure is difficult to recognize. Co-staining of EVs with the nucleus is necessary.
#1. In figure 3a-c, the uptake of EVs labeled with PKH into rat IEC6 cells was shown using fluorescence microscopy.
#2. In addition to #1, in figure 3a, how many hours after the addition of EVs is this photograph taken? The morphology of the cytoplasm looks very different compared to Fig. 3b at 1 hour after EV addition. The authors should explain this difference between figure 3a and 3b.
Answer:
Thank you for your valuable comments. IEC6 cells are shown with an overlay, differential interference contrast (DIC), and fluorescence from PKH67.
We indicate the nuclei with black arrows in Figs.3a ~ 3c, and an enlarged view was added to Fig. 3c. We also added the following sentence to the figure legend (lines 156 -157): “ Black arrows: nuclei of IEC cells are shown with differential interference contrast (DIC). The inset in c shows an enlarged boxed area.”
We think that the IEC6 cells in Fig. 3a had no phagosome containing PKH67-labeled EVs, and the number of phagosomes was increased in 1h-cells. We added an explanation to the text to the effect that control experiment showed no phagosome containing PKH67-labeled EVs (Fig. 3a) (line 139). We also added "0 h" to Figure 3a and the legend (line 154).
Reviewer’s comments 3
#3. The RT-PCR results showed that the hand-squeezed juice contained more miRNAs in its EVs than the commercial juice. Conversely, in Fig. 1d, the number of EVs in commercial grapefruit juice was about four times the number of EVs in hand-squeezed paprika juice. If the authors claimed that these miRNAs are ubiquitous, they should explain why there is such a discrepancy between the number of EVs and the amount of miRNAs in those EVs. Please add the number of EVs of hand-squeezed grapefruit juice to Figure 1d.
Answer:
The concentration of SJ-grapefruits juice was 339.6 x 108 /mL. It was lower than that of commercial grapefruits juice (please find an attached file). We think EVs of commercial grapefruits juice and SJ-paprika juice contained pigment granules. Hence, we used filter paper to remove pigment granules through squeezing and pre-filtration. We do not think that every EV contained miRNAs, and we consider that the number of EVs did not correspond to the volume containing miRNA.
We added these points to the Discussion section as follows (line 205 - 207):
“Although the concentration of grapefruits SJ (339.6 x 108 /ml) was lower than that of grapefruits J (Fig. 1d), expression of miRNAs for grapefruits SJ were higher than that of grapefruits J (Table 1).”
Reviewer’s comments 4
#4. From the result of table 2, EVs derived from hand-squeezed grapefruit juice were taken up by IEC6 cells. Have you checked to see if EVs taken from commercial juices are taken up by IEC6 cells? Please add data to see if there was any difference in the uptake capability.
Answer:
Thank you for your comments. We did not check the differences in uptake capability. We conducted the uptaking experiments using IEC6 cells for grapefruits SJ because their expression of miRNAs was higher than that of grapefruits J, as shown in Table 1. We will evaluate uptake capability and its effects on enterocytes in our subsequent work. Thank you for the suggestion.
Reviewer’s comments 5
#5. Although the authors discussed the limitation of this manuscript, it should be confirmed the characterization of the collected EVs by western blotting. International Society of Extracellular Vesicles has published a position paper: Minimal information for studies of extracellular vesicles 2018 (MISEV2018). It is stated that one should always show that the isolated EVs contain EV specific protein. Refs. 8 and 19 in the references are papers on Sunflowers and Arabidopsis. Therefore, it is very informative to show expression of these protein levels in plant juice-derived EVs.
Answer:
Thank you for your comments. We tried to perform western blots using the plant EV markers TET-8 and Rab 11 as previously described (8). However, because of animal antigens, we did not observe any signal. We think that antibodies suitable for plant EVs are not commercially available. We are preparing antibodies against some plant EVs, and we will show the results of grapefruit EVs in a future publication.
We modified the text regarding this point in the Discussion section as follows (lines 248 - 252):
“First, we did not use any specific plant EV marker, such as Rab11a and TET8,9 that have been suggested in previous studies [8,22], nor detected the expression of proteins. We mainly detected plant EVs based on morphology. Further experiments are needed to more easily observe plant EVs.”
Reviewer’s comments 6
#6. Generally speaking, if EVs are co-cultured with cells, a certain amount will be taken up by endocytosis or phagocytosis. If the authors assume that enterocytes can take up EVs because of their ability to absorb nutrients, they need to confirm that EVs accumulate specifically in enterocytes in animal experiments with oral administration.
Answer:
Thank you for your comments. Of course, your opinion is valid. The subject of this project was to perform in vitro experiments before the animal experiments. We are now designing in vitro experiments as the next project. We now mention this point in the Discussion section as follows (lines 228 - 230):
“Further studies, especially using in vivo systems, are needed to demonstrate the fate of EVs incorporated into enterocytes.”
Reviewer’s comments 7
#7. Please add the scale size in figure legends 1f-h.
Answer:
Thank you for your comments. As suggested, we added scale bars to Figure 1f-1h and in the legend as follows (line 91):
“Scale bars: f, 667 nm; g, 600 nm; h, 750 nm.”
Reviewer 2
In this manuscript, the authors focused on plant juice-derived exosome like nanoparticles, which are taken up by enterocyte. Although Plant-derived exosomes are attracting attention as carriers for drug delivery, their biological significance is still poorly understood. So, what the authors are arguing in this paper is very important. However, the data in this manuscript are poor and there is little novelty in this paper. There are several concerns which authors should address before they publish this paper.
Answer:
We appreciate the valuable comments of the reviewer and agree with them. Here we address the comments one by one:
Reviewer’s comments 1 and 2
Firstly, the figures were rough. It should be changed to something more clear. Furthermore, the authors described that EVs were localized in the cytoplasm, especially in the perinuclear area of IEC6 cells; however, the nucleus in these figure is difficult to recognize. Co-staining of EVs with the nucleus is necessary.
#1. In figure 3a-c, the uptake of EVs labeled with PKH into rat IEC6 cells was shown using fluorescence microscopy.
#2. In addition to #1, in figure 3a, how many hours after the addition of EVs is this photograph taken? The morphology of the cytoplasm looks very different compared to Fig. 3b at 1 hour after EV addition. The authors should explain this difference between figure 3a and 3b.
Answer:
Thank you for your valuable comments. IEC6 cells are shown with an overlay, differential interference contrast (DIC), and fluorescence from PKH67.
We indicate the nuclei with black arrows in Figs.3a ~ 3c, and an enlarged view was added to Fig. 3c. We also added the following sentence to the figure legend (lines 156 -157): “ Black arrows: nuclei of IEC cells are shown with differential interference contrast (DIC). The inset in c shows an enlarged boxed area.”
We think that the IEC6 cells in Fig. 3a had no phagosome containing PKH67-labeled EVs, and the number of phagosomes was increased in 1h-cells. We added an explanation to the text to the effect that control experiment showed no phagosome containing PKH67-labeled EVs (Fig. 3a) (line 139). We also added "0 h" to Figure 3a and the legend (line 154).
Reviewer’s comments 3
#3. The RT-PCR results showed that the hand-squeezed juice contained more miRNAs in its EVs than the commercial juice. Conversely, in Fig. 1d, the number of EVs in commercial grapefruit juice was about four times the number of EVs in hand-squeezed paprika juice. If the authors claimed that these miRNAs are ubiquitous, they should explain why there is such a discrepancy between the number of EVs and the amount of miRNAs in those EVs. Please add the number of EVs of hand-squeezed grapefruit juice to Figure 1d.
Answer:
The concentration of SJ-grapefruits juice was 339.6 x 108 /mL. It was lower than that of commercial grapefruits juice (please find an attached file). We think EVs of commercial grapefruits juice and SJ-paprika juice contained pigment granules. Hence, we used filter paper to remove pigment granules through squeezing and pre-filtration. We do not think that every EV contained miRNAs, and we consider that the number of EVs did not correspond to the volume containing miRNA.
We added these points to the Discussion section as follows (line 205 - 207):
“Although the concentration of grapefruits SJ (339.6 x 108 /ml) was lower than that of grapefruits J (Fig. 1d), expression of miRNAs for grapefruits SJ were higher than that of grapefruits J (Table 1).”
Reviewer’s comments 4
#4. From the result of table 2, EVs derived from hand-squeezed grapefruit juice were taken up by IEC6 cells. Have you checked to see if EVs taken from commercial juices are taken up by IEC6 cells? Please add data to see if there was any difference in the uptake capability.
Answer:
Thank you for your comments. We did not check the differences in uptake capability. We conducted the uptaking experiments using IEC6 cells for grapefruits SJ because their expression of miRNAs was higher than that of grapefruits J, as shown in Table 1. We will evaluate uptake capability and its effects on enterocytes in our subsequent work. Thank you for the suggestion.
Reviewer’s comments 5
#5. Although the authors discussed the limitation of this manuscript, it should be confirmed the characterization of the collected EVs by western blotting. International Society of Extracellular Vesicles has published a position paper: Minimal information for studies of extracellular vesicles 2018 (MISEV2018). It is stated that one should always show that the isolated EVs contain EV specific protein. Refs. 8 and 19 in the references are papers on Sunflowers and Arabidopsis. Therefore, it is very informative to show expression of these protein levels in plant juice-derived EVs.
Answer:
Thank you for your comments. We tried to perform western blots using the plant EV markers TET-8 and Rab 11 as previously described (8). However, because of animal antigens, we did not observe any signal. We think that antibodies suitable for plant EVs are not commercially available. We are preparing antibodies against some plant EVs, and we will show the results of grapefruit EVs in a future publication.
We modified the text regarding this point in the Discussion section as follows (lines 248 - 252):
“First, we did not use any specific plant EV marker, such as Rab11a and TET8,9 that have been suggested in previous studies [8,22], nor detected the expression of proteins. We mainly detected plant EVs based on morphology. Further experiments are needed to more easily observe plant EVs.”
Reviewer’s comments 6
#6. Generally speaking, if EVs are co-cultured with cells, a certain amount will be taken up by endocytosis or phagocytosis. If the authors assume that enterocytes can take up EVs because of their ability to absorb nutrients, they need to confirm that EVs accumulate specifically in enterocytes in animal experiments with oral administration.
Answer:
Thank you for your comments. Of course, your opinion is valid. The subject of this project was to perform in vitro experiments before the animal experiments. We are now designing in vitro experiments as the next project. We now mention this point in the Discussion section as follows (lines 228 - 230):
“Further studies, especially using in vivo systems, are needed to demonstrate the fate of EVs incorporated into enterocytes.”
Reviewer’s comments 7
#7. Please add the scale size in figure legends 1f-h.
Answer:
Thank you for your comments. As suggested, we added scale bars to Figure 1f-1h and in the legend as follows (line 91):
“Scale bars: f, 667 nm; g, 600 nm; h, 750 nm.”
Reviewer 2
In this manuscript, the authors focused on plant juice-derived exosome like nanoparticles, which are taken up by enterocyte. Although Plant-derived exosomes are attracting attention as carriers for drug delivery, their biological significance is still poorly understood. So, what the authors are arguing in this paper is very important. However, the data in this manuscript are poor and there is little novelty in this paper. There are several concerns which authors should address before they publish this paper.
Answer:
We appreciate the valuable comments of the reviewer and agree with them. Here we address the comments one by one:
Reviewer’s comments 1 and 2
Firstly, the figures were rough. It should be changed to something more clear. Furthermore, the authors described that EVs were localized in the cytoplasm, especially in the perinuclear area of IEC6 cells; however, the nucleus in these figure is difficult to recognize. Co-staining of EVs with the nucleus is necessary.
#1. In figure 3a-c, the uptake of EVs labeled with PKH into rat IEC6 cells was shown using fluorescence microscopy.
#2. In addition to #1, in figure 3a, how many hours after the addition of EVs is this photograph taken? The morphology of the cytoplasm looks very different compared to Fig. 3b at 1 hour after EV addition. The authors should explain this difference between figure 3a and 3b.
Answer:
Thank you for your valuable comments. IEC6 cells are shown with an overlay, differential interference contrast (DIC), and fluorescence from PKH67.
We indicate the nuclei with black arrows in Figs.3a ~ 3c, and an enlarged view was added to Fig. 3c. We also added the following sentence to the figure legend (lines 156 -157): “ Black arrows: nuclei of IEC cells are shown with differential interference contrast (DIC). The inset in c shows an enlarged boxed area.”
We think that the IEC6 cells in Fig. 3a had no phagosome containing PKH67-labeled EVs, and the number of phagosomes was increased in 1h-cells. We added an explanation to the text to the effect that control experiment showed no phagosome containing PKH67-labeled EVs (Fig. 3a) (line 139). We also added "0 h" to Figure 3a and the legend (line 154).
Reviewer’s comments 3
#3. The RT-PCR results showed that the hand-squeezed juice contained more miRNAs in its EVs than the commercial juice. Conversely, in Fig. 1d, the number of EVs in commercial grapefruit juice was about four times the number of EVs in hand-squeezed paprika juice. If the authors claimed that these miRNAs are ubiquitous, they should explain why there is such a discrepancy between the number of EVs and the amount of miRNAs in those EVs. Please add the number of EVs of hand-squeezed grapefruit juice to Figure 1d.
Answer:
The concentration of SJ-grapefruits juice was 339.6 x 108 /mL. It was lower than that of commercial grapefruits juice (please find an attached file). We think EVs of commercial grapefruits juice and SJ-paprika juice contained pigment granules. Hence, we used filter paper to remove pigment granules through squeezing and pre-filtration. We do not think that every EV contained miRNAs, and we consider that the number of EVs did not correspond to the volume containing miRNA.
We added these points to the Discussion section as follows (line 205 - 207):
“Although the concentration of grapefruits SJ (339.6 x 108 /ml) was lower than that of grapefruits J (Fig. 1d), expression of miRNAs for grapefruits SJ were higher than that of grapefruits J (Table 1).”
Reviewer’s comments 4
#4. From the result of table 2, EVs derived from hand-squeezed grapefruit juice were taken up by IEC6 cells. Have you checked to see if EVs taken from commercial juices are taken up by IEC6 cells? Please add data to see if there was any difference in the uptake capability.
Answer:
Thank you for your comments. We did not check the differences in uptake capability. We conducted the uptaking experiments using IEC6 cells for grapefruits SJ because their expression of miRNAs was higher than that of grapefruits J, as shown in Table 1. We will evaluate uptake capability and its effects on enterocytes in our subsequent work. Thank you for the suggestion.
Reviewer’s comments 5
#5. Although the authors discussed the limitation of this manuscript, it should be confirmed the characterization of the collected EVs by western blotting. International Society of Extracellular Vesicles has published a position paper: Minimal information for studies of extracellular vesicles 2018 (MISEV2018). It is stated that one should always show that the isolated EVs contain EV specific protein. Refs. 8 and 19 in the references are papers on Sunflowers and Arabidopsis. Therefore, it is very informative to show expression of these protein levels in plant juice-derived EVs.
Answer:
Thank you for your comments. We tried to perform western blots using the plant EV markers TET-8 and Rab 11 as previously described (8). However, because of animal antigens, we did not observe any signal. We think that antibodies suitable for plant EVs are not commercially available. We are preparing antibodies against some plant EVs, and we will show the results of grapefruit EVs in a future publication.
We modified the text regarding this point in the Discussion section as follows (lines 248 - 252):
“First, we did not use any specific plant EV marker, such as Rab11a and TET8,9 that have been suggested in previous studies [8,22], nor detected the expression of proteins. We mainly detected plant EVs based on morphology. Further experiments are needed to more easily observe plant EVs.”
Reviewer’s comments 6
#6. Generally speaking, if EVs are co-cultured with cells, a certain amount will be taken up by endocytosis or phagocytosis. If the authors assume that enterocytes can take up EVs because of their ability to absorb nutrients, they need to confirm that EVs accumulate specifically in enterocytes in animal experiments with oral administration.
Answer:
Thank you for your comments. Of course, your opinion is valid. The subject of this project was to perform in vitro experiments before the animal experiments. We are now designing in vitro experiments as the next project. We now mention this point in the Discussion section as follows (lines 228 - 230):
“Further studies, especially using in vivo systems, are needed to demonstrate the fate of EVs incorporated into enterocytes.”
Reviewer’s comments 7
#7. Please add the scale size in figure legends 1f-h.
Answer:
Thank you for your comments. As suggested, we added scale bars to Figure 1f-1h and in the legend as follows (line 91):
“Scale bars: f, 667 nm; g, 600 nm; h, 750 nm.”
Reviewer 2
In this manuscript, the authors focused on plant juice-derived exosome like nanoparticles, which are taken up by enterocyte. Although Plant-derived exosomes are attracting attention as carriers for drug delivery, their biological significance is still poorly understood. So, what the authors are arguing in this paper is very important. However, the data in this manuscript are poor and there is little novelty in this paper. There are several concerns which authors should address before they publish this paper.
Answer:
We appreciate the valuable comments of the reviewer and agree with them. Here we address the comments one by one:
Reviewer’s comments 1 and 2
Firstly, the figures were rough. It should be changed to something more clear. Furthermore, the authors described that EVs were localized in the cytoplasm, especially in the perinuclear area of IEC6 cells; however, the nucleus in these figure is difficult to recognize. Co-staining of EVs with the nucleus is necessary.
#1. In figure 3a-c, the uptake of EVs labeled with PKH into rat IEC6 cells was shown using fluorescence microscopy.
#2. In addition to #1, in figure 3a, how many hours after the addition of EVs is this photograph taken? The morphology of the cytoplasm looks very different compared to Fig. 3b at 1 hour after EV addition. The authors should explain this difference between figure 3a and 3b.
Answer:
Thank you for your valuable comments. IEC6 cells are shown with an overlay, differential interference contrast (DIC), and fluorescence from PKH67.
We indicate the nuclei with black arrows in Figs.3a ~ 3c, and an enlarged view was added to Fig. 3c. We also added the following sentence to the figure legend (lines 156 -157): “ Black arrows: nuclei of IEC cells are shown with differential interference contrast (DIC). The inset in c shows an enlarged boxed area.”
We think that the IEC6 cells in Fig. 3a had no phagosome containing PKH67-labeled EVs, and the number of phagosomes was increased in 1h-cells. We added an explanation to the text to the effect that control experiment showed no phagosome containing PKH67-labeled EVs (Fig. 3a) (line 139). We also added "0 h" to Figure 3a and the legend (line 154).
Reviewer’s comments 3
#3. The RT-PCR results showed that the hand-squeezed juice contained more miRNAs in its EVs than the commercial juice. Conversely, in Fig. 1d, the number of EVs in commercial grapefruit juice was about four times the number of EVs in hand-squeezed paprika juice. If the authors claimed that these miRNAs are ubiquitous, they should explain why there is such a discrepancy between the number of EVs and the amount of miRNAs in those EVs. Please add the number of EVs of hand-squeezed grapefruit juice to Figure 1d.
Answer:
The concentration of SJ-grapefruits juice was 339.6 x 108 /mL. It was lower than that of commercial grapefruits juice (please find an attached file). We think EVs of commercial grapefruits juice and SJ-paprika juice contained pigment granules. Hence, we used filter paper to remove pigment granules through squeezing and pre-filtration. We do not think that every EV contained miRNAs, and we consider that the number of EVs did not correspond to the volume containing miRNA.
We added these points to the Discussion section as follows (line 205 - 207):
“Although the concentration of grapefruits SJ (339.6 x 108 /ml) was lower than that of grapefruits J (Fig. 1d), expression of miRNAs for grapefruits SJ were higher than that of grapefruits J (Table 1).”
Reviewer’s comments 4
#4. From the result of table 2, EVs derived from hand-squeezed grapefruit juice were taken up by IEC6 cells. Have you checked to see if EVs taken from commercial juices are taken up by IEC6 cells? Please add data to see if there was any difference in the uptake capability.
Answer:
Thank you for your comments. We did not check the differences in uptake capability. We conducted the uptaking experiments using IEC6 cells for grapefruits SJ because their expression of miRNAs was higher than that of grapefruits J, as shown in Table 1. We will evaluate uptake capability and its effects on enterocytes in our subsequent work. Thank you for the suggestion.
Reviewer’s comments 5
#5. Although the authors discussed the limitation of this manuscript, it should be confirmed the characterization of the collected EVs by western blotting. International Society of Extracellular Vesicles has published a position paper: Minimal information for studies of extracellular vesicles 2018 (MISEV2018). It is stated that one should always show that the isolated EVs contain EV specific protein. Refs. 8 and 19 in the references are papers on Sunflowers and Arabidopsis. Therefore, it is very informative to show expression of these protein levels in plant juice-derived EVs.
Answer:
Thank you for your comments. We tried to perform western blots using the plant EV markers TET-8 and Rab 11 as previously described (8). However, because of animal antigens, we did not observe any signal. We think that antibodies suitable for plant EVs are not commercially available. We are preparing antibodies against some plant EVs, and we will show the results of grapefruit EVs in a future publication.
We modified the text regarding this point in the Discussion section as follows (lines 248 - 252):
“First, we did not use any specific plant EV marker, such as Rab11a and TET8,9 that have been suggested in previous studies [8,22], nor detected the expression of proteins. We mainly detected plant EVs based on morphology. Further experiments are needed to more easily observe plant EVs.”
Reviewer’s comments 6
#6. Generally speaking, if EVs are co-cultured with cells, a certain amount will be taken up by endocytosis or phagocytosis. If the authors assume that enterocytes can take up EVs because of their ability to absorb nutrients, they need to confirm that EVs accumulate specifically in enterocytes in animal experiments with oral administration.
Answer:
Thank you for your comments. Of course, your opinion is valid. The subject of this project was to perform in vitro experiments before the animal experiments. We are now designing in vitro experiments as the next project. We now mention this point in the Discussion section as follows (lines 228 - 230):
“Further studies, especially using in vivo systems, are needed to demonstrate the fate of EVs incorporated into enterocytes.”
Reviewer’s comments 7
#7. Please add the scale size in figure legends 1f-h.
Answer:
Thank you for your comments. As suggested, we added scale bars to Figure 1f-1h and in the legend as follows (line 91):
“Scale bars: f, 667 nm; g, 600 nm; h, 750 nm.”
Reviewer 2
In this manuscript, the authors focused on plant juice-derived exosome like nanoparticles, which are taken up by enterocyte. Although Plant-derived exosomes are attracting attention as carriers for drug delivery, their biological significance is still poorly understood. So, what the authors are arguing in this paper is very important. However, the data in this manuscript are poor and there is little novelty in this paper. There are several concerns which authors should address before they publish this paper.
Answer:
We appreciate the valuable comments of the reviewer and agree with them. Here we address the comments one by one:
Reviewer’s comments 1 and 2
Firstly, the figures were rough. It should be changed to something more clear. Furthermore, the authors described that EVs were localized in the cytoplasm, especially in the perinuclear area of IEC6 cells; however, the nucleus in these figure is difficult to recognize. Co-staining of EVs with the nucleus is necessary.
#1. In figure 3a-c, the uptake of EVs labeled with PKH into rat IEC6 cells was shown using fluorescence microscopy.
#2. In addition to #1, in figure 3a, how many hours after the addition of EVs is this photograph taken? The morphology of the cytoplasm looks very different compared to Fig. 3b at 1 hour after EV addition. The authors should explain this difference between figure 3a and 3b.
Answer:
Thank you for your valuable comments. IEC6 cells are shown with an overlay, differential interference contrast (DIC), and fluorescence from PKH67.
We indicate the nuclei with black arrows in Figs.3a ~ 3c, and an enlarged view was added to Fig. 3c. We also added the following sentence to the figure legend (lines 156 -157): “ Black arrows: nuclei of IEC cells are shown with differential interference contrast (DIC). The inset in c shows an enlarged boxed area.”
We think that the IEC6 cells in Fig. 3a had no phagosome containing PKH67-labeled EVs, and the number of phagosomes was increased in 1h-cells. We added an explanation to the text to the effect that control experiment showed no phagosome containing PKH67-labeled EVs (Fig. 3a) (line 139). We also added "0 h" to Figure 3a and the legend (line 154).
Reviewer’s comments 3
#3. The RT-PCR results showed that the hand-squeezed juice contained more miRNAs in its EVs than the commercial juice. Conversely, in Fig. 1d, the number of EVs in commercial grapefruit juice was about four times the number of EVs in hand-squeezed paprika juice. If the authors claimed that these miRNAs are ubiquitous, they should explain why there is such a discrepancy between the number of EVs and the amount of miRNAs in those EVs. Please add the number of EVs of hand-squeezed grapefruit juice to Figure 1d.
Answer:
The concentration of SJ-grapefruits juice was 339.6 x 108 /mL. It was lower than that of commercial grapefruits juice (please find an attached file). We think EVs of commercial grapefruits juice and SJ-paprika juice contained pigment granules. Hence, we used filter paper to remove pigment granules through squeezing and pre-filtration. We do not think that every EV contained miRNAs, and we consider that the number of EVs did not correspond to the volume containing miRNA.
We added these points to the Discussion section as follows (line 205 - 207):
“Although the concentration of grapefruits SJ (339.6 x 108 /ml) was lower than that of grapefruits J (Fig. 1d), expression of miRNAs for grapefruits SJ were higher than that of grapefruits J (Table 1).”
Reviewer’s comments 4
#4. From the result of table 2, EVs derived from hand-squeezed grapefruit juice were taken up by IEC6 cells. Have you checked to see if EVs taken from commercial juices are taken up by IEC6 cells? Please add data to see if there was any difference in the uptake capability.
Answer:
Thank you for your comments. We did not check the differences in uptake capability. We conducted the uptaking experiments using IEC6 cells for grapefruits SJ because their expression of miRNAs was higher than that of grapefruits J, as shown in Table 1. We will evaluate uptake capability and its effects on enterocytes in our subsequent work. Thank you for the suggestion.
Reviewer’s comments 5
#5. Although the authors discussed the limitation of this manuscript, it should be confirmed the characterization of the collected EVs by western blotting. International Society of Extracellular Vesicles has published a position paper: Minimal information for studies of extracellular vesicles 2018 (MISEV2018). It is stated that one should always show that the isolated EVs contain EV specific protein. Refs. 8 and 19 in the references are papers on Sunflowers and Arabidopsis. Therefore, it is very informative to show expression of these protein levels in plant juice-derived EVs.
Answer:
Thank you for your comments. We tried to perform western blots using the plant EV markers TET-8 and Rab 11 as previously described (8). However, because of animal antigens, we did not observe any signal. We think that antibodies suitable for plant EVs are not commercially available. We are preparing antibodies against some plant EVs, and we will show the results of grapefruit EVs in a future publication.
We modified the text regarding this point in the Discussion section as follows (lines 248 - 252):
“First, we did not use any specific plant EV marker, such as Rab11a and TET8,9 that have been suggested in previous studies [8,22], nor detected the expression of proteins. We mainly detected plant EVs based on morphology. Further experiments are needed to more easily observe plant EVs.”
Reviewer’s comments 6
#6. Generally speaking, if EVs are co-cultured with cells, a certain amount will be taken up by endocytosis or phagocytosis. If the authors assume that enterocytes can take up EVs because of their ability to absorb nutrients, they need to confirm that EVs accumulate specifically in enterocytes in animal experiments with oral administration.
Answer:
Thank you for your comments. Of course, your opinion is valid. The subject of this project was to perform in vitro experiments before the animal experiments. We are now designing in vitro experiments as the next project. We now mention this point in the Discussion section as follows (lines 228 - 230):
“Further studies, especially using in vivo systems, are needed to demonstrate the fate of EVs incorporated into enterocytes.”
Reviewer’s comments 7
#7. Please add the scale size in figure legends 1f-h.
Answer:
Thank you for your comments. As suggested, we added scale bars to Figure 1f-1h and in the legend as follows (line 91):
“Scale bars: f, 667 nm; g, 600 nm; h, 750 nm.”
Reviewer 2
In this manuscript, the authors focused on plant juice-derived exosome like nanoparticles, which are taken up by enterocyte. Although Plant-derived exosomes are attracting attention as carriers for drug delivery, their biological significance is still poorly understood. So, what the authors are arguing in this paper is very important. However, the data in this manuscript are poor and there is little novelty in this paper. There are several concerns which authors should address before they publish this paper.
Answer:
We appreciate the valuable comments of the reviewer and agree with them. Here we address the comments one by one:
Reviewer’s comments 1 and 2
Firstly, the figures were rough. It should be changed to something more clear. Furthermore, the authors described that EVs were localized in the cytoplasm, especially in the perinuclear area of IEC6 cells; however, the nucleus in these figure is difficult to recognize. Co-staining of EVs with the nucleus is necessary.
#1. In figure 3a-c, the uptake of EVs labeled with PKH into rat IEC6 cells was shown using fluorescence microscopy.
#2. In addition to #1, in figure 3a, how many hours after the addition of EVs is this photograph taken? The morphology of the cytoplasm looks very different compared to Fig. 3b at 1 hour after EV addition. The authors should explain this difference between figure 3a and 3b.
Answer:
Thank you for your valuable comments. IEC6 cells are shown with an overlay, differential interference contrast (DIC), and fluorescence from PKH67.
We indicate the nuclei with black arrows in Figs.3a ~ 3c, and an enlarged view was added to Fig. 3c. We also added the following sentence to the figure legend (lines 156 -157): “ Black arrows: nuclei of IEC cells are shown with differential interference contrast (DIC). The inset in c shows an enlarged boxed area.”
We think that the IEC6 cells in Fig. 3a had no phagosome containing PKH67-labeled EVs, and the number of phagosomes was increased in 1h-cells. We added an explanation to the text to the effect that control experiment showed no phagosome containing PKH67-labeled EVs (Fig. 3a) (line 139). We also added "0 h" to Figure 3a and the legend (line 154).
Reviewer’s comments 3
#3. The RT-PCR results showed that the hand-squeezed juice contained more miRNAs in its EVs than the commercial juice. Conversely, in Fig. 1d, the number of EVs in commercial grapefruit juice was about four times the number of EVs in hand-squeezed paprika juice. If the authors claimed that these miRNAs are ubiquitous, they should explain why there is such a discrepancy between the number of EVs and the amount of miRNAs in those EVs. Please add the number of EVs of hand-squeezed grapefruit juice to Figure 1d.
Answer:
The concentration of SJ-grapefruits juice was 339.6 x 108 /mL. It was lower than that of commercial grapefruits juice (please find an attached file). We think EVs of commercial grapefruits juice and SJ-paprika juice contained pigment granules. Hence, we used filter paper to remove pigment granules through squeezing and pre-filtration. We do not think that every EV contained miRNAs, and we consider that the number of EVs did not correspond to the volume containing miRNA.
We added these points to the Discussion section as follows (line 205 - 207):
“Although the concentration of grapefruits SJ (339.6 x 108 /ml) was lower than that of grapefruits J (Fig. 1d), expression of miRNAs for grapefruits SJ were higher than that of grapefruits J (Table 1).”
Reviewer’s comments 4
#4. From the result of table 2, EVs derived from hand-squeezed grapefruit juice were taken up by IEC6 cells. Have you checked to see if EVs taken from commercial juices are taken up by IEC6 cells? Please add data to see if there was any difference in the uptake capability.
Answer:
Thank you for your comments. We did not check the differences in uptake capability. We conducted the uptaking experiments using IEC6 cells for grapefruits SJ because their expression of miRNAs was higher than that of grapefruits J, as shown in Table 1. We will evaluate uptake capability and its effects on enterocytes in our subsequent work. Thank you for the suggestion.
Reviewer’s comments 5
#5. Although the authors discussed the limitation of this manuscript, it should be confirmed the characterization of the collected EVs by western blotting. International Society of Extracellular Vesicles has published a position paper: Minimal information for studies of extracellular vesicles 2018 (MISEV2018). It is stated that one should always show that the isolated EVs contain EV specific protein. Refs. 8 and 19 in the references are papers on Sunflowers and Arabidopsis. Therefore, it is very informative to show expression of these protein levels in plant juice-derived EVs.
Answer:
Thank you for your comments. We tried to perform western blots using the plant EV markers TET-8 and Rab 11 as previously described (8). However, because of animal antigens, we did not observe any signal. We think that antibodies suitable for plant EVs are not commercially available. We are preparing antibodies against some plant EVs, and we will show the results of grapefruit EVs in a future publication.
We modified the text regarding this point in the Discussion section as follows (lines 248 - 252):
“First, we did not use any specific plant EV marker, such as Rab11a and TET8,9 that have been suggested in previous studies [8,22], nor detected the expression of proteins. We mainly detected plant EVs based on morphology. Further experiments are needed to more easily observe plant EVs.”
Reviewer’s comments 6
#6. Generally speaking, if EVs are co-cultured with cells, a certain amount will be taken up by endocytosis or phagocytosis. If the authors assume that enterocytes can take up EVs because of their ability to absorb nutrients, they need to confirm that EVs accumulate specifically in enterocytes in animal experiments with oral administration.
Answer:
Thank you for your comments. Of course, your opinion is valid. The subject of this project was to perform in vitro experiments before the animal experiments. We are now designing in vitro experiments as the next project. We now mention this point in the Discussion section as follows (lines 228 - 230):
“Further studies, especially using in vivo systems, are needed to demonstrate the fate of EVs incorporated into enterocytes.”
Reviewer’s comments 7
#7. Please add the scale size in figure legends 1f-h.
Answer:
Thank you for your comments. As suggested, we added scale bars to Figure 1f-1h and in the legend as follows (line 91):
“Scale bars: f, 667 nm; g, 600 nm; h, 750 nm.”
Reviewer 2
In this manuscript, the authors focused on plant juice-derived exosome like nanoparticles, which are taken up by enterocyte. Although Plant-derived exosomes are attracting attention as carriers for drug delivery, their biological significance is still poorly understood. So, what the authors are arguing in this paper is very important. However, the data in this manuscript are poor and there is little novelty in this paper. There are several concerns which authors should address before they publish this paper.
Answer:
We appreciate the valuable comments of the reviewer and agree with them. Here we address the comments one by one:
Reviewer’s comments 1 and 2
Firstly, the figures were rough. It should be changed to something more clear. Furthermore, the authors described that EVs were localized in the cytoplasm, especially in the perinuclear area of IEC6 cells; however, the nucleus in these figure is difficult to recognize. Co-staining of EVs with the nucleus is necessary.
#1. In figure 3a-c, the uptake of EVs labeled with PKH into rat IEC6 cells was shown using fluorescence microscopy.
#2. In addition to #1, in figure 3a, how many hours after the addition of EVs is this photograph taken? The morphology of the cytoplasm looks very different compared to Fig. 3b at 1 hour after EV addition. The authors should explain this difference between figure 3a and 3b.
Answer:
Thank you for your valuable comments. IEC6 cells are shown with an overlay, differential interference contrast (DIC), and fluorescence from PKH67.
We indicate the nuclei with black arrows in Figs.3a ~ 3c, and an enlarged view was added to Fig. 3c. We also added the following sentence to the figure legend (lines 156 -157): “ Black arrows: nuclei of IEC cells are shown with differential interference contrast (DIC). The inset in c shows an enlarged boxed area.”
We think that the IEC6 cells in Fig. 3a had no phagosome containing PKH67-labeled EVs, and the number of phagosomes was increased in 1h-cells. We added an explanation to the text to the effect that control experiment showed no phagosome containing PKH67-labeled EVs (Fig. 3a) (line 139). We also added "0 h" to Figure 3a and the legend (line 154).
Reviewer’s comments 3
#3. The RT-PCR results showed that the hand-squeezed juice contained more miRNAs in its EVs than the commercial juice. Conversely, in Fig. 1d, the number of EVs in commercial grapefruit juice was about four times the number of EVs in hand-squeezed paprika juice. If the authors claimed that these miRNAs are ubiquitous, they should explain why there is such a discrepancy between the number of EVs and the amount of miRNAs in those EVs. Please add the number of EVs of hand-squeezed grapefruit juice to Figure 1d.
Answer:
The concentration of SJ-grapefruits juice was 339.6 x 108 /mL. It was lower than that of commercial grapefruits juice (please find an attached file). We think EVs of commercial grapefruits juice and SJ-paprika juice contained pigment granules. Hence, we used filter paper to remove pigment granules through squeezing and pre-filtration. We do not think that every EV contained miRNAs, and we consider that the number of EVs did not correspond to the volume containing miRNA.
We added these points to the Discussion section as follows (line 205 - 207):
“Although the concentration of grapefruits SJ (339.6 x 108 /ml) was lower than that of grapefruits J (Fig. 1d), expression of miRNAs for grapefruits SJ were higher than that of grapefruits J (Table 1).”
Reviewer’s comments 4
#4. From the result of table 2, EVs derived from hand-squeezed grapefruit juice were taken up by IEC6 cells. Have you checked to see if EVs taken from commercial juices are taken up by IEC6 cells? Please add data to see if there was any difference in the uptake capability.
Answer:
Thank you for your comments. We did not check the differences in uptake capability. We conducted the uptaking experiments using IEC6 cells for grapefruits SJ because their expression of miRNAs was higher than that of grapefruits J, as shown in Table 1. We will evaluate uptake capability and its effects on enterocytes in our subsequent work. Thank you for the suggestion.
Reviewer’s comments 5
#5. Although the authors discussed the limitation of this manuscript, it should be confirmed the characterization of the collected EVs by western blotting. International Society of Extracellular Vesicles has published a position paper: Minimal information for studies of extracellular vesicles 2018 (MISEV2018). It is stated that one should always show that the isolated EVs contain EV specific protein. Refs. 8 and 19 in the references are papers on Sunflowers and Arabidopsis. Therefore, it is very informative to show expression of these protein levels in plant juice-derived EVs.
Answer:
Thank you for your comments. We tried to perform western blots using the plant EV markers TET-8 and Rab 11 as previously described (8). However, because of animal antigens, we did not observe any signal. We think that antibodies suitable for plant EVs are not commercially available. We are preparing antibodies against some plant EVs, and we will show the results of grapefruit EVs in a future publication.
We modified the text regarding this point in the Discussion section as follows (lines 248 - 252):
“First, we did not use any specific plant EV marker, such as Rab11a and TET8,9 that have been suggested in previous studies [8,22], nor detected the expression of proteins. We mainly detected plant EVs based on morphology. Further experiments are needed to more easily observe plant EVs.”
Reviewer’s comments 6
#6. Generally speaking, if EVs are co-cultured with cells, a certain amount will be taken up by endocytosis or phagocytosis. If the authors assume that enterocytes can take up EVs because of their ability to absorb nutrients, they need to confirm that EVs accumulate specifically in enterocytes in animal experiments with oral administration.
Answer:
Thank you for your comments. Of course, your opinion is valid. The subject of this project was to perform in vitro experiments before the animal experiments. We are now designing in vitro experiments as the next project. We now mention this point in the Discussion section as follows (lines 228 - 230):
“Further studies, especially using in vivo systems, are needed to demonstrate the fate of EVs incorporated into enterocytes.”
Reviewer’s comments 7
#7. Please add the scale size in figure legends 1f-h.
Answer:
Thank you for your comments. As suggested, we added scale bars to Figure 1f-1h and in the legend as follows (line 91):
“Scale bars: f, 667 nm; g, 600 nm; h, 750 nm.”
Reviewer 2
In this manuscript, the authors focused on plant juice-derived exosome like nanoparticles, which are taken up by enterocyte. Although Plant-derived exosomes are attracting attention as carriers for drug delivery, their biological significance is still poorly understood. So, what the authors are arguing in this paper is very important. However, the data in this manuscript are poor and there is little novelty in this paper. There are several concerns which authors should address before they publish this paper.
Answer:
We appreciate the valuable comments of the reviewer and agree with them. Here we address the comments one by one:
Reviewer’s comments 1 and 2
Firstly, the figures were rough. It should be changed to something more clear. Furthermore, the authors described that EVs were localized in the cytoplasm, especially in the perinuclear area of IEC6 cells; however, the nucleus in these figure is difficult to recognize. Co-staining of EVs with the nucleus is necessary.
#1. In figure 3a-c, the uptake of EVs labeled with PKH into rat IEC6 cells was shown using fluorescence microscopy.
#2. In addition to #1, in figure 3a, how many hours after the addition of EVs is this photograph taken? The morphology of the cytoplasm looks very different compared to Fig. 3b at 1 hour after EV addition. The authors should explain this difference between figure 3a and 3b.
Answer:
Thank you for your valuable comments. IEC6 cells are shown with an overlay, differential interference contrast (DIC), and fluorescence from PKH67.
We indicate the nuclei with black arrows in Figs.3a ~ 3c, and an enlarged view was added to Fig. 3c. We also added the following sentence to the figure legend (lines 156 -157): “ Black arrows: nuclei of IEC cells are shown with differential interference contrast (DIC). The inset in c shows an enlarged boxed area.”
We think that the IEC6 cells in Fig. 3a had no phagosome containing PKH67-labeled EVs, and the number of phagosomes was increased in 1h-cells. We added an explanation to the text to the effect that control experiment showed no phagosome containing PKH67-labeled EVs (Fig. 3a) (line 139). We also added "0 h" to Figure 3a and the legend (line 154).
Reviewer’s comments 3
#3. The RT-PCR results showed that the hand-squeezed juice contained more miRNAs in its EVs than the commercial juice. Conversely, in Fig. 1d, the number of EVs in commercial grapefruit juice was about four times the number of EVs in hand-squeezed paprika juice. If the authors claimed that these miRNAs are ubiquitous, they should explain why there is such a discrepancy between the number of EVs and the amount of miRNAs in those EVs. Please add the number of EVs of hand-squeezed grapefruit juice to Figure 1d.
Answer:
The concentration of SJ-grapefruits juice was 339.6 x 108 /mL. It was lower than that of commercial grapefruits juice (please find an attached file). We think EVs of commercial grapefruits juice and SJ-paprika juice contained pigment granules. Hence, we used filter paper to remove pigment granules through squeezing and pre-filtration. We do not think that every EV contained miRNAs, and we consider that the number of EVs did not correspond to the volume containing miRNA.
We added these points to the Discussion section as follows (line 205 - 207):
“Although the concentration of grapefruits SJ (339.6 x 108 /ml) was lower than that of grapefruits J (Fig. 1d), expression of miRNAs for grapefruits SJ were higher than that of grapefruits J (Table 1).”
Reviewer’s comments 4
#4. From the result of table 2, EVs derived from hand-squeezed grapefruit juice were taken up by IEC6 cells. Have you checked to see if EVs taken from commercial juices are taken up by IEC6 cells? Please add data to see if there was any difference in the uptake capability.
Answer:
Thank you for your comments. We did not check the differences in uptake capability. We conducted the uptaking experiments using IEC6 cells for grapefruits SJ because their expression of miRNAs was higher than that of grapefruits J, as shown in Table 1. We will evaluate uptake capability and its effects on enterocytes in our subsequent work. Thank you for the suggestion.
Reviewer’s comments 5
#5. Although the authors discussed the limitation of this manuscript, it should be confirmed the characterization of the collected EVs by western blotting. International Society of Extracellular Vesicles has published a position paper: Minimal information for studies of extracellular vesicles 2018 (MISEV2018). It is stated that one should always show that the isolated EVs contain EV specific protein. Refs. 8 and 19 in the references are papers on Sunflowers and Arabidopsis. Therefore, it is very informative to show expression of these protein levels in plant juice-derived EVs.
Answer:
Thank you for your comments. We tried to perform western blots using the plant EV markers TET-8 and Rab 11 as previously described (8). However, because of animal antigens, we did not observe any signal. We think that antibodies suitable for plant EVs are not commercially available. We are preparing antibodies against some plant EVs, and we will show the results of grapefruit EVs in a future publication.
We modified the text regarding this point in the Discussion section as follows (lines 248 - 252):
“First, we did not use any specific plant EV marker, such as Rab11a and TET8,9 that have been suggested in previous studies [8,22], nor detected the expression of proteins. We mainly detected plant EVs based on morphology. Further experiments are needed to more easily observe plant EVs.”
Reviewer’s comments 6
#6. Generally speaking, if EVs are co-cultured with cells, a certain amount will be taken up by endocytosis or phagocytosis. If the authors assume that enterocytes can take up EVs because of their ability to absorb nutrients, they need to confirm that EVs accumulate specifically in enterocytes in animal experiments with oral administration.
Answer:
Thank you for your comments. Of course, your opinion is valid. The subject of this project was to perform in vitro experiments before the animal experiments. We are now designing in vitro experiments as the next project. We now mention this point in the Discussion section as follows (lines 228 - 230):
“Further studies, especially using in vivo systems, are needed to demonstrate the fate of EVs incorporated into enterocytes.”
Reviewer’s comments 7
#7. Please add the scale size in figure legends 1f-h.
Answer:
Thank you for your comments. As suggested, we added scale bars to Figure 1f-1h and in the legend as follows (line 91):
“Scale bars: f, 667 nm; g, 600 nm; h, 750 nm.”
Reviewer 2
In this manuscript, the authors focused on plant juice-derived exosome like nanoparticles, which are taken up by enterocyte. Although Plant-derived exosomes are attracting attention as carriers for drug delivery, their biological significance is still poorly understood. So, what the authors are arguing in this paper is very important. However, the data in this manuscript are poor and there is little novelty in this paper. There are several concerns which authors should address before they publish this paper.
Answer:
We appreciate the valuable comments of the reviewer and agree with them. Here we address the comments one by one:
Reviewer’s comments 1 and 2
Firstly, the figures were rough. It should be changed to something more clear. Furthermore, the authors described that EVs were localized in the cytoplasm, especially in the perinuclear area of IEC6 cells; however, the nucleus in these figure is difficult to recognize. Co-staining of EVs with the nucleus is necessary.
#1. In figure 3a-c, the uptake of EVs labeled with PKH into rat IEC6 cells was shown using fluorescence microscopy.
#2. In addition to #1, in figure 3a, how many hours after the addition of EVs is this photograph taken? The morphology of the cytoplasm looks very different compared to Fig. 3b at 1 hour after EV addition. The authors should explain this difference between figure 3a and 3b.
Answer:
Thank you for your valuable comments. IEC6 cells are shown with an overlay, differential interference contrast (DIC), and fluorescence from PKH67.
We indicate the nuclei with black arrows in Figs.3a ~ 3c, and an enlarged view was added to Fig. 3c. We also added the following sentence to the figure legend (lines 156 -157): “ Black arrows: nuclei of IEC cells are shown with differential interference contrast (DIC). The inset in c shows an enlarged boxed area.”
We think that the IEC6 cells in Fig. 3a had no phagosome containing PKH67-labeled EVs, and the number of phagosomes was increased in 1h-cells. We added an explanation to the text to the effect that control experiment showed no phagosome containing PKH67-labeled EVs (Fig. 3a) (line 139). We also added "0 h" to Figure 3a and the legend (line 154).
Reviewer’s comments 3
#3. The RT-PCR results showed that the hand-squeezed juice contained more miRNAs in its EVs than the commercial juice. Conversely, in Fig. 1d, the number of EVs in commercial grapefruit juice was about four times the number of EVs in hand-squeezed paprika juice. If the authors claimed that these miRNAs are ubiquitous, they should explain why there is such a discrepancy between the number of EVs and the amount of miRNAs in those EVs. Please add the number of EVs of hand-squeezed grapefruit juice to Figure 1d.
Answer:
The concentration of SJ-grapefruits juice was 339.6 x 108 /mL. It was lower than that of commercial grapefruits juice (please find an attached file). We think EVs of commercial grapefruits juice and SJ-paprika juice contained pigment granules. Hence, we used filter paper to remove pigment granules through squeezing and pre-filtration. We do not think that every EV contained miRNAs, and we consider that the number of EVs did not correspond to the volume containing miRNA.
We added these points to the Discussion section as follows (line 205 - 207):
“Although the concentration of grapefruits SJ (339.6 x 108 /ml) was lower than that of grapefruits J (Fig. 1d), expression of miRNAs for grapefruits SJ were higher than that of grapefruits J (Table 1).”
Reviewer’s comments 4
#4. From the result of table 2, EVs derived from hand-squeezed grapefruit juice were taken up by IEC6 cells. Have you checked to see if EVs taken from commercial juices are taken up by IEC6 cells? Please add data to see if there was any difference in the uptake capability.
Answer:
Thank you for your comments. We did not check the differences in uptake capability. We conducted the uptaking experiments using IEC6 cells for grapefruits SJ because their expression of miRNAs was higher than that of grapefruits J, as shown in Table 1. We will evaluate uptake capability and its effects on enterocytes in our subsequent work. Thank you for the suggestion.
Reviewer’s comments 5
#5. Although the authors discussed the limitation of this manuscript, it should be confirmed the characterization of the collected EVs by western blotting. International Society of Extracellular Vesicles has published a position paper: Minimal information for studies of extracellular vesicles 2018 (MISEV2018). It is stated that one should always show that the isolated EVs contain EV specific protein. Refs. 8 and 19 in the references are papers on Sunflowers and Arabidopsis. Therefore, it is very informative to show expression of these protein levels in plant juice-derived EVs.
Answer:
Thank you for your comments. We tried to perform western blots using the plant EV markers TET-8 and Rab 11 as previously described (8). However, because of animal antigens, we did not observe any signal. We think that antibodies suitable for plant EVs are not commercially available. We are preparing antibodies against some plant EVs, and we will show the results of grapefruit EVs in a future publication.
We modified the text regarding this point in the Discussion section as follows (lines 248 - 252):
“First, we did not use any specific plant EV marker, such as Rab11a and TET8,9 that have been suggested in previous studies [8,22], nor detected the expression of proteins. We mainly detected plant EVs based on morphology. Further experiments are needed to more easily observe plant EVs.”
Reviewer’s comments 6
#6. Generally speaking, if EVs are co-cultured with cells, a certain amount will be taken up by endocytosis or phagocytosis. If the authors assume that enterocytes can take up EVs because of their ability to absorb nutrients, they need to confirm that EVs accumulate specifically in enterocytes in animal experiments with oral administration.
Answer:
Thank you for your comments. Of course, your opinion is valid. The subject of this project was to perform in vitro experiments before the animal experiments. We are now designing in vitro experiments as the next project. We now mention this point in the Discussion section as follows (lines 228 - 230):
“Further studies, especially using in vivo systems, are needed to demonstrate the fate of EVs incorporated into enterocytes.”
Reviewer’s comments 7
#7. Please add the scale size in figure legends 1f-h.
Answer:
Thank you for your comments. As suggested, we added scale bars to Figure 1f-1h and in the legend as follows (line 91):
“Scale bars: f, 667 nm; g, 600 nm; h, 750 nm.”
Reviewer 2
In this manuscript, the authors focused on plant juice-derived exosome like nanoparticles, which are taken up by enterocyte. Although Plant-derived exosomes are attracting attention as carriers for drug delivery, their biological significance is still poorly understood. So, what the authors are arguing in this paper is very important. However, the data in this manuscript are poor and there is little novelty in this paper. There are several concerns which authors should address before they publish this paper.
Answer:
We appreciate the valuable comments of the reviewer and agree with them. Here we address the comments one by one:
Reviewer’s comments 1 and 2
Firstly, the figures were rough. It should be changed to something more clear. Furthermore, the authors described that EVs were localized in the cytoplasm, especially in the perinuclear area of IEC6 cells; however, the nucleus in these figure is difficult to recognize. Co-staining of EVs with the nucleus is necessary.
#1. In figure 3a-c, the uptake of EVs labeled with PKH into rat IEC6 cells was shown using fluorescence microscopy.
#2. In addition to #1, in figure 3a, how many hours after the addition of EVs is this photograph taken? The morphology of the cytoplasm looks very different compared to Fig. 3b at 1 hour after EV addition. The authors should explain this difference between figure 3a and 3b.
Answer:
Thank you for your valuable comments. IEC6 cells are shown with an overlay, differential interference contrast (DIC), and fluorescence from PKH67.
We indicate the nuclei with black arrows in Figs.3a ~ 3c, and an enlarged view was added to Fig. 3c. We also added the following sentence to the figure legend (lines 156 -157): “ Black arrows: nuclei of IEC cells are shown with differential interference contrast (DIC). The inset in c shows an enlarged boxed area.”
We think that the IEC6 cells in Fig. 3a had no phagosome containing PKH67-labeled EVs, and the number of phagosomes was increased in 1h-cells. We added an explanation to the text to the effect that control experiment showed no phagosome containing PKH67-labeled EVs (Fig. 3a) (line 139). We also added "0 h" to Figure 3a and the legend (line 154).
Reviewer’s comments 3
#3. The RT-PCR results showed that the hand-squeezed juice contained more miRNAs in its EVs than the commercial juice. Conversely, in Fig. 1d, the number of EVs in commercial grapefruit juice was about four times the number of EVs in hand-squeezed paprika juice. If the authors claimed that these miRNAs are ubiquitous, they should explain why there is such a discrepancy between the number of EVs and the amount of miRNAs in those EVs. Please add the number of EVs of hand-squeezed grapefruit juice to Figure 1d.
Answer:
The concentration of SJ-grapefruits juice was 339.6 x 108 /mL. It was lower than that of commercial grapefruits juice (please find an attached file). We think EVs of commercial grapefruits juice and SJ-paprika juice contained pigment granules. Hence, we used filter paper to remove pigment granules through squeezing and pre-filtration. We do not think that every EV contained miRNAs, and we consider that the number of EVs did not correspond to the volume containing miRNA.
We added these points to the Discussion section as follows (line 205 - 207):
“Although the concentration of grapefruits SJ (339.6 x 108 /ml) was lower than that of grapefruits J (Fig. 1d), expression of miRNAs for grapefruits SJ were higher than that of grapefruits J (Table 1).”
Reviewer’s comments 4
#4. From the result of table 2, EVs derived from hand-squeezed grapefruit juice were taken up by IEC6 cells. Have you checked to see if EVs taken from commercial juices are taken up by IEC6 cells? Please add data to see if there was any difference in the uptake capability.
Answer:
Thank you for your comments. We did not check the differences in uptake capability. We conducted the uptaking experiments using IEC6 cells for grapefruits SJ because their expression of miRNAs was higher than that of grapefruits J, as shown in Table 1. We will evaluate uptake capability and its effects on enterocytes in our subsequent work. Thank you for the suggestion.
Reviewer’s comments 5
#5. Although the authors discussed the limitation of this manuscript, it should be confirmed the characterization of the collected EVs by western blotting. International Society of Extracellular Vesicles has published a position paper: Minimal information for studies of extracellular vesicles 2018 (MISEV2018). It is stated that one should always show that the isolated EVs contain EV specific protein. Refs. 8 and 19 in the references are papers on Sunflowers and Arabidopsis. Therefore, it is very informative to show expression of these protein levels in plant juice-derived EVs.
Answer:
Thank you for your comments. We tried to perform western blots using the plant EV markers TET-8 and Rab 11 as previously described (8). However, because of animal antigens, we did not observe any signal. We think that antibodies suitable for plant EVs are not commercially available. We are preparing antibodies against some plant EVs, and we will show the results of grapefruit EVs in a future publication.
We modified the text regarding this point in the Discussion section as follows (lines 248 - 252):
“First, we did not use any specific plant EV marker, such as Rab11a and TET8,9 that have been suggested in previous studies [8,22], nor detected the expression of proteins. We mainly detected plant EVs based on morphology. Further experiments are needed to more easily observe plant EVs.”
Reviewer’s comments 6
#6. Generally speaking, if EVs are co-cultured with cells, a certain amount will be taken up by endocytosis or phagocytosis. If the authors assume that enterocytes can take up EVs because of their ability to absorb nutrients, they need to confirm that EVs accumulate specifically in enterocytes in animal experiments with oral administration.
Answer:
Thank you for your comments. Of course, your opinion is valid. The subject of this project was to perform in vitro experiments before the animal experiments. We are now designing in vitro experiments as the next project. We now mention this point in the Discussion section as follows (lines 228 - 230):
“Further studies, especially using in vivo systems, are needed to demonstrate the fate of EVs incorporated into enterocytes.”
Reviewer’s comments 7
#7. Please add the scale size in figure legends 1f-h.
Answer:
Thank you for your comments. As suggested, we added scale bars to Figure 1f-1h and in the legend as follows (line 91):
“Scale bars: f, 667 nm; g, 600 nm; h, 750 nm.”
Reviewer 2
In this manuscript, the authors focused on plant juice-derived exosome like nanoparticles, which are taken up by enterocyte. Although Plant-derived exosomes are attracting attention as carriers for drug delivery, their biological significance is still poorly understood. So, what the authors are arguing in this paper is very important. However, the data in this manuscript are poor and there is little novelty in this paper. There are several concerns which authors should address before they publish this paper.
Answer:
We appreciate the valuable comments of the reviewer and agree with them. Here we address the comments one by one:
Reviewer’s comments 1 and 2
Firstly, the figures were rough. It should be changed to something more clear. Furthermore, the authors described that EVs were localized in the cytoplasm, especially in the perinuclear area of IEC6 cells; however, the nucleus in these figure is difficult to recognize. Co-staining of EVs with the nucleus is necessary.
#1. In figure 3a-c, the uptake of EVs labeled with PKH into rat IEC6 cells was shown using fluorescence microscopy.
#2. In addition to #1, in figure 3a, how many hours after the addition of EVs is this photograph taken? The morphology of the cytoplasm looks very different compared to Fig. 3b at 1 hour after EV addition. The authors should explain this difference between figure 3a and 3b.
Answer:
Thank you for your valuable comments. IEC6 cells are shown with an overlay, differential interference contrast (DIC), and fluorescence from PKH67.
We indicate the nuclei with black arrows in Figs.3a ~ 3c, and an enlarged view was added to Fig. 3c. We also added the following sentence to the figure legend (lines 156 -157): “ Black arrows: nuclei of IEC cells are shown with differential interference contrast (DIC). The inset in c shows an enlarged boxed area.”
We think that the IEC6 cells in Fig. 3a had no phagosome containing PKH67-labeled EVs, and the number of phagosomes was increased in 1h-cells. We added an explanation to the text to the effect that control experiment showed no phagosome containing PKH67-labeled EVs (Fig. 3a) (line 139). We also added "0 h" to Figure 3a and the legend (line 154).
Reviewer’s comments 3
#3. The RT-PCR results showed that the hand-squeezed juice contained more miRNAs in its EVs than the commercial juice. Conversely, in Fig. 1d, the number of EVs in commercial grapefruit juice was about four times the number of EVs in hand-squeezed paprika juice. If the authors claimed that these miRNAs are ubiquitous, they should explain why there is such a discrepancy between the number of EVs and the amount of miRNAs in those EVs. Please add the number of EVs of hand-squeezed grapefruit juice to Figure 1d.
Answer:
The concentration of SJ-grapefruits juice was 339.6 x 108 /mL. It was lower than that of commercial grapefruits juice (please find an attached file). We think EVs of commercial grapefruits juice and SJ-paprika juice contained pigment granules. Hence, we used filter paper to remove pigment granules through squeezing and pre-filtration. We do not think that every EV contained miRNAs, and we consider that the number of EVs did not correspond to the volume containing miRNA.
We added these points to the Discussion section as follows (line 205 - 207):
“Although the concentration of grapefruits SJ (339.6 x 108 /ml) was lower than that of grapefruits J (Fig. 1d), expression of miRNAs for grapefruits SJ were higher than that of grapefruits J (Table 1).”
Reviewer’s comments 4
#4. From the result of table 2, EVs derived from hand-squeezed grapefruit juice were taken up by IEC6 cells. Have you checked to see if EVs taken from commercial juices are taken up by IEC6 cells? Please add data to see if there was any difference in the uptake capability.
Answer:
Thank you for your comments. We did not check the differences in uptake capability. We conducted the uptaking experiments using IEC6 cells for grapefruits SJ because their expression of miRNAs was higher than that of grapefruits J, as shown in Table 1. We will evaluate uptake capability and its effects on enterocytes in our subsequent work. Thank you for the suggestion.
Reviewer’s comments 5
#5. Although the authors discussed the limitation of this manuscript, it should be confirmed the characterization of the collected EVs by western blotting. International Society of Extracellular Vesicles has published a position paper: Minimal information for studies of extracellular vesicles 2018 (MISEV2018). It is stated that one should always show that the isolated EVs contain EV specific protein. Refs. 8 and 19 in the references are papers on Sunflowers and Arabidopsis. Therefore, it is very informative to show expression of these protein levels in plant juice-derived EVs.
Answer:
Thank you for your comments. We tried to perform western blots using the plant EV markers TET-8 and Rab 11 as previously described (8). However, because of animal antigens, we did not observe any signal. We think that antibodies suitable for plant EVs are not commercially available. We are preparing antibodies against some plant EVs, and we will show the results of grapefruit EVs in a future publication.
We modified the text regarding this point in the Discussion section as follows (lines 248 - 252):
“First, we did not use any specific plant EV marker, such as Rab11a and TET8,9 that have been suggested in previous studies [8,22], nor detected the expression of proteins. We mainly detected plant EVs based on morphology. Further experiments are needed to more easily observe plant EVs.”
Reviewer’s comments 6
#6. Generally speaking, if EVs are co-cultured with cells, a certain amount will be taken up by endocytosis or phagocytosis. If the authors assume that enterocytes can take up EVs because of their ability to absorb nutrients, they need to confirm that EVs accumulate specifically in enterocytes in animal experiments with oral administration.
Answer:
Thank you for your comments. Of course, your opinion is valid. The subject of this project was to perform in vitro experiments before the animal experiments. We are now designing in vitro experiments as the next project. We now mention this point in the Discussion section as follows (lines 228 - 230):
“Further studies, especially using in vivo systems, are needed to demonstrate the fate of EVs incorporated into enterocytes.”
Reviewer’s comments 7
#7. Please add the scale size in figure legends 1f-h.
Answer:
Thank you for your comments. As suggested, we added scale bars to Figure 1f-1h and in the legend as follows (line 91):
“Scale bars: f, 667 nm; g, 600 nm; h, 750 nm.”
Reviewer 2
In this manuscript, the authors focused on plant juice-derived exosome like nanoparticles, which are taken up by enterocyte. Although Plant-derived exosomes are attracting attention as carriers for drug delivery, their biological significance is still poorly understood. So, what the authors are arguing in this paper is very important. However, the data in this manuscript are poor and there is little novelty in this paper. There are several concerns which authors should address before they publish this paper.
Answer:
We appreciate the valuable comments of the reviewer and agree with them. Here we address the comments one by one:
Reviewer’s comments 1 and 2
Firstly, the figures were rough. It should be changed to something more clear. Furthermore, the authors described that EVs were localized in the cytoplasm, especially in the perinuclear area of IEC6 cells; however, the nucleus in these figure is difficult to recognize. Co-staining of EVs with the nucleus is necessary.
#1. In figure 3a-c, the uptake of EVs labeled with PKH into rat IEC6 cells was shown using fluorescence microscopy.
#2. In addition to #1, in figure 3a, how many hours after the addition of EVs is this photograph taken? The morphology of the cytoplasm looks very different compared to Fig. 3b at 1 hour after EV addition. The authors should explain this difference between figure 3a and 3b.
Answer:
Thank you for your valuable comments. IEC6 cells are shown with an overlay, differential interference contrast (DIC), and fluorescence from PKH67.
We indicate the nuclei with black arrows in Figs.3a ~ 3c, and an enlarged view was added to Fig. 3c. We also added the following sentence to the figure legend (lines 156 -157): “ Black arrows: nuclei of IEC cells are shown with differential interference contrast (DIC). The inset in c shows an enlarged boxed area.”
We think that the IEC6 cells in Fig. 3a had no phagosome containing PKH67-labeled EVs, and the number of phagosomes was increased in 1h-cells. We added an explanation to the text to the effect that control experiment showed no phagosome containing PKH67-labeled EVs (Fig. 3a) (line 139). We also added "0 h" to Figure 3a and the legend (line 154).
Reviewer’s comments 3
#3. The RT-PCR results showed that the hand-squeezed juice contained more miRNAs in its EVs than the commercial juice. Conversely, in Fig. 1d, the number of EVs in commercial grapefruit juice was about four times the number of EVs in hand-squeezed paprika juice. If the authors claimed that these miRNAs are ubiquitous, they should explain why there is such a discrepancy between the number of EVs and the amount of miRNAs in those EVs. Please add the number of EVs of hand-squeezed grapefruit juice to Figure 1d.
Answer:
The concentration of SJ-grapefruits juice was 339.6 x 108 /mL. It was lower than that of commercial grapefruits juice (please find an attached file). We think EVs of commercial grapefruits juice and SJ-paprika juice contained pigment granules. Hence, we used filter paper to remove pigment granules through squeezing and pre-filtration. We do not think that every EV contained miRNAs, and we consider that the number of EVs did not correspond to the volume containing miRNA.
We added these points to the Discussion section as follows (line 205 - 207):
“Although the concentration of grapefruits SJ (339.6 x 108 /ml) was lower than that of grapefruits J (Fig. 1d), expression of miRNAs for grapefruits SJ were higher than that of grapefruits J (Table 1).”
Reviewer’s comments 4
#4. From the result of table 2, EVs derived from hand-squeezed grapefruit juice were taken up by IEC6 cells. Have you checked to see if EVs taken from commercial juices are taken up by IEC6 cells? Please add data to see if there was any difference in the uptake capability.
Answer:
Thank you for your comments. We did not check the differences in uptake capability. We conducted the uptaking experiments using IEC6 cells for grapefruits SJ because their expression of miRNAs was higher than that of grapefruits J, as shown in Table 1. We will evaluate uptake capability and its effects on enterocytes in our subsequent work. Thank you for the suggestion.
Reviewer’s comments 5
#5. Although the authors discussed the limitation of this manuscript, it should be confirmed the characterization of the collected EVs by western blotting. International Society of Extracellular Vesicles has published a position paper: Minimal information for studies of extracellular vesicles 2018 (MISEV2018). It is stated that one should always show that the isolated EVs contain EV specific protein. Refs. 8 and 19 in the references are papers on Sunflowers and Arabidopsis. Therefore, it is very informative to show expression of these protein levels in plant juice-derived EVs.
Answer:
Thank you for your comments. We tried to perform western blots using the plant EV markers TET-8 and Rab 11 as previously described (8). However, because of animal antigens, we did not observe any signal. We think that antibodies suitable for plant EVs are not commercially available. We are preparing antibodies against some plant EVs, and we will show the results of grapefruit EVs in a future publication.
We modified the text regarding this point in the Discussion section as follows (lines 248 - 252):
“First, we did not use any specific plant EV marker, such as Rab11a and TET8,9 that have been suggested in previous studies [8,22], nor detected the expression of proteins. We mainly detected plant EVs based on morphology. Further experiments are needed to more easily observe plant EVs.”
Reviewer’s comments 6
#6. Generally speaking, if EVs are co-cultured with cells, a certain amount will be taken up by endocytosis or phagocytosis. If the authors assume that enterocytes can take up EVs because of their ability to absorb nutrients, they need to confirm that EVs accumulate specifically in enterocytes in animal experiments with oral administration.
Answer:
Thank you for your comments. Of course, your opinion is valid. The subject of this project was to perform in vitro experiments before the animal experiments. We are now designing in vitro experiments as the next project. We now mention this point in the Discussion section as follows (lines 228 - 230):
“Further studies, especially using in vivo systems, are needed to demonstrate the fate of EVs incorporated into enterocytes.”
Reviewer’s comments 7
#7. Please add the scale size in figure legends 1f-h.
Answer:
Thank you for your comments. As suggested, we added scale bars to Figure 1f-1h and in the legend as follows (line 91):
“Scale bars: f, 667 nm; g, 600 nm; h, 750 nm.”
Reviewer 2
In this manuscript, the authors focused on plant juice-derived exosome like nanoparticles, which are taken up by enterocyte. Although Plant-derived exosomes are attracting attention as carriers for drug delivery, their biological significance is still poorly understood. So, what the authors are arguing in this paper is very important. However, the data in this manuscript are poor and there is little novelty in this paper. There are several concerns which authors should address before they publish this paper.
Answer:
We appreciate the valuable comments of the reviewer and agree with them. Here we address the comments one by one:
Reviewer’s comments 1 and 2
Firstly, the figures were rough. It should be changed to something more clear. Furthermore, the authors described that EVs were localized in the cytoplasm, especially in the perinuclear area of IEC6 cells; however, the nucleus in these figure is difficult to recognize. Co-staining of EVs with the nucleus is necessary.
#1. In figure 3a-c, the uptake of EVs labeled with PKH into rat IEC6 cells was shown using fluorescence microscopy.
#2. In addition to #1, in figure 3a, how many hours after the addition of EVs is this photograph taken? The morphology of the cytoplasm looks very different compared to Fig. 3b at 1 hour after EV addition. The authors should explain this difference between figure 3a and 3b.
Answer:
Thank you for your valuable comments. IEC6 cells are shown with an overlay, differential interference contrast (DIC), and fluorescence from PKH67.
We indicate the nuclei with black arrows in Figs.3a ~ 3c, and an enlarged view was added to Fig. 3c. We also added the following sentence to the figure legend (lines 156 -157): “ Black arrows: nuclei of IEC cells are shown with differential interference contrast (DIC). The inset in c shows an enlarged boxed area.”
We think that the IEC6 cells in Fig. 3a had no phagosome containing PKH67-labeled EVs, and the number of phagosomes was increased in 1h-cells. We added an explanation to the text to the effect that control experiment showed no phagosome containing PKH67-labeled EVs (Fig. 3a) (line 139). We also added "0 h" to Figure 3a and the legend (line 154).
Reviewer’s comments 3
#3. The RT-PCR results showed that the hand-squeezed juice contained more miRNAs in its EVs than the commercial juice. Conversely, in Fig. 1d, the number of EVs in commercial grapefruit juice was about four times the number of EVs in hand-squeezed paprika juice. If the authors claimed that these miRNAs are ubiquitous, they should explain why there is such a discrepancy between the number of EVs and the amount of miRNAs in those EVs. Please add the number of EVs of hand-squeezed grapefruit juice to Figure 1d.
Answer:
The concentration of SJ-grapefruits juice was 339.6 x 108 /mL. It was lower than that of commercial grapefruits juice (please find an attached file). We think EVs of commercial grapefruits juice and SJ-paprika juice contained pigment granules. Hence, we used filter paper to remove pigment granules through squeezing and pre-filtration. We do not think that every EV contained miRNAs, and we consider that the number of EVs did not correspond to the volume containing miRNA.
We added these points to the Discussion section as follows (line 205 - 207):
“Although the concentration of grapefruits SJ (339.6 x 108 /ml) was lower than that of grapefruits J (Fig. 1d), expression of miRNAs for grapefruits SJ were higher than that of grapefruits J (Table 1).”
Reviewer’s comments 4
#4. From the result of table 2, EVs derived from hand-squeezed grapefruit juice were taken up by IEC6 cells. Have you checked to see if EVs taken from commercial juices are taken up by IEC6 cells? Please add data to see if there was any difference in the uptake capability.
Answer:
Thank you for your comments. We did not check the differences in uptake capability. We conducted the uptaking experiments using IEC6 cells for grapefruits SJ because their expression of miRNAs was higher than that of grapefruits J, as shown in Table 1. We will evaluate uptake capability and its effects on enterocytes in our subsequent work. Thank you for the suggestion.
Reviewer’s comments 5
#5. Although the authors discussed the limitation of this manuscript, it should be confirmed the characterization of the collected EVs by western blotting. International Society of Extracellular Vesicles has published a position paper: Minimal information for studies of extracellular vesicles 2018 (MISEV2018). It is stated that one should always show that the isolated EVs contain EV specific protein. Refs. 8 and 19 in the references are papers on Sunflowers and Arabidopsis. Therefore, it is very informative to show expression of these protein levels in plant juice-derived EVs.
Answer:
Thank you for your comments. We tried to perform western blots using the plant EV markers TET-8 and Rab 11 as previously described (8). However, because of animal antigens, we did not observe any signal. We think that antibodies suitable for plant EVs are not commercially available. We are preparing antibodies against some plant EVs, and we will show the results of grapefruit EVs in a future publication.
We modified the text regarding this point in the Discussion section as follows (lines 248 - 252):
“First, we did not use any specific plant EV marker, such as Rab11a and TET8,9 that have been suggested in previous studies [8,22], nor detected the expression of proteins. We mainly detected plant EVs based on morphology. Further experiments are needed to more easily observe plant EVs.”
Reviewer’s comments 6
#6. Generally speaking, if EVs are co-cultured with cells, a certain amount will be taken up by endocytosis or phagocytosis. If the authors assume that enterocytes can take up EVs because of their ability to absorb nutrients, they need to confirm that EVs accumulate specifically in enterocytes in animal experiments with oral administration.
Answer:
Thank you for your comments. Of course, your opinion is valid. The subject of this project was to perform in vitro experiments before the animal experiments. We are now designing in vitro experiments as the next project. We now mention this point in the Discussion section as follows (lines 228 - 230):
“Further studies, especially using in vivo systems, are needed to demonstrate the fate of EVs incorporated into enterocytes.”
Reviewer’s comments 7
#7. Please add the scale size in figure legends 1f-h.
Answer:
Thank you for your comments. As suggested, we added scale bars to Figure 1f-1h and in the legend as follows (line 91):
“Scale bars: f, 667 nm; g, 600 nm; h, 750 nm.”
Reviewer 2
In this manuscript, the authors focused on plant juice-derived exosome like nanoparticles, which are taken up by enterocyte. Although Plant-derived exosomes are attracting attention as carriers for drug delivery, their biological significance is still poorly understood. So, what the authors are arguing in this paper is very important. However, the data in this manuscript are poor and there is little novelty in this paper. There are several concerns which authors should address before they publish this paper.
Answer:
We appreciate the valuable comments of the reviewer and agree with them. Here we address the comments one by one:
Reviewer’s comments 1 and 2
Firstly, the figures were rough. It should be changed to something more clear. Furthermore, the authors described that EVs were localized in the cytoplasm, especially in the perinuclear area of IEC6 cells; however, the nucleus in these figure is difficult to recognize. Co-staining of EVs with the nucleus is necessary.
#1. In figure 3a-c, the uptake of EVs labeled with PKH into rat IEC6 cells was shown using fluorescence microscopy.
#2. In addition to #1, in figure 3a, how many hours after the addition of EVs is this photograph taken? The morphology of the cytoplasm looks very different compared to Fig. 3b at 1 hour after EV addition. The authors should explain this difference between figure 3a and 3b.
Answer:
Thank you for your valuable comments. IEC6 cells are shown with an overlay, differential interference contrast (DIC), and fluorescence from PKH67.
We indicate the nuclei with black arrows in Figs.3a ~ 3c, and an enlarged view was added to Fig. 3c. We also added the following sentence to the figure legend (lines 156 -157): “ Black arrows: nuclei of IEC cells are shown with differential interference contrast (DIC). The inset in c shows an enlarged boxed area.”
We think that the IEC6 cells in Fig. 3a had no phagosome containing PKH67-labeled EVs, and the number of phagosomes was increased in 1h-cells. We added an explanation to the text to the effect that control experiment showed no phagosome containing PKH67-labeled EVs (Fig. 3a) (line 139). We also added "0 h" to Figure 3a and the legend (line 154).
Reviewer’s comments 3
#3. The RT-PCR results showed that the hand-squeezed juice contained more miRNAs in its EVs than the commercial juice. Conversely, in Fig. 1d, the number of EVs in commercial grapefruit juice was about four times the number of EVs in hand-squeezed paprika juice. If the authors claimed that these miRNAs are ubiquitous, they should explain why there is such a discrepancy between the number of EVs and the amount of miRNAs in those EVs. Please add the number of EVs of hand-squeezed grapefruit juice to Figure 1d.
Answer:
The concentration of SJ-grapefruits juice was 339.6 x 108 /mL. It was lower than that of commercial grapefruits juice (please find an attached file). We think EVs of commercial grapefruits juice and SJ-paprika juice contained pigment granules. Hence, we used filter paper to remove pigment granules through squeezing and pre-filtration. We do not think that every EV contained miRNAs, and we consider that the number of EVs did not correspond to the volume containing miRNA.
We added these points to the Discussion section as follows (line 205 - 207):
“Although the concentration of grapefruits SJ (339.6 x 108 /ml) was lower than that of grapefruits J (Fig. 1d), expression of miRNAs for grapefruits SJ were higher than that of grapefruits J (Table 1).”
Reviewer’s comments 4
#4. From the result of table 2, EVs derived from hand-squeezed grapefruit juice were taken up by IEC6 cells. Have you checked to see if EVs taken from commercial juices are taken up by IEC6 cells? Please add data to see if there was any difference in the uptake capability.
Answer:
Thank you for your comments. We did not check the differences in uptake capability. We conducted the uptaking experiments using IEC6 cells for grapefruits SJ because their expression of miRNAs was higher than that of grapefruits J, as shown in Table 1. We will evaluate uptake capability and its effects on enterocytes in our subsequent work. Thank you for the suggestion.
Reviewer’s comments 5
#5. Although the authors discussed the limitation of this manuscript, it should be confirmed the characterization of the collected EVs by western blotting. International Society of Extracellular Vesicles has published a position paper: Minimal information for studies of extracellular vesicles 2018 (MISEV2018). It is stated that one should always show that the isolated EVs contain EV specific protein. Refs. 8 and 19 in the references are papers on Sunflowers and Arabidopsis. Therefore, it is very informative to show expression of these protein levels in plant juice-derived EVs.
Answer:
Thank you for your comments. We tried to perform western blots using the plant EV markers TET-8 and Rab 11 as previously described (8). However, because of animal antigens, we did not observe any signal. We think that antibodies suitable for plant EVs are not commercially available. We are preparing antibodies against some plant EVs, and we will show the results of grapefruit EVs in a future publication.
We modified the text regarding this point in the Discussion section as follows (lines 248 - 252):
“First, we did not use any specific plant EV marker, such as Rab11a and TET8,9 that have been suggested in previous studies [8,22], nor detected the expression of proteins. We mainly detected plant EVs based on morphology. Further experiments are needed to more easily observe plant EVs.”
Reviewer’s comments 6
#6. Generally speaking, if EVs are co-cultured with cells, a certain amount will be taken up by endocytosis or phagocytosis. If the authors assume that enterocytes can take up EVs because of their ability to absorb nutrients, they need to confirm that EVs accumulate specifically in enterocytes in animal experiments with oral administration.
Answer:
Thank you for your comments. Of course, your opinion is valid. The subject of this project was to perform in vitro experiments before the animal experiments. We are now designing in vitro experiments as the next project. We now mention this point in the Discussion section as follows (lines 228 - 230):
“Further studies, especially using in vivo systems, are needed to demonstrate the fate of EVs incorporated into enterocytes.”
Reviewer’s comments 7
#7. Please add the scale size in figure legends 1f-h.
Answer:
Thank you for your comments. As suggested, we added scale bars to Figure 1f-1h and in the legend as follows (line 91):
“Scale bars: f, 667 nm; g, 600 nm; h, 750 nm.”
Reviewer 2
In this manuscript, the authors focused on plant juice-derived exosome like nanoparticles, which are taken up by enterocyte. Although Plant-derived exosomes are attracting attention as carriers for drug delivery, their biological significance is still poorly understood. So, what the authors are arguing in this paper is very important. However, the data in this manuscript are poor and there is little novelty in this paper. There are several concerns which authors should address before they publish this paper.
Answer:
We appreciate the valuable comments of the reviewer and agree with them. Here we address the comments one by one:
Reviewer’s comments 1 and 2
Firstly, the figures were rough. It should be changed to something more clear. Furthermore, the authors described that EVs were localized in the cytoplasm, especially in the perinuclear area of IEC6 cells; however, the nucleus in these figure is difficult to recognize. Co-staining of EVs with the nucleus is necessary.
#1. In figure 3a-c, the uptake of EVs labeled with PKH into rat IEC6 cells was shown using fluorescence microscopy.
#2. In addition to #1, in figure 3a, how many hours after the addition of EVs is this photograph taken? The morphology of the cytoplasm looks very different compared to Fig. 3b at 1 hour after EV addition. The authors should explain this difference between figure 3a and 3b.
Answer:
Thank you for your valuable comments. IEC6 cells are shown with an overlay, differential interference contrast (DIC), and fluorescence from PKH67.
We indicate the nuclei with black arrows in Figs.3a ~ 3c, and an enlarged view was added to Fig. 3c. We also added the following sentence to the figure legend (lines 156 -157): “ Black arrows: nuclei of IEC cells are shown with differential interference contrast (DIC). The inset in c shows an enlarged boxed area.”
We think that the IEC6 cells in Fig. 3a had no phagosome containing PKH67-labeled EVs, and the number of phagosomes was increased in 1h-cells. We added an explanation to the text to the effect that control experiment showed no phagosome containing PKH67-labeled EVs (Fig. 3a) (line 139). We also added "0 h" to Figure 3a and the legend (line 154).
Reviewer’s comments 3
#3. The RT-PCR results showed that the hand-squeezed juice contained more miRNAs in its EVs than the commercial juice. Conversely, in Fig. 1d, the number of EVs in commercial grapefruit juice was about four times the number of EVs in hand-squeezed paprika juice. If the authors claimed that these miRNAs are ubiquitous, they should explain why there is such a discrepancy between the number of EVs and the amount of miRNAs in those EVs. Please add the number of EVs of hand-squeezed grapefruit juice to Figure 1d.
Answer:
The concentration of SJ-grapefruits juice was 339.6 x 108 /mL. It was lower than that of commercial grapefruits juice (please find an attached file). We think EVs of commercial grapefruits juice and SJ-paprika juice contained pigment granules. Hence, we used filter paper to remove pigment granules through squeezing and pre-filtration. We do not think that every EV contained miRNAs, and we consider that the number of EVs did not correspond to the volume containing miRNA.
We added these points to the Discussion section as follows (line 205 - 207):
“Although the concentration of grapefruits SJ (339.6 x 108 /ml) was lower than that of grapefruits J (Fig. 1d), expression of miRNAs for grapefruits SJ were higher than that of grapefruits J (Table 1).”
Reviewer’s comments 4
#4. From the result of table 2, EVs derived from hand-squeezed grapefruit juice were taken up by IEC6 cells. Have you checked to see if EVs taken from commercial juices are taken up by IEC6 cells? Please add data to see if there was any difference in the uptake capability.
Answer:
Thank you for your comments. We did not check the differences in uptake capability. We conducted the uptaking experiments using IEC6 cells for grapefruits SJ because their expression of miRNAs was higher than that of grapefruits J, as shown in Table 1. We will evaluate uptake capability and its effects on enterocytes in our subsequent work. Thank you for the suggestion.
Reviewer’s comments 5
#5. Although the authors discussed the limitation of this manuscript, it should be confirmed the characterization of the collected EVs by western blotting. International Society of Extracellular Vesicles has published a position paper: Minimal information for studies of extracellular vesicles 2018 (MISEV2018). It is stated that one should always show that the isolated EVs contain EV specific protein. Refs. 8 and 19 in the references are papers on Sunflowers and Arabidopsis. Therefore, it is very informative to show expression of these protein levels in plant juice-derived EVs.
Answer:
Thank you for your comments. We tried to perform western blots using the plant EV markers TET-8 and Rab 11 as previously described (8). However, because of animal antigens, we did not observe any signal. We think that antibodies suitable for plant EVs are not commercially available. We are preparing antibodies against some plant EVs, and we will show the results of grapefruit EVs in a future publication.
We modified the text regarding this point in the Discussion section as follows (lines 248 - 252):
“First, we did not use any specific plant EV marker, such as Rab11a and TET8,9 that have been suggested in previous studies [8,22], nor detected the expression of proteins. We mainly detected plant EVs based on morphology. Further experiments are needed to more easily observe plant EVs.”
Reviewer’s comments 6
#6. Generally speaking, if EVs are co-cultured with cells, a certain amount will be taken up by endocytosis or phagocytosis. If the authors assume that enterocytes can take up EVs because of their ability to absorb nutrients, they need to confirm that EVs accumulate specifically in enterocytes in animal experiments with oral administration.
Answer:
Thank you for your comments. Of course, your opinion is valid. The subject of this project was to perform in vitro experiments before the animal experiments. We are now designing in vitro experiments as the next project. We now mention this point in the Discussion section as follows (lines 228 - 230):
“Further studies, especially using in vivo systems, are needed to demonstrate the fate of EVs incorporated into enterocytes.”
Reviewer’s comments 7
#7. Please add the scale size in figure legends 1f-h.
Answer:
Thank you for your comments. As suggested, we added scale bars to Figure 1f-1h and in the legend as follows (line 91):
“Scale bars: f, 667 nm; g, 600 nm; h, 750 nm.”
Reviewer 3 Report
Hello, first of all I appreciated this chance to comment about your study.
#1. Anyway I seen an article about specific marker for plant EVs, they said Patellins 1-3, Clathrin heavy chain, and heat shock proteins are the marker of plant EVs. Can you try these marker or only one of these or any other exosomes marker? I think you guys showed good quality of TEM results. However If you contains some results of western blot using extracellular marker, strengthen your arguments.
#2. I had never seen any studies using same method(about isolation of exosome from plants or fruits) of yours. So, If possible, can you add the pictures of that steps, respectively?
#3. According to your Fig.1 and Fig.2 I think the main particle size of your exosome is over 150 nm. And their size distribution also desirable. But, I think the TEM results are little confused to me. Because the difference between particles seems to be lack of homogenousity. Can you explain why did you decide the particles which seems to different to be the same EVs?
#4. How about change the Table. 1 and .2 to graphs? I think schematic can improve what you intended.
Author Response
Reviewer 3
Hello, first of all I appreciated this chance to comment about your study.
Answer:
Thank you for the kind reading and valuable comments of an expert on extracellular vesicles.
Reviewer comment #1. Anyway I seen an article about specific marker for plant EVs, they said Patellins 1-3, Clathrin heavy chain, and heat shock proteins are the marker of plant EVs. Can you try these marker or only one of these or any other exosomes marker? I think you guys showed good quality of TEM results. However If you contains some results of western blot using extracellular marker, strengthen your arguments.
Answer:
Thank you for your comments. We know one of your suggested markers. We already performed western blotting for plant EV markers such as TET-8 and Rab 11 as previously described (8), but we did not detect any signals. We consider that suitable antibodies for plant EVs with cross-reactivity for grapefruits are not commercially available. We are preparing antibodies against some plant EVs, and we will show the results of grapefruit EVs in a future publication. Thank you for the kind suggestion.
Reviewer comment #2. I had never seen any studies using same method(about isolation of exosome from plants or fruits) of yours. So, If possible, can you add the pictures of that steps, respectively?
Answer:
Thank you for your comments. We used centrifugation (at 2000 ´ g for 5 min and at 100,000 ´ g for 4 h) and filtration; this method is not uncommon. However, to remove some debris, we wrapped segments of grapefruits and squeezed them by hand. We think that this filtration was effective to remove some fibrous debris and pigment granules. Therefore, we added an explanation regarding this procedure in the Materials and Methods section as follows (lines 261):
“For SJ, segments were squeezed through filter papers.”
Reviewer comment #3. According to your Fig.1 and Fig.2 I think the main particle size of your exosome is over 150 nm. And their size distribution also desirable. But, I think the TEM results are little confused to me. Because the difference between particles seems to be lack of homogenousity. Can you explain why did you decide the particles which seems to different to be the same EVs?
Answer:
Thank you for your comments. We apologize for the confusion. TEM and SEM were used to observe EV structure and sometimes to determine EV diameter (Yong C, et al., Plant extracellular vesicles. Protoplasma 2020, 2573-12). TEM, SEM, and Nano Sight are used to obtain information for different purposes because of their various capacities. We performed the usual TEM (by ultrathin sections) for Figure 1e to demonstrate that plant EVs had membranous structures similar to those in Figure 2c, 2d, and 2e. SEM pictures indicate that EVs are spherical, even though their diameter was increased by a metal coat of about 10 nm. We think that the mean diameter of EVs cannot be obtained with TEM and SEM, but the analysis with Nano Sight was suitable to determine their mean diameter and size distribution. In any case, 150 nm (as you mentioned above) was close to the value of 162 nm shown by Nano Sight analysis (Fig. 2f).
Reviewer comment #4. How about change the Table. 1 and .2 to graphs? I think schematic can improve what you intended.
Answer:
Thank you for your comments. We show RT-PCR results by threshold cycle values because we could not obtain an adequate internal control for miRNAs in plants. Threshold cycle values should be shown in tables (please, see our reference 19) in such a situation. If we show these results with graphs, threshold cycle values may be confused with the expression of miRNAs.
Reviewer 3
Hello, first of all I appreciated this chance to comment about your study.
Answer:
Thank you for the kind reading and valuable comments of an expert on extracellular vesicles.
Reviewer comment #1. Anyway I seen an article about specific marker for plant EVs, they said Patellins 1-3, Clathrin heavy chain, and heat shock proteins are the marker of plant EVs. Can you try these marker or only one of these or any other exosomes marker? I think you guys showed good quality of TEM results. However If you contains some results of western blot using extracellular marker, strengthen your arguments.
Answer:
Thank you for your comments. We know one of your suggested markers. We already performed western blotting for plant EV markers such as TET-8 and Rab 11 as previously described (8), but we did not detect any signals. We consider that suitable antibodies for plant EVs with cross-reactivity for grapefruits are not commercially available. We are preparing antibodies against some plant EVs, and we will show the results of grapefruit EVs in a future publication. Thank you for the kind suggestion.
Reviewer comment #2. I had never seen any studies using same method(about isolation of exosome from plants or fruits) of yours. So, If possible, can you add the pictures of that steps, respectively?
Answer:
Thank you for your comments. We used centrifugation (at 2000 ´ g for 5 min and at 100,000 ´ g for 4 h) and filtration; this method is not uncommon. However, to remove some debris, we wrapped segments of grapefruits and squeezed them by hand. We think that this filtration was effective to remove some fibrous debris and pigment granules. Therefore, we added an explanation regarding this procedure in the Materials and Methods section as follows (lines 261):
“For SJ, segments were squeezed through filter papers.”
Reviewer comment #3. According to your Fig.1 and Fig.2 I think the main particle size of your exosome is over 150 nm. And their size distribution also desirable. But, I think the TEM results are little confused to me. Because the difference between particles seems to be lack of homogenousity. Can you explain why did you decide the particles which seems to different to be the same EVs?
Answer:
Thank you for your comments. We apologize for the confusion. TEM and SEM were used to observe EV structure and sometimes to determine EV diameter (Yong C, et al., Plant extracellular vesicles. Protoplasma 2020, 2573-12). TEM, SEM, and Nano Sight are used to obtain information for different purposes because of their various capacities. We performed the usual TEM (by ultrathin sections) for Figure 1e to demonstrate that plant EVs had membranous structures similar to those in Figure 2c, 2d, and 2e. SEM pictures indicate that EVs are spherical, even though their diameter was increased by a metal coat of about 10 nm. We think that the mean diameter of EVs cannot be obtained with TEM and SEM, but the analysis with Nano Sight was suitable to determine their mean diameter and size distribution. In any case, 150 nm (as you mentioned above) was close to the value of 162 nm shown by Nano Sight analysis (Fig. 2f).
Reviewer comment #4. How about change the Table. 1 and .2 to graphs? I think schematic can improve what you intended.
Answer:
Thank you for your comments. We show RT-PCR results by threshold cycle values because we could not obtain an adequate internal control for miRNAs in plants. Threshold cycle values should be shown in tables (please, see our reference 19) in such a situation. If we show these results with graphs, threshold cycle values may be confused with the expression of miRNAs.
Reviewer 3
Hello, first of all I appreciated this chance to comment about your study.
Answer:
Thank you for the kind reading and valuable comments of an expert on extracellular vesicles.
Reviewer comment #1. Anyway I seen an article about specific marker for plant EVs, they said Patellins 1-3, Clathrin heavy chain, and heat shock proteins are the marker of plant EVs. Can you try these marker or only one of these or any other exosomes marker? I think you guys showed good quality of TEM results. However If you contains some results of western blot using extracellular marker, strengthen your arguments.
Answer:
Thank you for your comments. We know one of your suggested markers. We already performed western blotting for plant EV markers such as TET-8 and Rab 11 as previously described (8), but we did not detect any signals. We consider that suitable antibodies for plant EVs with cross-reactivity for grapefruits are not commercially available. We are preparing antibodies against some plant EVs, and we will show the results of grapefruit EVs in a future publication. Thank you for the kind suggestion.
Reviewer comment #2. I had never seen any studies using same method(about isolation of exosome from plants or fruits) of yours. So, If possible, can you add the pictures of that steps, respectively?
Answer:
Thank you for your comments. We used centrifugation (at 2000 ´ g for 5 min and at 100,000 ´ g for 4 h) and filtration; this method is not uncommon. However, to remove some debris, we wrapped segments of grapefruits and squeezed them by hand. We think that this filtration was effective to remove some fibrous debris and pigment granules. Therefore, we added an explanation regarding this procedure in the Materials and Methods section as follows (lines 261):
“For SJ, segments were squeezed through filter papers.”
Reviewer comment #3. According to your Fig.1 and Fig.2 I think the main particle size of your exosome is over 150 nm. And their size distribution also desirable. But, I think the TEM results are little confused to me. Because the difference between particles seems to be lack of homogenousity. Can you explain why did you decide the particles which seems to different to be the same EVs?
Answer:
Thank you for your comments. We apologize for the confusion. TEM and SEM were used to observe EV structure and sometimes to determine EV diameter (Yong C, et al., Plant extracellular vesicles. Protoplasma 2020, 2573-12). TEM, SEM, and Nano Sight are used to obtain information for different purposes because of their various capacities. We performed the usual TEM (by ultrathin sections) for Figure 1e to demonstrate that plant EVs had membranous structures similar to those in Figure 2c, 2d, and 2e. SEM pictures indicate that EVs are spherical, even though their diameter was increased by a metal coat of about 10 nm. We think that the mean diameter of EVs cannot be obtained with TEM and SEM, but the analysis with Nano Sight was suitable to determine their mean diameter and size distribution. In any case, 150 nm (as you mentioned above) was close to the value of 162 nm shown by Nano Sight analysis (Fig. 2f).
Reviewer comment #4. How about change the Table. 1 and .2 to graphs? I think schematic can improve what you intended.
Answer:
Thank you for your comments. We show RT-PCR results by threshold cycle values because we could not obtain an adequate internal control for miRNAs in plants. Threshold cycle values should be shown in tables (please, see our reference 19) in such a situation. If we show these results with graphs, threshold cycle values may be confused with the expression of miRNAs.
Reviewer 3
Hello, first of all I appreciated this chance to comment about your study.
Answer:
Thank you for the kind reading and valuable comments of an expert on extracellular vesicles.
Reviewer comment #1. Anyway I seen an article about specific marker for plant EVs, they said Patellins 1-3, Clathrin heavy chain, and heat shock proteins are the marker of plant EVs. Can you try these marker or only one of these or any other exosomes marker? I think you guys showed good quality of TEM results. However If you contains some results of western blot using extracellular marker, strengthen your arguments.
Answer:
Thank you for your comments. We know one of your suggested markers. We already performed western blotting for plant EV markers such as TET-8 and Rab 11 as previously described (8), but we did not detect any signals. We consider that suitable antibodies for plant EVs with cross-reactivity for grapefruits are not commercially available. We are preparing antibodies against some plant EVs, and we will show the results of grapefruit EVs in a future publication. Thank you for the kind suggestion.
Reviewer comment #2. I had never seen any studies using same method(about isolation of exosome from plants or fruits) of yours. So, If possible, can you add the pictures of that steps, respectively?
Answer:
Thank you for your comments. We used centrifugation (at 2000 ´ g for 5 min and at 100,000 ´ g for 4 h) and filtration; this method is not uncommon. However, to remove some debris, we wrapped segments of grapefruits and squeezed them by hand. We think that this filtration was effective to remove some fibrous debris and pigment granules. Therefore, we added an explanation regarding this procedure in the Materials and Methods section as follows (lines 261):
“For SJ, segments were squeezed through filter papers.”
Reviewer comment #3. According to your Fig.1 and Fig.2 I think the main particle size of your exosome is over 150 nm. And their size distribution also desirable. But, I think the TEM results are little confused to me. Because the difference between particles seems to be lack of homogenousity. Can you explain why did you decide the particles which seems to different to be the same EVs?
Answer:
Thank you for your comments. We apologize for the confusion. TEM and SEM were used to observe EV structure and sometimes to determine EV diameter (Yong C, et al., Plant extracellular vesicles. Protoplasma 2020, 2573-12). TEM, SEM, and Nano Sight are used to obtain information for different purposes because of their various capacities. We performed the usual TEM (by ultrathin sections) for Figure 1e to demonstrate that plant EVs had membranous structures similar to those in Figure 2c, 2d, and 2e. SEM pictures indicate that EVs are spherical, even though their diameter was increased by a metal coat of about 10 nm. We think that the mean diameter of EVs cannot be obtained with TEM and SEM, but the analysis with Nano Sight was suitable to determine their mean diameter and size distribution. In any case, 150 nm (as you mentioned above) was close to the value of 162 nm shown by Nano Sight analysis (Fig. 2f).
Reviewer comment #4. How about change the Table. 1 and .2 to graphs? I think schematic can improve what you intended.
Answer:
Thank you for your comments. We show RT-PCR results by threshold cycle values because we could not obtain an adequate internal control for miRNAs in plants. Threshold cycle values should be shown in tables (please, see our reference 19) in such a situation. If we show these results with graphs, threshold cycle values may be confused with the expression of miRNAs.
Reviewer 3
Hello, first of all I appreciated this chance to comment about your study.
Answer:
Thank you for the kind reading and valuable comments of an expert on extracellular vesicles.
Reviewer comment #1. Anyway I seen an article about specific marker for plant EVs, they said Patellins 1-3, Clathrin heavy chain, and heat shock proteins are the marker of plant EVs. Can you try these marker or only one of these or any other exosomes marker? I think you guys showed good quality of TEM results. However If you contains some results of western blot using extracellular marker, strengthen your arguments.
Answer:
Thank you for your comments. We know one of your suggested markers. We already performed western blotting for plant EV markers such as TET-8 and Rab 11 as previously described (8), but we did not detect any signals. We consider that suitable antibodies for plant EVs with cross-reactivity for grapefruits are not commercially available. We are preparing antibodies against some plant EVs, and we will show the results of grapefruit EVs in a future publication. Thank you for the kind suggestion.
Reviewer comment #2. I had never seen any studies using same method(about isolation of exosome from plants or fruits) of yours. So, If possible, can you add the pictures of that steps, respectively?
Answer:
Thank you for your comments. We used centrifugation (at 2000 ´ g for 5 min and at 100,000 ´ g for 4 h) and filtration; this method is not uncommon. However, to remove some debris, we wrapped segments of grapefruits and squeezed them by hand. We think that this filtration was effective to remove some fibrous debris and pigment granules. Therefore, we added an explanation regarding this procedure in the Materials and Methods section as follows (lines 261):
“For SJ, segments were squeezed through filter papers.”
Reviewer comment #3. According to your Fig.1 and Fig.2 I think the main particle size of your exosome is over 150 nm. And their size distribution also desirable. But, I think the TEM results are little confused to me. Because the difference between particles seems to be lack of homogenousity. Can you explain why did you decide the particles which seems to different to be the same EVs?
Answer:
Thank you for your comments. We apologize for the confusion. TEM and SEM were used to observe EV structure and sometimes to determine EV diameter (Yong C, et al., Plant extracellular vesicles. Protoplasma 2020, 2573-12). TEM, SEM, and Nano Sight are used to obtain information for different purposes because of their various capacities. We performed the usual TEM (by ultrathin sections) for Figure 1e to demonstrate that plant EVs had membranous structures similar to those in Figure 2c, 2d, and 2e. SEM pictures indicate that EVs are spherical, even though their diameter was increased by a metal coat of about 10 nm. We think that the mean diameter of EVs cannot be obtained with TEM and SEM, but the analysis with Nano Sight was suitable to determine their mean diameter and size distribution. In any case, 150 nm (as you mentioned above) was close to the value of 162 nm shown by Nano Sight analysis (Fig. 2f).
Reviewer comment #4. How about change the Table. 1 and .2 to graphs? I think schematic can improve what you intended.
Answer:
Thank you for your comments. We show RT-PCR results by threshold cycle values because we could not obtain an adequate internal control for miRNAs in plants. Threshold cycle values should be shown in tables (please, see our reference 19) in such a situation. If we show these results with graphs, threshold cycle values may be confused with the expression of miRNAs.
Reviewer 3
Hello, first of all I appreciated this chance to comment about your study.
Answer:
Thank you for the kind reading and valuable comments of an expert on extracellular vesicles.
Reviewer comment #1. Anyway I seen an article about specific marker for plant EVs, they said Patellins 1-3, Clathrin heavy chain, and heat shock proteins are the marker of plant EVs. Can you try these marker or only one of these or any other exosomes marker? I think you guys showed good quality of TEM results. However If you contains some results of western blot using extracellular marker, strengthen your arguments.
Answer:
Thank you for your comments. We know one of your suggested markers. We already performed western blotting for plant EV markers such as TET-8 and Rab 11 as previously described (8), but we did not detect any signals. We consider that suitable antibodies for plant EVs with cross-reactivity for grapefruits are not commercially available. We are preparing antibodies against some plant EVs, and we will show the results of grapefruit EVs in a future publication. Thank you for the kind suggestion.
Reviewer comment #2. I had never seen any studies using same method(about isolation of exosome from plants or fruits) of yours. So, If possible, can you add the pictures of that steps, respectively?
Answer:
Thank you for your comments. We used centrifugation (at 2000 ´ g for 5 min and at 100,000 ´ g for 4 h) and filtration; this method is not uncommon. However, to remove some debris, we wrapped segments of grapefruits and squeezed them by hand. We think that this filtration was effective to remove some fibrous debris and pigment granules. Therefore, we added an explanation regarding this procedure in the Materials and Methods section as follows (lines 261):
“For SJ, segments were squeezed through filter papers.”
Reviewer comment #3. According to your Fig.1 and Fig.2 I think the main particle size of your exosome is over 150 nm. And their size distribution also desirable. But, I think the TEM results are little confused to me. Because the difference between particles seems to be lack of homogenousity. Can you explain why did you decide the particles which seems to different to be the same EVs?
Answer:
Thank you for your comments. We apologize for the confusion. TEM and SEM were used to observe EV structure and sometimes to determine EV diameter (Yong C, et al., Plant extracellular vesicles. Protoplasma 2020, 2573-12). TEM, SEM, and Nano Sight are used to obtain information for different purposes because of their various capacities. We performed the usual TEM (by ultrathin sections) for Figure 1e to demonstrate that plant EVs had membranous structures similar to those in Figure 2c, 2d, and 2e. SEM pictures indicate that EVs are spherical, even though their diameter was increased by a metal coat of about 10 nm. We think that the mean diameter of EVs cannot be obtained with TEM and SEM, but the analysis with Nano Sight was suitable to determine their mean diameter and size distribution. In any case, 150 nm (as you mentioned above) was close to the value of 162 nm shown by Nano Sight analysis (Fig. 2f).
Reviewer comment #4. How about change the Table. 1 and .2 to graphs? I think schematic can improve what you intended.
Answer:
Thank you for your comments. We show RT-PCR results by threshold cycle values because we could not obtain an adequate internal control for miRNAs in plants. Threshold cycle values should be shown in tables (please, see our reference 19) in such a situation. If we show these results with graphs, threshold cycle values may be confused with the expression of miRNAs.
Reviewer 3
Hello, first of all I appreciated this chance to comment about your study.
Answer:
Thank you for the kind reading and valuable comments of an expert on extracellular vesicles.
Reviewer comment #1. Anyway I seen an article about specific marker for plant EVs, they said Patellins 1-3, Clathrin heavy chain, and heat shock proteins are the marker of plant EVs. Can you try these marker or only one of these or any other exosomes marker? I think you guys showed good quality of TEM results. However If you contains some results of western blot using extracellular marker, strengthen your arguments.
Answer:
Thank you for your comments. We know one of your suggested markers. We already performed western blotting for plant EV markers such as TET-8 and Rab 11 as previously described (8), but we did not detect any signals. We consider that suitable antibodies for plant EVs with cross-reactivity for grapefruits are not commercially available. We are preparing antibodies against some plant EVs, and we will show the results of grapefruit EVs in a future publication. Thank you for the kind suggestion.
Reviewer comment #2. I had never seen any studies using same method(about isolation of exosome from plants or fruits) of yours. So, If possible, can you add the pictures of that steps, respectively?
Answer:
Thank you for your comments. We used centrifugation (at 2000 ´ g for 5 min and at 100,000 ´ g for 4 h) and filtration; this method is not uncommon. However, to remove some debris, we wrapped segments of grapefruits and squeezed them by hand. We think that this filtration was effective to remove some fibrous debris and pigment granules. Therefore, we added an explanation regarding this procedure in the Materials and Methods section as follows (lines 261):
“For SJ, segments were squeezed through filter papers.”
Reviewer comment #3. According to your Fig.1 and Fig.2 I think the main particle size of your exosome is over 150 nm. And their size distribution also desirable. But, I think the TEM results are little confused to me. Because the difference between particles seems to be lack of homogenousity. Can you explain why did you decide the particles which seems to different to be the same EVs?
Answer:
Thank you for your comments. We apologize for the confusion. TEM and SEM were used to observe EV structure and sometimes to determine EV diameter (Yong C, et al., Plant extracellular vesicles. Protoplasma 2020, 2573-12). TEM, SEM, and Nano Sight are used to obtain information for different purposes because of their various capacities. We performed the usual TEM (by ultrathin sections) for Figure 1e to demonstrate that plant EVs had membranous structures similar to those in Figure 2c, 2d, and 2e. SEM pictures indicate that EVs are spherical, even though their diameter was increased by a metal coat of about 10 nm. We think that the mean diameter of EVs cannot be obtained with TEM and SEM, but the analysis with Nano Sight was suitable to determine their mean diameter and size distribution. In any case, 150 nm (as you mentioned above) was close to the value of 162 nm shown by Nano Sight analysis (Fig. 2f).
Reviewer comment #4. How about change the Table. 1 and .2 to graphs? I think schematic can improve what you intended.
Answer:
Thank you for your comments. We show RT-PCR results by threshold cycle values because we could not obtain an adequate internal control for miRNAs in plants. Threshold cycle values should be shown in tables (please, see our reference 19) in such a situation. If we show these results with graphs, threshold cycle values may be confused with the expression of miRNAs.
Reviewer 3
Hello, first of all I appreciated this chance to comment about your study.
Answer:
Thank you for the kind reading and valuable comments of an expert on extracellular vesicles.
Reviewer comment #1. Anyway I seen an article about specific marker for plant EVs, they said Patellins 1-3, Clathrin heavy chain, and heat shock proteins are the marker of plant EVs. Can you try these marker or only one of these or any other exosomes marker? I think you guys showed good quality of TEM results. However If you contains some results of western blot using extracellular marker, strengthen your arguments.
Answer:
Thank you for your comments. We know one of your suggested markers. We already performed western blotting for plant EV markers such as TET-8 and Rab 11 as previously described (8), but we did not detect any signals. We consider that suitable antibodies for plant EVs with cross-reactivity for grapefruits are not commercially available. We are preparing antibodies against some plant EVs, and we will show the results of grapefruit EVs in a future publication. Thank you for the kind suggestion.
Reviewer comment #2. I had never seen any studies using same method(about isolation of exosome from plants or fruits) of yours. So, If possible, can you add the pictures of that steps, respectively?
Answer:
Thank you for your comments. We used centrifugation (at 2000 ´ g for 5 min and at 100,000 ´ g for 4 h) and filtration; this method is not uncommon. However, to remove some debris, we wrapped segments of grapefruits and squeezed them by hand. We think that this filtration was effective to remove some fibrous debris and pigment granules. Therefore, we added an explanation regarding this procedure in the Materials and Methods section as follows (lines 261):
“For SJ, segments were squeezed through filter papers.”
Reviewer comment #3. According to your Fig.1 and Fig.2 I think the main particle size of your exosome is over 150 nm. And their size distribution also desirable. But, I think the TEM results are little confused to me. Because the difference between particles seems to be lack of homogenousity. Can you explain why did you decide the particles which seems to different to be the same EVs?
Answer:
Thank you for your comments. We apologize for the confusion. TEM and SEM were used to observe EV structure and sometimes to determine EV diameter (Yong C, et al., Plant extracellular vesicles. Protoplasma 2020, 2573-12). TEM, SEM, and Nano Sight are used to obtain information for different purposes because of their various capacities. We performed the usual TEM (by ultrathin sections) for Figure 1e to demonstrate that plant EVs had membranous structures similar to those in Figure 2c, 2d, and 2e. SEM pictures indicate that EVs are spherical, even though their diameter was increased by a metal coat of about 10 nm. We think that the mean diameter of EVs cannot be obtained with TEM and SEM, but the analysis with Nano Sight was suitable to determine their mean diameter and size distribution. In any case, 150 nm (as you mentioned above) was close to the value of 162 nm shown by Nano Sight analysis (Fig. 2f).
Reviewer comment #4. How about change the Table. 1 and .2 to graphs? I think schematic can improve what you intended.
Answer:
Thank you for your comments. We show RT-PCR results by threshold cycle values because we could not obtain an adequate internal control for miRNAs in plants. Threshold cycle values should be shown in tables (please, see our reference 19) in such a situation. If we show these results with graphs, threshold cycle values may be confused with the expression of miRNAs.
Reviewer 3
Hello, first of all I appreciated this chance to comment about your study.
Answer:
Thank you for the kind reading and valuable comments of an expert on extracellular vesicles.
Reviewer comment #1. Anyway I seen an article about specific marker for plant EVs, they said Patellins 1-3, Clathrin heavy chain, and heat shock proteins are the marker of plant EVs. Can you try these marker or only one of these or any other exosomes marker? I think you guys showed good quality of TEM results. However If you contains some results of western blot using extracellular marker, strengthen your arguments.
Answer:
Thank you for your comments. We know one of your suggested markers. We already performed western blotting for plant EV markers such as TET-8 and Rab 11 as previously described (8), but we did not detect any signals. We consider that suitable antibodies for plant EVs with cross-reactivity for grapefruits are not commercially available. We are preparing antibodies against some plant EVs, and we will show the results of grapefruit EVs in a future publication. Thank you for the kind suggestion.
Reviewer comment #2. I had never seen any studies using same method(about isolation of exosome from plants or fruits) of yours. So, If possible, can you add the pictures of that steps, respectively?
Answer:
Thank you for your comments. We used centrifugation (at 2000 ´ g for 5 min and at 100,000 ´ g for 4 h) and filtration; this method is not uncommon. However, to remove some debris, we wrapped segments of grapefruits and squeezed them by hand. We think that this filtration was effective to remove some fibrous debris and pigment granules. Therefore, we added an explanation regarding this procedure in the Materials and Methods section as follows (lines 261):
“For SJ, segments were squeezed through filter papers.”
Reviewer comment #3. According to your Fig.1 and Fig.2 I think the main particle size of your exosome is over 150 nm. And their size distribution also desirable. But, I think the TEM results are little confused to me. Because the difference between particles seems to be lack of homogenousity. Can you explain why did you decide the particles which seems to different to be the same EVs?
Answer:
Thank you for your comments. We apologize for the confusion. TEM and SEM were used to observe EV structure and sometimes to determine EV diameter (Yong C, et al., Plant extracellular vesicles. Protoplasma 2020, 2573-12). TEM, SEM, and Nano Sight are used to obtain information for different purposes because of their various capacities. We performed the usual TEM (by ultrathin sections) for Figure 1e to demonstrate that plant EVs had membranous structures similar to those in Figure 2c, 2d, and 2e. SEM pictures indicate that EVs are spherical, even though their diameter was increased by a metal coat of about 10 nm. We think that the mean diameter of EVs cannot be obtained with TEM and SEM, but the analysis with Nano Sight was suitable to determine their mean diameter and size distribution. In any case, 150 nm (as you mentioned above) was close to the value of 162 nm shown by Nano Sight analysis (Fig. 2f).
Reviewer comment #4. How about change the Table. 1 and .2 to graphs? I think schematic can improve what you intended.
Answer:
Thank you for your comments. We show RT-PCR results by threshold cycle values because we could not obtain an adequate internal control for miRNAs in plants. Threshold cycle values should be shown in tables (please, see our reference 19) in such a situation. If we show these results with graphs, threshold cycle values may be confused with the expression of miRNAs.
Reviewer 3
Hello, first of all I appreciated this chance to comment about your study.
Answer:
Thank you for the kind reading and valuable comments of an expert on extracellular vesicles.
Reviewer comment #1. Anyway I seen an article about specific marker for plant EVs, they said Patellins 1-3, Clathrin heavy chain, and heat shock proteins are the marker of plant EVs. Can you try these marker or only one of these or any other exosomes marker? I think you guys showed good quality of TEM results. However If you contains some results of western blot using extracellular marker, strengthen your arguments.
Answer:
Thank you for your comments. We know one of your suggested markers. We already performed western blotting for plant EV markers such as TET-8 and Rab 11 as previously described (8), but we did not detect any signals. We consider that suitable antibodies for plant EVs with cross-reactivity for grapefruits are not commercially available. We are preparing antibodies against some plant EVs, and we will show the results of grapefruit EVs in a future publication. Thank you for the kind suggestion.
Reviewer comment #2. I had never seen any studies using same method(about isolation of exosome from plants or fruits) of yours. So, If possible, can you add the pictures of that steps, respectively?
Answer:
Thank you for your comments. We used centrifugation (at 2000 ´ g for 5 min and at 100,000 ´ g for 4 h) and filtration; this method is not uncommon. However, to remove some debris, we wrapped segments of grapefruits and squeezed them by hand. We think that this filtration was effective to remove some fibrous debris and pigment granules. Therefore, we added an explanation regarding this procedure in the Materials and Methods section as follows (lines 261):
“For SJ, segments were squeezed through filter papers.”
Reviewer comment #3. According to your Fig.1 and Fig.2 I think the main particle size of your exosome is over 150 nm. And their size distribution also desirable. But, I think the TEM results are little confused to me. Because the difference between particles seems to be lack of homogenousity. Can you explain why did you decide the particles which seems to different to be the same EVs?
Answer:
Thank you for your comments. We apologize for the confusion. TEM and SEM were used to observe EV structure and sometimes to determine EV diameter (Yong C, et al., Plant extracellular vesicles. Protoplasma 2020, 2573-12). TEM, SEM, and Nano Sight are used to obtain information for different purposes because of their various capacities. We performed the usual TEM (by ultrathin sections) for Figure 1e to demonstrate that plant EVs had membranous structures similar to those in Figure 2c, 2d, and 2e. SEM pictures indicate that EVs are spherical, even though their diameter was increased by a metal coat of about 10 nm. We think that the mean diameter of EVs cannot be obtained with TEM and SEM, but the analysis with Nano Sight was suitable to determine their mean diameter and size distribution. In any case, 150 nm (as you mentioned above) was close to the value of 162 nm shown by Nano Sight analysis (Fig. 2f).
Reviewer comment #4. How about change the Table. 1 and .2 to graphs? I think schematic can improve what you intended.
Answer:
Thank you for your comments. We show RT-PCR results by threshold cycle values because we could not obtain an adequate internal control for miRNAs in plants. Threshold cycle values should be shown in tables (please, see our reference 19) in such a situation. If we show these results with graphs, threshold cycle values may be confused with the expression of miRNAs.
Reviewer 3
Hello, first of all I appreciated this chance to comment about your study.
Answer:
Thank you for the kind reading and valuable comments of an expert on extracellular vesicles.
Reviewer comment #1. Anyway I seen an article about specific marker for plant EVs, they said Patellins 1-3, Clathrin heavy chain, and heat shock proteins are the marker of plant EVs. Can you try these marker or only one of these or any other exosomes marker? I think you guys showed good quality of TEM results. However If you contains some results of western blot using extracellular marker, strengthen your arguments.
Answer:
Thank you for your comments. We know one of your suggested markers. We already performed western blotting for plant EV markers such as TET-8 and Rab 11 as previously described (8), but we did not detect any signals. We consider that suitable antibodies for plant EVs with cross-reactivity for grapefruits are not commercially available. We are preparing antibodies against some plant EVs, and we will show the results of grapefruit EVs in a future publication. Thank you for the kind suggestion.
Reviewer comment #2. I had never seen any studies using same method(about isolation of exosome from plants or fruits) of yours. So, If possible, can you add the pictures of that steps, respectively?
Answer:
Thank you for your comments. We used centrifugation (at 2000 ´ g for 5 min and at 100,000 ´ g for 4 h) and filtration; this method is not uncommon. However, to remove some debris, we wrapped segments of grapefruits and squeezed them by hand. We think that this filtration was effective to remove some fibrous debris and pigment granules. Therefore, we added an explanation regarding this procedure in the Materials and Methods section as follows (lines 261):
“For SJ, segments were squeezed through filter papers.”
Reviewer comment #3. According to your Fig.1 and Fig.2 I think the main particle size of your exosome is over 150 nm. And their size distribution also desirable. But, I think the TEM results are little confused to me. Because the difference between particles seems to be lack of homogenousity. Can you explain why did you decide the particles which seems to different to be the same EVs?
Answer:
Thank you for your comments. We apologize for the confusion. TEM and SEM were used to observe EV structure and sometimes to determine EV diameter (Yong C, et al., Plant extracellular vesicles. Protoplasma 2020, 2573-12). TEM, SEM, and Nano Sight are used to obtain information for different purposes because of their various capacities. We performed the usual TEM (by ultrathin sections) for Figure 1e to demonstrate that plant EVs had membranous structures similar to those in Figure 2c, 2d, and 2e. SEM pictures indicate that EVs are spherical, even though their diameter was increased by a metal coat of about 10 nm. We think that the mean diameter of EVs cannot be obtained with TEM and SEM, but the analysis with Nano Sight was suitable to determine their mean diameter and size distribution. In any case, 150 nm (as you mentioned above) was close to the value of 162 nm shown by Nano Sight analysis (Fig. 2f).
Reviewer comment #4. How about change the Table. 1 and .2 to graphs? I think schematic can improve what you intended.
Answer:
Thank you for your comments. We show RT-PCR results by threshold cycle values because we could not obtain an adequate internal control for miRNAs in plants. Threshold cycle values should be shown in tables (please, see our reference 19) in such a situation. If we show these results with graphs, threshold cycle values may be confused with the expression of miRNAs.
Reviewer 3
Hello, first of all I appreciated this chance to comment about your study.
Answer:
Thank you for the kind reading and valuable comments of an expert on extracellular vesicles.
Reviewer comment #1. Anyway I seen an article about specific marker for plant EVs, they said Patellins 1-3, Clathrin heavy chain, and heat shock proteins are the marker of plant EVs. Can you try these marker or only one of these or any other exosomes marker? I think you guys showed good quality of TEM results. However If you contains some results of western blot using extracellular marker, strengthen your arguments.
Answer:
Thank you for your comments. We know one of your suggested markers. We already performed western blotting for plant EV markers such as TET-8 and Rab 11 as previously described (8), but we did not detect any signals. We consider that suitable antibodies for plant EVs with cross-reactivity for grapefruits are not commercially available. We are preparing antibodies against some plant EVs, and we will show the results of grapefruit EVs in a future publication. Thank you for the kind suggestion.
Reviewer comment #2. I had never seen any studies using same method(about isolation of exosome from plants or fruits) of yours. So, If possible, can you add the pictures of that steps, respectively?
Answer:
Thank you for your comments. We used centrifugation (at 2000 ´ g for 5 min and at 100,000 ´ g for 4 h) and filtration; this method is not uncommon. However, to remove some debris, we wrapped segments of grapefruits and squeezed them by hand. We think that this filtration was effective to remove some fibrous debris and pigment granules. Therefore, we added an explanation regarding this procedure in the Materials and Methods section as follows (lines 261):
“For SJ, segments were squeezed through filter papers.”
Reviewer comment #3. According to your Fig.1 and Fig.2 I think the main particle size of your exosome is over 150 nm. And their size distribution also desirable. But, I think the TEM results are little confused to me. Because the difference between particles seems to be lack of homogenousity. Can you explain why did you decide the particles which seems to different to be the same EVs?
Answer:
Thank you for your comments. We apologize for the confusion. TEM and SEM were used to observe EV structure and sometimes to determine EV diameter (Yong C, et al., Plant extracellular vesicles. Protoplasma 2020, 2573-12). TEM, SEM, and Nano Sight are used to obtain information for different purposes because of their various capacities. We performed the usual TEM (by ultrathin sections) for Figure 1e to demonstrate that plant EVs had membranous structures similar to those in Figure 2c, 2d, and 2e. SEM pictures indicate that EVs are spherical, even though their diameter was increased by a metal coat of about 10 nm. We think that the mean diameter of EVs cannot be obtained with TEM and SEM, but the analysis with Nano Sight was suitable to determine their mean diameter and size distribution. In any case, 150 nm (as you mentioned above) was close to the value of 162 nm shown by Nano Sight analysis (Fig. 2f).
Reviewer comment #4. How about change the Table. 1 and .2 to graphs? I think schematic can improve what you intended.
Answer:
Thank you for your comments. We show RT-PCR results by threshold cycle values because we could not obtain an adequate internal control for miRNAs in plants. Threshold cycle values should be shown in tables (please, see our reference 19) in such a situation. If we show these results with graphs, threshold cycle values may be confused with the expression of miRNAs.
Reviewer 3
Hello, first of all I appreciated this chance to comment about your study.
Answer:
Thank you for the kind reading and valuable comments of an expert on extracellular vesicles.
Reviewer comment #1. Anyway I seen an article about specific marker for plant EVs, they said Patellins 1-3, Clathrin heavy chain, and heat shock proteins are the marker of plant EVs. Can you try these marker or only one of these or any other exosomes marker? I think you guys showed good quality of TEM results. However If you contains some results of western blot using extracellular marker, strengthen your arguments.
Answer:
Thank you for your comments. We know one of your suggested markers. We already performed western blotting for plant EV markers such as TET-8 and Rab 11 as previously described (8), but we did not detect any signals. We consider that suitable antibodies for plant EVs with cross-reactivity for grapefruits are not commercially available. We are preparing antibodies against some plant EVs, and we will show the results of grapefruit EVs in a future publication. Thank you for the kind suggestion.
Reviewer comment #2. I had never seen any studies using same method(about isolation of exosome from plants or fruits) of yours. So, If possible, can you add the pictures of that steps, respectively?
Answer:
Thank you for your comments. We used centrifugation (at 2000 ´ g for 5 min and at 100,000 ´ g for 4 h) and filtration; this method is not uncommon. However, to remove some debris, we wrapped segments of grapefruits and squeezed them by hand. We think that this filtration was effective to remove some fibrous debris and pigment granules. Therefore, we added an explanation regarding this procedure in the Materials and Methods section as follows (lines 261):
“For SJ, segments were squeezed through filter papers.”
Reviewer comment #3. According to your Fig.1 and Fig.2 I think the main particle size of your exosome is over 150 nm. And their size distribution also desirable. But, I think the TEM results are little confused to me. Because the difference between particles seems to be lack of homogenousity. Can you explain why did you decide the particles which seems to different to be the same EVs?
Answer:
Thank you for your comments. We apologize for the confusion. TEM and SEM were used to observe EV structure and sometimes to determine EV diameter (Yong C, et al., Plant extracellular vesicles. Protoplasma 2020, 2573-12). TEM, SEM, and Nano Sight are used to obtain information for different purposes because of their various capacities. We performed the usual TEM (by ultrathin sections) for Figure 1e to demonstrate that plant EVs had membranous structures similar to those in Figure 2c, 2d, and 2e. SEM pictures indicate that EVs are spherical, even though their diameter was increased by a metal coat of about 10 nm. We think that the mean diameter of EVs cannot be obtained with TEM and SEM, but the analysis with Nano Sight was suitable to determine their mean diameter and size distribution. In any case, 150 nm (as you mentioned above) was close to the value of 162 nm shown by Nano Sight analysis (Fig. 2f).
Reviewer comment #4. How about change the Table. 1 and .2 to graphs? I think schematic can improve what you intended.
Answer:
Thank you for your comments. We show RT-PCR results by threshold cycle values because we could not obtain an adequate internal control for miRNAs in plants. Threshold cycle values should be shown in tables (please, see our reference 19) in such a situation. If we show these results with graphs, threshold cycle values may be confused with the expression of miRNAs.
Reviewer 3
Hello, first of all I appreciated this chance to comment about your study.
Answer:
Thank you for the kind reading and valuable comments of an expert on extracellular vesicles.
Reviewer comment #1. Anyway I seen an article about specific marker for plant EVs, they said Patellins 1-3, Clathrin heavy chain, and heat shock proteins are the marker of plant EVs. Can you try these marker or only one of these or any other exosomes marker? I think you guys showed good quality of TEM results. However If you contains some results of western blot using extracellular marker, strengthen your arguments.
Answer:
Thank you for your comments. We know one of your suggested markers. We already performed western blotting for plant EV markers such as TET-8 and Rab 11 as previously described (8), but we did not detect any signals. We consider that suitable antibodies for plant EVs with cross-reactivity for grapefruits are not commercially available. We are preparing antibodies against some plant EVs, and we will show the results of grapefruit EVs in a future publication. Thank you for the kind suggestion.
Reviewer comment #2. I had never seen any studies using same method(about isolation of exosome from plants or fruits) of yours. So, If possible, can you add the pictures of that steps, respectively?
Answer:
Thank you for your comments. We used centrifugation (at 2000 ´ g for 5 min and at 100,000 ´ g for 4 h) and filtration; this method is not uncommon. However, to remove some debris, we wrapped segments of grapefruits and squeezed them by hand. We think that this filtration was effective to remove some fibrous debris and pigment granules. Therefore, we added an explanation regarding this procedure in the Materials and Methods section as follows (lines 261):
“For SJ, segments were squeezed through filter papers.”
Reviewer comment #3. According to your Fig.1 and Fig.2 I think the main particle size of your exosome is over 150 nm. And their size distribution also desirable. But, I think the TEM results are little confused to me. Because the difference between particles seems to be lack of homogenousity. Can you explain why did you decide the particles which seems to different to be the same EVs?
Answer:
Thank you for your comments. We apologize for the confusion. TEM and SEM were used to observe EV structure and sometimes to determine EV diameter (Yong C, et al., Plant extracellular vesicles. Protoplasma 2020, 2573-12). TEM, SEM, and Nano Sight are used to obtain information for different purposes because of their various capacities. We performed the usual TEM (by ultrathin sections) for Figure 1e to demonstrate that plant EVs had membranous structures similar to those in Figure 2c, 2d, and 2e. SEM pictures indicate that EVs are spherical, even though their diameter was increased by a metal coat of about 10 nm. We think that the mean diameter of EVs cannot be obtained with TEM and SEM, but the analysis with Nano Sight was suitable to determine their mean diameter and size distribution. In any case, 150 nm (as you mentioned above) was close to the value of 162 nm shown by Nano Sight analysis (Fig. 2f).
Reviewer comment #4. How about change the Table. 1 and .2 to graphs? I think schematic can improve what you intended.
Answer:
Thank you for your comments. We show RT-PCR results by threshold cycle values because we could not obtain an adequate internal control for miRNAs in plants. Threshold cycle values should be shown in tables (please, see our reference 19) in such a situation. If we show these results with graphs, threshold cycle values may be confused with the expression of miRNAs.
Reviewer 3
Hello, first of all I appreciated this chance to comment about your study.
Answer:
Thank you for the kind reading and valuable comments of an expert on extracellular vesicles.
Reviewer comment #1. Anyway I seen an article about specific marker for plant EVs, they said Patellins 1-3, Clathrin heavy chain, and heat shock proteins are the marker of plant EVs. Can you try these marker or only one of these or any other exosomes marker? I think you guys showed good quality of TEM results. However If you contains some results of western blot using extracellular marker, strengthen your arguments.
Answer:
Thank you for your comments. We know one of your suggested markers. We already performed western blotting for plant EV markers such as TET-8 and Rab 11 as previously described (8), but we did not detect any signals. We consider that suitable antibodies for plant EVs with cross-reactivity for grapefruits are not commercially available. We are preparing antibodies against some plant EVs, and we will show the results of grapefruit EVs in a future publication. Thank you for the kind suggestion.
Reviewer comment #2. I had never seen any studies using same method(about isolation of exosome from plants or fruits) of yours. So, If possible, can you add the pictures of that steps, respectively?
Answer:
Thank you for your comments. We used centrifugation (at 2000 ´ g for 5 min and at 100,000 ´ g for 4 h) and filtration; this method is not uncommon. However, to remove some debris, we wrapped segments of grapefruits and squeezed them by hand. We think that this filtration was effective to remove some fibrous debris and pigment granules. Therefore, we added an explanation regarding this procedure in the Materials and Methods section as follows (lines 261):
“For SJ, segments were squeezed through filter papers.”
Reviewer comment #3. According to your Fig.1 and Fig.2 I think the main particle size of your exosome is over 150 nm. And their size distribution also desirable. But, I think the TEM results are little confused to me. Because the difference between particles seems to be lack of homogenousity. Can you explain why did you decide the particles which seems to different to be the same EVs?
Answer:
Thank you for your comments. We apologize for the confusion. TEM and SEM were used to observe EV structure and sometimes to determine EV diameter (Yong C, et al., Plant extracellular vesicles. Protoplasma 2020, 2573-12). TEM, SEM, and Nano Sight are used to obtain information for different purposes because of their various capacities. We performed the usual TEM (by ultrathin sections) for Figure 1e to demonstrate that plant EVs had membranous structures similar to those in Figure 2c, 2d, and 2e. SEM pictures indicate that EVs are spherical, even though their diameter was increased by a metal coat of about 10 nm. We think that the mean diameter of EVs cannot be obtained with TEM and SEM, but the analysis with Nano Sight was suitable to determine their mean diameter and size distribution. In any case, 150 nm (as you mentioned above) was close to the value of 162 nm shown by Nano Sight analysis (Fig. 2f).
Reviewer comment #4. How about change the Table. 1 and .2 to graphs? I think schematic can improve what you intended.
Answer:
Thank you for your comments. We show RT-PCR results by threshold cycle values because we could not obtain an adequate internal control for miRNAs in plants. Threshold cycle values should be shown in tables (please, see our reference 19) in such a situation. If we show these results with graphs, threshold cycle values may be confused with the expression of miRNAs.
Reviewer 4 Report
The manuscript entitled “Edible Plant Juice Contains Exosome-like Nanovesicles that Harbor MicroRNAs and Are Taken up by Rat Enterocytes” an original research article is focussed on Plant miRNAs regulate the expression of certain mammalian gene and its biological functions.The exogenous plant miRNAs carried by EVs can be absorbed via the gas trointestinal tract was dealt in details. The manuscript is prepared in well mannered. Materials and Methods are described in details and in comprehensive way. The results are well elaborate. Though an Manuscript is well presented, may some minor mistake need to be addressed.
- Title needs to be revised and made more intriguing to the readers
- I think introduction can be improved by exiting pool of knowledge through recent published papers.
- In abstract section, line #21 please corrects the form of verb after sentence “subcellular localization”
- Terms like " edible plant; exosome and MicroRNAs" are already mentioned in tiltle. Authors may delete these and replace with more appropriate ones.
- In line 161, please remove “in” after the words “and” in table 2
- In line 173, Please make sure the verb used after “which”
- In line 74, please add the verb after word “ EVs”
- In line 177, please add “the” before the word “Previous”
- In line 188, please make sure the sentence is/are before “analogous”
- In line 190, Please add the “are” before the word “frequently”
Author Response
Reviewer 4
The manuscript entitled “Edible Plant Juice Contains Exosome-like Nanovesicles that Harbor MicroRNAs and Are Taken up by Rat Enterocytes” an original research article is focussed on Plant miRNAs regulate the expression of certain mammalian gene and its biological functions.The exogenous plant miRNAs carried by EVs can be absorbed via the gastrointestinal tract was dealt in details. The manuscript is prepared in well mannered. Materials and Methods are described in details and in comprehensive way. The results are well elaborate. Though an Manuscript is well presented, may some minor mistake need to be addressed.
Answer:
We thank the reviewer for the valuable comments and suggestions. Our point-by-point responses to all the comments are presented below.
Reviewer’s comment 1
Title needs to be revised and made more intriguing to the readers
Answer:
Thank you for your suggestion. We changed the title as follows: line 1 - 2
“Uptake of microRNAs from Exosome-like Nanovesicles of Edible Plant Juice by Rat Enterocytes.”
Reviewer’s comment 2
I think introduction can be improved by exiting pool of knowledge through recent published papers.
Answer:
Thank you for your comments. As suggested, we added recent publications to the Introduction section as follows: line 50 - 57
" However, several lines of evidence suggest that plants also release EVs having various functions including symbiosis and defense against pathogens [13]. EVs from different eukaryotic and prokaryotic organisms are involved in many processes such as host-pathogen interactions, resistance transmission, and plant diseases, which may help in the prevention and treatment of pests and pathogens [14]. Interestingly, a growing body of evidence indicates that autophagy-related proteins are involved in exosome biogenesis, which indicates that there are pathways for the interaction between autophagy and EVs that may be important in the context of cancer and neurodegenerative diseases [15]."
In line 377 - 381
[13] Cui Y, Gao J, He Y, Jiang L. Plant extracellular vesicles. Protoplasma. 2020, 257, 3-12. DOI: 10.1007/s00709-019-01435-6.
[14] Woith E, Fuhrmann G, Melzig MF. Extracellular Vesicles-Connecting Kingdoms. Int J Mol Sci. 2019, 20, 5695. DOI: 10.3390/ijms20225695.
[15] Xu J, Camfield R, Gorski SM. The interplay between exosomes and autophagy - partners in crime. J Cell Sci. 2018, 131, jcs215210. DOI: 10.1242/jcs.215210.
Reviewer’s comment 3
In abstract section, line #21 please corrects the form of verb after sentence “subcellular localization”
Answer:
Thank you for your comments. As suggested, we revised the sentence as follows:
in line 21
“EV morphological features and subcellular localization were analyzed…..”
Reviewer’s comment 4
Terms like " edible plant; exosome and MicroRNAs" are already mentioned in tiltle. Authors may delete these and replace with more appropriate ones.
Answer:
Thank you for your comments. Do you recommend using abbreviations for these terms?
Reviewer’s comment 5
In line 161, please remove “in” after the words “and” in table 2
Answer:
Thank you for your comments. We revised the text as suggested (line 171).
Reviewer’s comment 6
In line 173, Please make sure the verb used after “which”
Answer:
Thank you for your comments. As suggested, we replaced “are” with “were” (line 175).
Reviewer’s comment 7
In line 74, please add the verb after word “ EVs”
Answer:
Thank you for your comments. We changed the text (line 82) from “Representative particle size distributions are shown for EVs “ to “Representative particle size distributions for EVs are as follows:"
Reviewer’s comment 8
In line 177, please add “the” before the word “Previous”
Answer:
Thank you for your comments. We added “the” before “previous” (line 187).
Reviewer’s comment 9
In line 188, please make sure the sentence is/are before “analogous”
Answer:
Thank you for your comments. We replaced “is” with “are” (line 190).
Reviewer’s comment 10
In line 190, Please add the “are” before the word “frequently”
Answer:
Thank you for your comments. We replaced “which are among the 26 most frequently observed miRNAs” with “which are frequently observed miRNAs” (line 202).
Thank you for your careful evaluation and kind comments.
Reviewer 4
The manuscript entitled “Edible Plant Juice Contains Exosome-like Nanovesicles that Harbor MicroRNAs and Are Taken up by Rat Enterocytes” an original research article is focussed on Plant miRNAs regulate the expression of certain mammalian gene and its biological functions.The exogenous plant miRNAs carried by EVs can be absorbed via the gastrointestinal tract was dealt in details. The manuscript is prepared in well mannered. Materials and Methods are described in details and in comprehensive way. The results are well elaborate. Though an Manuscript is well presented, may some minor mistake need to be addressed.
Answer:
We thank the reviewer for the valuable comments and suggestions. Our point-by-point responses to all the comments are presented below.
Reviewer’s comment 1
Title needs to be revised and made more intriguing to the readers
Answer:
Thank you for your suggestion. We changed the title as follows: line 1 - 2
“Uptake of microRNAs from Exosome-like Nanovesicles of Edible Plant Juice by Rat Enterocytes.”
Reviewer’s comment 2
I think introduction can be improved by exiting pool of knowledge through recent published papers.
Answer:
Thank you for your comments. As suggested, we added recent publications to the Introduction section as follows: line 50 - 57
" However, several lines of evidence suggest that plants also release EVs having various functions including symbiosis and defense against pathogens [13]. EVs from different eukaryotic and prokaryotic organisms are involved in many processes such as host-pathogen interactions, resistance transmission, and plant diseases, which may help in the prevention and treatment of pests and pathogens [14]. Interestingly, a growing body of evidence indicates that autophagy-related proteins are involved in exosome biogenesis, which indicates that there are pathways for the interaction between autophagy and EVs that may be important in the context of cancer and neurodegenerative diseases [15]."
In line 377 - 381
[13] Cui Y, Gao J, He Y, Jiang L. Plant extracellular vesicles. Protoplasma. 2020, 257, 3-12. DOI: 10.1007/s00709-019-01435-6.
[14] Woith E, Fuhrmann G, Melzig MF. Extracellular Vesicles-Connecting Kingdoms. Int J Mol Sci. 2019, 20, 5695. DOI: 10.3390/ijms20225695.
[15] Xu J, Camfield R, Gorski SM. The interplay between exosomes and autophagy - partners in crime. J Cell Sci. 2018, 131, jcs215210. DOI: 10.1242/jcs.215210.
Reviewer’s comment 3
In abstract section, line #21 please corrects the form of verb after sentence “subcellular localization”
Answer:
Thank you for your comments. As suggested, we revised the sentence as follows:
in line 21
“EV morphological features and subcellular localization were analyzed…..”
Reviewer’s comment 4
Terms like " edible plant; exosome and MicroRNAs" are already mentioned in tiltle. Authors may delete these and replace with more appropriate ones.
Answer:
Thank you for your comments. Do you recommend using abbreviations for these terms?
Reviewer’s comment 5
In line 161, please remove “in” after the words “and” in table 2
Answer:
Thank you for your comments. We revised the text as suggested (line 171).
Reviewer’s comment 6
In line 173, Please make sure the verb used after “which”
Answer:
Thank you for your comments. As suggested, we replaced “are” with “were” (line 175).
Reviewer’s comment 7
In line 74, please add the verb after word “ EVs”
Answer:
Thank you for your comments. We changed the text (line 82) from “Representative particle size distributions are shown for EVs “ to “Representative particle size distributions for EVs are as follows:"
Reviewer’s comment 8
In line 177, please add “the” before the word “Previous”
Answer:
Thank you for your comments. We added “the” before “previous” (line 187).
Reviewer’s comment 9
In line 188, please make sure the sentence is/are before “analogous”
Answer:
Thank you for your comments. We replaced “is” with “are” (line 190).
Reviewer’s comment 10
In line 190, Please add the “are” before the word “frequently”
Answer:
Thank you for your comments. We replaced “which are among the 26 most frequently observed miRNAs” with “which are frequently observed miRNAs” (line 202).
Thank you for your careful evaluation and kind comments.
Reviewer 4
The manuscript entitled “Edible Plant Juice Contains Exosome-like Nanovesicles that Harbor MicroRNAs and Are Taken up by Rat Enterocytes” an original research article is focussed on Plant miRNAs regulate the expression of certain mammalian gene and its biological functions.The exogenous plant miRNAs carried by EVs can be absorbed via the gastrointestinal tract was dealt in details. The manuscript is prepared in well mannered. Materials and Methods are described in details and in comprehensive way. The results are well elaborate. Though an Manuscript is well presented, may some minor mistake need to be addressed.
Answer:
We thank the reviewer for the valuable comments and suggestions. Our point-by-point responses to all the comments are presented below.
Reviewer’s comment 1
Title needs to be revised and made more intriguing to the readers
Answer:
Thank you for your suggestion. We changed the title as follows: line 1 - 2
“Uptake of microRNAs from Exosome-like Nanovesicles of Edible Plant Juice by Rat Enterocytes.”
Reviewer’s comment 2
I think introduction can be improved by exiting pool of knowledge through recent published papers.
Answer:
Thank you for your comments. As suggested, we added recent publications to the Introduction section as follows: line 50 - 57
" However, several lines of evidence suggest that plants also release EVs having various functions including symbiosis and defense against pathogens [13]. EVs from different eukaryotic and prokaryotic organisms are involved in many processes such as host-pathogen interactions, resistance transmission, and plant diseases, which may help in the prevention and treatment of pests and pathogens [14]. Interestingly, a growing body of evidence indicates that autophagy-related proteins are involved in exosome biogenesis, which indicates that there are pathways for the interaction between autophagy and EVs that may be important in the context of cancer and neurodegenerative diseases [15]."
In line 377 - 381
[13] Cui Y, Gao J, He Y, Jiang L. Plant extracellular vesicles. Protoplasma. 2020, 257, 3-12. DOI: 10.1007/s00709-019-01435-6.
[14] Woith E, Fuhrmann G, Melzig MF. Extracellular Vesicles-Connecting Kingdoms. Int J Mol Sci. 2019, 20, 5695. DOI: 10.3390/ijms20225695.
[15] Xu J, Camfield R, Gorski SM. The interplay between exosomes and autophagy - partners in crime. J Cell Sci. 2018, 131, jcs215210. DOI: 10.1242/jcs.215210.
Reviewer’s comment 3
In abstract section, line #21 please corrects the form of verb after sentence “subcellular localization”
Answer:
Thank you for your comments. As suggested, we revised the sentence as follows:
in line 21
“EV morphological features and subcellular localization were analyzed…..”
Reviewer’s comment 4
Terms like " edible plant; exosome and MicroRNAs" are already mentioned in tiltle. Authors may delete these and replace with more appropriate ones.
Answer:
Thank you for your comments. Do you recommend using abbreviations for these terms?
Reviewer’s comment 5
In line 161, please remove “in” after the words “and” in table 2
Answer:
Thank you for your comments. We revised the text as suggested (line 171).
Reviewer’s comment 6
In line 173, Please make sure the verb used after “which”
Answer:
Thank you for your comments. As suggested, we replaced “are” with “were” (line 175).
Reviewer’s comment 7
In line 74, please add the verb after word “ EVs”
Answer:
Thank you for your comments. We changed the text (line 82) from “Representative particle size distributions are shown for EVs “ to “Representative particle size distributions for EVs are as follows:"
Reviewer’s comment 8
In line 177, please add “the” before the word “Previous”
Answer:
Thank you for your comments. We added “the” before “previous” (line 187).
Reviewer’s comment 9
In line 188, please make sure the sentence is/are before “analogous”
Answer:
Thank you for your comments. We replaced “is” with “are” (line 190).
Reviewer’s comment 10
In line 190, Please add the “are” before the word “frequently”
Answer:
Thank you for your comments. We replaced “which are among the 26 most frequently observed miRNAs” with “which are frequently observed miRNAs” (line 202).
Thank you for your careful evaluation and kind comments.
Reviewer 4
The manuscript entitled “Edible Plant Juice Contains Exosome-like Nanovesicles that Harbor MicroRNAs and Are Taken up by Rat Enterocytes” an original research article is focussed on Plant miRNAs regulate the expression of certain mammalian gene and its biological functions.The exogenous plant miRNAs carried by EVs can be absorbed via the gastrointestinal tract was dealt in details. The manuscript is prepared in well mannered. Materials and Methods are described in details and in comprehensive way. The results are well elaborate. Though an Manuscript is well presented, may some minor mistake need to be addressed.
Answer:
We thank the reviewer for the valuable comments and suggestions. Our point-by-point responses to all the comments are presented below.
Reviewer’s comment 1
Title needs to be revised and made more intriguing to the readers
Answer:
Thank you for your suggestion. We changed the title as follows: line 1 - 2
“Uptake of microRNAs from Exosome-like Nanovesicles of Edible Plant Juice by Rat Enterocytes.”
Reviewer’s comment 2
I think introduction can be improved by exiting pool of knowledge through recent published papers.
Answer:
Thank you for your comments. As suggested, we added recent publications to the Introduction section as follows: line 50 - 57
" However, several lines of evidence suggest that plants also release EVs having various functions including symbiosis and defense against pathogens [13]. EVs from different eukaryotic and prokaryotic organisms are involved in many processes such as host-pathogen interactions, resistance transmission, and plant diseases, which may help in the prevention and treatment of pests and pathogens [14]. Interestingly, a growing body of evidence indicates that autophagy-related proteins are involved in exosome biogenesis, which indicates that there are pathways for the interaction between autophagy and EVs that may be important in the context of cancer and neurodegenerative diseases [15]."
In line 377 - 381
[13] Cui Y, Gao J, He Y, Jiang L. Plant extracellular vesicles. Protoplasma. 2020, 257, 3-12. DOI: 10.1007/s00709-019-01435-6.
[14] Woith E, Fuhrmann G, Melzig MF. Extracellular Vesicles-Connecting Kingdoms. Int J Mol Sci. 2019, 20, 5695. DOI: 10.3390/ijms20225695.
[15] Xu J, Camfield R, Gorski SM. The interplay between exosomes and autophagy - partners in crime. J Cell Sci. 2018, 131, jcs215210. DOI: 10.1242/jcs.215210.
Reviewer’s comment 3
In abstract section, line #21 please corrects the form of verb after sentence “subcellular localization”
Answer:
Thank you for your comments. As suggested, we revised the sentence as follows:
in line 21
“EV morphological features and subcellular localization were analyzed…..”
Reviewer’s comment 4
Terms like " edible plant; exosome and MicroRNAs" are already mentioned in tiltle. Authors may delete these and replace with more appropriate ones.
Answer:
Thank you for your comments. Do you recommend using abbreviations for these terms?
Reviewer’s comment 5
In line 161, please remove “in” after the words “and” in table 2
Answer:
Thank you for your comments. We revised the text as suggested (line 171).
Reviewer’s comment 6
In line 173, Please make sure the verb used after “which”
Answer:
Thank you for your comments. As suggested, we replaced “are” with “were” (line 175).
Reviewer’s comment 7
In line 74, please add the verb after word “ EVs”
Answer:
Thank you for your comments. We changed the text (line 82) from “Representative particle size distributions are shown for EVs “ to “Representative particle size distributions for EVs are as follows:"
Reviewer’s comment 8
In line 177, please add “the” before the word “Previous”
Answer:
Thank you for your comments. We added “the” before “previous” (line 187).
Reviewer’s comment 9
In line 188, please make sure the sentence is/are before “analogous”
Answer:
Thank you for your comments. We replaced “is” with “are” (line 190).
Reviewer’s comment 10
In line 190, Please add the “are” before the word “frequently”
Answer:
Thank you for your comments. We replaced “which are among the 26 most frequently observed miRNAs” with “which are frequently observed miRNAs” (line 202).
Thank you for your careful evaluation and kind comments.
Reviewer 4
The manuscript entitled “Edible Plant Juice Contains Exosome-like Nanovesicles that Harbor MicroRNAs and Are Taken up by Rat Enterocytes” an original research article is focussed on Plant miRNAs regulate the expression of certain mammalian gene and its biological functions.The exogenous plant miRNAs carried by EVs can be absorbed via the gastrointestinal tract was dealt in details. The manuscript is prepared in well mannered. Materials and Methods are described in details and in comprehensive way. The results are well elaborate. Though an Manuscript is well presented, may some minor mistake need to be addressed.
Answer:
We thank the reviewer for the valuable comments and suggestions. Our point-by-point responses to all the comments are presented below.
Reviewer’s comment 1
Title needs to be revised and made more intriguing to the readers
Answer:
Thank you for your suggestion. We changed the title as follows: line 1 - 2
“Uptake of microRNAs from Exosome-like Nanovesicles of Edible Plant Juice by Rat Enterocytes.”
Reviewer’s comment 2
I think introduction can be improved by exiting pool of knowledge through recent published papers.
Answer:
Thank you for your comments. As suggested, we added recent publications to the Introduction section as follows: line 50 - 57
" However, several lines of evidence suggest that plants also release EVs having various functions including symbiosis and defense against pathogens [13]. EVs from different eukaryotic and prokaryotic organisms are involved in many processes such as host-pathogen interactions, resistance transmission, and plant diseases, which may help in the prevention and treatment of pests and pathogens [14]. Interestingly, a growing body of evidence indicates that autophagy-related proteins are involved in exosome biogenesis, which indicates that there are pathways for the interaction between autophagy and EVs that may be important in the context of cancer and neurodegenerative diseases [15]."
In line 377 - 381
[13] Cui Y, Gao J, He Y, Jiang L. Plant extracellular vesicles. Protoplasma. 2020, 257, 3-12. DOI: 10.1007/s00709-019-01435-6.
[14] Woith E, Fuhrmann G, Melzig MF. Extracellular Vesicles-Connecting Kingdoms. Int J Mol Sci. 2019, 20, 5695. DOI: 10.3390/ijms20225695.
[15] Xu J, Camfield R, Gorski SM. The interplay between exosomes and autophagy - partners in crime. J Cell Sci. 2018, 131, jcs215210. DOI: 10.1242/jcs.215210.
Reviewer’s comment 3
In abstract section, line #21 please corrects the form of verb after sentence “subcellular localization”
Answer:
Thank you for your comments. As suggested, we revised the sentence as follows:
in line 21
“EV morphological features and subcellular localization were analyzed…..”
Reviewer’s comment 4
Terms like " edible plant; exosome and MicroRNAs" are already mentioned in tiltle. Authors may delete these and replace with more appropriate ones.
Answer:
Thank you for your comments. Do you recommend using abbreviations for these terms?
Reviewer’s comment 5
In line 161, please remove “in” after the words “and” in table 2
Answer:
Thank you for your comments. We revised the text as suggested (line 171).
Reviewer’s comment 6
In line 173, Please make sure the verb used after “which”
Answer:
Thank you for your comments. As suggested, we replaced “are” with “were” (line 175).
Reviewer’s comment 7
In line 74, please add the verb after word “ EVs”
Answer:
Thank you for your comments. We changed the text (line 82) from “Representative particle size distributions are shown for EVs “ to “Representative particle size distributions for EVs are as follows:"
Reviewer’s comment 8
In line 177, please add “the” before the word “Previous”
Answer:
Thank you for your comments. We added “the” before “previous” (line 187).
Reviewer’s comment 9
In line 188, please make sure the sentence is/are before “analogous”
Answer:
Thank you for your comments. We replaced “is” with “are” (line 190).
Reviewer’s comment 10
In line 190, Please add the “are” before the word “frequently”
Answer:
Thank you for your comments. We replaced “which are among the 26 most frequently observed miRNAs” with “which are frequently observed miRNAs” (line 202).
Thank you for your careful evaluation and kind comments.
Reviewer 4
The manuscript entitled “Edible Plant Juice Contains Exosome-like Nanovesicles that Harbor MicroRNAs and Are Taken up by Rat Enterocytes” an original research article is focussed on Plant miRNAs regulate the expression of certain mammalian gene and its biological functions.The exogenous plant miRNAs carried by EVs can be absorbed via the gastrointestinal tract was dealt in details. The manuscript is prepared in well mannered. Materials and Methods are described in details and in comprehensive way. The results are well elaborate. Though an Manuscript is well presented, may some minor mistake need to be addressed.
Answer:
We thank the reviewer for the valuable comments and suggestions. Our point-by-point responses to all the comments are presented below.
Reviewer’s comment 1
Title needs to be revised and made more intriguing to the readers
Answer:
Thank you for your suggestion. We changed the title as follows: line 1 - 2
“Uptake of microRNAs from Exosome-like Nanovesicles of Edible Plant Juice by Rat Enterocytes.”
Reviewer’s comment 2
I think introduction can be improved by exiting pool of knowledge through recent published papers.
Answer:
Thank you for your comments. As suggested, we added recent publications to the Introduction section as follows: line 50 - 57
" However, several lines of evidence suggest that plants also release EVs having various functions including symbiosis and defense against pathogens [13]. EVs from different eukaryotic and prokaryotic organisms are involved in many processes such as host-pathogen interactions, resistance transmission, and plant diseases, which may help in the prevention and treatment of pests and pathogens [14]. Interestingly, a growing body of evidence indicates that autophagy-related proteins are involved in exosome biogenesis, which indicates that there are pathways for the interaction between autophagy and EVs that may be important in the context of cancer and neurodegenerative diseases [15]."
In line 377 - 381
[13] Cui Y, Gao J, He Y, Jiang L. Plant extracellular vesicles. Protoplasma. 2020, 257, 3-12. DOI: 10.1007/s00709-019-01435-6.
[14] Woith E, Fuhrmann G, Melzig MF. Extracellular Vesicles-Connecting Kingdoms. Int J Mol Sci. 2019, 20, 5695. DOI: 10.3390/ijms20225695.
[15] Xu J, Camfield R, Gorski SM. The interplay between exosomes and autophagy - partners in crime. J Cell Sci. 2018, 131, jcs215210. DOI: 10.1242/jcs.215210.
Reviewer’s comment 3
In abstract section, line #21 please corrects the form of verb after sentence “subcellular localization”
Answer:
Thank you for your comments. As suggested, we revised the sentence as follows:
in line 21
“EV morphological features and subcellular localization were analyzed…..”
Reviewer’s comment 4
Terms like " edible plant; exosome and MicroRNAs" are already mentioned in tiltle. Authors may delete these and replace with more appropriate ones.
Answer:
Thank you for your comments. Do you recommend using abbreviations for these terms?
Reviewer’s comment 5
In line 161, please remove “in” after the words “and” in table 2
Answer:
Thank you for your comments. We revised the text as suggested (line 171).
Reviewer’s comment 6
In line 173, Please make sure the verb used after “which”
Answer:
Thank you for your comments. As suggested, we replaced “are” with “were” (line 175).
Reviewer’s comment 7
In line 74, please add the verb after word “ EVs”
Answer:
Thank you for your comments. We changed the text (line 82) from “Representative particle size distributions are shown for EVs “ to “Representative particle size distributions for EVs are as follows:"
Reviewer’s comment 8
In line 177, please add “the” before the word “Previous”
Answer:
Thank you for your comments. We added “the” before “previous” (line 187).
Reviewer’s comment 9
In line 188, please make sure the sentence is/are before “analogous”
Answer:
Thank you for your comments. We replaced “is” with “are” (line 190).
Reviewer’s comment 10
In line 190, Please add the “are” before the word “frequently”
Answer:
Thank you for your comments. We replaced “which are among the 26 most frequently observed miRNAs” with “which are frequently observed miRNAs” (line 202).
Thank you for your careful evaluation and kind comments.
Reviewer 4
The manuscript entitled “Edible Plant Juice Contains Exosome-like Nanovesicles that Harbor MicroRNAs and Are Taken up by Rat Enterocytes” an original research article is focussed on Plant miRNAs regulate the expression of certain mammalian gene and its biological functions.The exogenous plant miRNAs carried by EVs can be absorbed via the gastrointestinal tract was dealt in details. The manuscript is prepared in well mannered. Materials and Methods are described in details and in comprehensive way. The results are well elaborate. Though an Manuscript is well presented, may some minor mistake need to be addressed.
Answer:
We thank the reviewer for the valuable comments and suggestions. Our point-by-point responses to all the comments are presented below.
Reviewer’s comment 1
Title needs to be revised and made more intriguing to the readers
Answer:
Thank you for your suggestion. We changed the title as follows: line 1 - 2
“Uptake of microRNAs from Exosome-like Nanovesicles of Edible Plant Juice by Rat Enterocytes.”
Reviewer’s comment 2
I think introduction can be improved by exiting pool of knowledge through recent published papers.
Answer:
Thank you for your comments. As suggested, we added recent publications to the Introduction section as follows: line 50 - 57
" However, several lines of evidence suggest that plants also release EVs having various functions including symbiosis and defense against pathogens [13]. EVs from different eukaryotic and prokaryotic organisms are involved in many processes such as host-pathogen interactions, resistance transmission, and plant diseases, which may help in the prevention and treatment of pests and pathogens [14]. Interestingly, a growing body of evidence indicates that autophagy-related proteins are involved in exosome biogenesis, which indicates that there are pathways for the interaction between autophagy and EVs that may be important in the context of cancer and neurodegenerative diseases [15]."
In line 377 - 381
[13] Cui Y, Gao J, He Y, Jiang L. Plant extracellular vesicles. Protoplasma. 2020, 257, 3-12. DOI: 10.1007/s00709-019-01435-6.
[14] Woith E, Fuhrmann G, Melzig MF. Extracellular Vesicles-Connecting Kingdoms. Int J Mol Sci. 2019, 20, 5695. DOI: 10.3390/ijms20225695.
[15] Xu J, Camfield R, Gorski SM. The interplay between exosomes and autophagy - partners in crime. J Cell Sci. 2018, 131, jcs215210. DOI: 10.1242/jcs.215210.
Reviewer’s comment 3
In abstract section, line #21 please corrects the form of verb after sentence “subcellular localization”
Answer:
Thank you for your comments. As suggested, we revised the sentence as follows:
in line 21
“EV morphological features and subcellular localization were analyzed…..”
Reviewer’s comment 4
Terms like " edible plant; exosome and MicroRNAs" are already mentioned in tiltle. Authors may delete these and replace with more appropriate ones.
Answer:
Thank you for your comments. Do you recommend using abbreviations for these terms?
Reviewer’s comment 5
In line 161, please remove “in” after the words “and” in table 2
Answer:
Thank you for your comments. We revised the text as suggested (line 171).
Reviewer’s comment 6
In line 173, Please make sure the verb used after “which”
Answer:
Thank you for your comments. As suggested, we replaced “are” with “were” (line 175).
Reviewer’s comment 7
In line 74, please add the verb after word “ EVs”
Answer:
Thank you for your comments. We changed the text (line 82) from “Representative particle size distributions are shown for EVs “ to “Representative particle size distributions for EVs are as follows:"
Reviewer’s comment 8
In line 177, please add “the” before the word “Previous”
Answer:
Thank you for your comments. We added “the” before “previous” (line 187).
Reviewer’s comment 9
In line 188, please make sure the sentence is/are before “analogous”
Answer:
Thank you for your comments. We replaced “is” with “are” (line 190).
Reviewer’s comment 10
In line 190, Please add the “are” before the word “frequently”
Answer:
Thank you for your comments. We replaced “which are among the 26 most frequently observed miRNAs” with “which are frequently observed miRNAs” (line 202).
Thank you for your careful evaluation and kind comments.
Reviewer 4
The manuscript entitled “Edible Plant Juice Contains Exosome-like Nanovesicles that Harbor MicroRNAs and Are Taken up by Rat Enterocytes” an original research article is focussed on Plant miRNAs regulate the expression of certain mammalian gene and its biological functions.The exogenous plant miRNAs carried by EVs can be absorbed via the gastrointestinal tract was dealt in details. The manuscript is prepared in well mannered. Materials and Methods are described in details and in comprehensive way. The results are well elaborate. Though an Manuscript is well presented, may some minor mistake need to be addressed.
Answer:
We thank the reviewer for the valuable comments and suggestions. Our point-by-point responses to all the comments are presented below.
Reviewer’s comment 1
Title needs to be revised and made more intriguing to the readers
Answer:
Thank you for your suggestion. We changed the title as follows: line 1 - 2
“Uptake of microRNAs from Exosome-like Nanovesicles of Edible Plant Juice by Rat Enterocytes.”
Reviewer’s comment 2
I think introduction can be improved by exiting pool of knowledge through recent published papers.
Answer:
Thank you for your comments. As suggested, we added recent publications to the Introduction section as follows: line 50 - 57
" However, several lines of evidence suggest that plants also release EVs having various functions including symbiosis and defense against pathogens [13]. EVs from different eukaryotic and prokaryotic organisms are involved in many processes such as host-pathogen interactions, resistance transmission, and plant diseases, which may help in the prevention and treatment of pests and pathogens [14]. Interestingly, a growing body of evidence indicates that autophagy-related proteins are involved in exosome biogenesis, which indicates that there are pathways for the interaction between autophagy and EVs that may be important in the context of cancer and neurodegenerative diseases [15]."
In line 377 - 381
[13] Cui Y, Gao J, He Y, Jiang L. Plant extracellular vesicles. Protoplasma. 2020, 257, 3-12. DOI: 10.1007/s00709-019-01435-6.
[14] Woith E, Fuhrmann G, Melzig MF. Extracellular Vesicles-Connecting Kingdoms. Int J Mol Sci. 2019, 20, 5695. DOI: 10.3390/ijms20225695.
[15] Xu J, Camfield R, Gorski SM. The interplay between exosomes and autophagy - partners in crime. J Cell Sci. 2018, 131, jcs215210. DOI: 10.1242/jcs.215210.
Reviewer’s comment 3
In abstract section, line #21 please corrects the form of verb after sentence “subcellular localization”
Answer:
Thank you for your comments. As suggested, we revised the sentence as follows:
in line 21
“EV morphological features and subcellular localization were analyzed…..”
Reviewer’s comment 4
Terms like " edible plant; exosome and MicroRNAs" are already mentioned in tiltle. Authors may delete these and replace with more appropriate ones.
Answer:
Thank you for your comments. Do you recommend using abbreviations for these terms?
Reviewer’s comment 5
In line 161, please remove “in” after the words “and” in table 2
Answer:
Thank you for your comments. We revised the text as suggested (line 171).
Reviewer’s comment 6
In line 173, Please make sure the verb used after “which”
Answer:
Thank you for your comments. As suggested, we replaced “are” with “were” (line 175).
Reviewer’s comment 7
In line 74, please add the verb after word “ EVs”
Answer:
Thank you for your comments. We changed the text (line 82) from “Representative particle size distributions are shown for EVs “ to “Representative particle size distributions for EVs are as follows:"
Reviewer’s comment 8
In line 177, please add “the” before the word “Previous”
Answer:
Thank you for your comments. We added “the” before “previous” (line 187).
Reviewer’s comment 9
In line 188, please make sure the sentence is/are before “analogous”
Answer:
Thank you for your comments. We replaced “is” with “are” (line 190).
Reviewer’s comment 10
In line 190, Please add the “are” before the word “frequently”
Answer:
Thank you for your comments. We replaced “which are among the 26 most frequently observed miRNAs” with “which are frequently observed miRNAs” (line 202).
Thank you for your careful evaluation and kind comments.
Reviewer 4
The manuscript entitled “Edible Plant Juice Contains Exosome-like Nanovesicles that Harbor MicroRNAs and Are Taken up by Rat Enterocytes” an original research article is focussed on Plant miRNAs regulate the expression of certain mammalian gene and its biological functions.The exogenous plant miRNAs carried by EVs can be absorbed via the gastrointestinal tract was dealt in details. The manuscript is prepared in well mannered. Materials and Methods are described in details and in comprehensive way. The results are well elaborate. Though an Manuscript is well presented, may some minor mistake need to be addressed.
Answer:
We thank the reviewer for the valuable comments and suggestions. Our point-by-point responses to all the comments are presented below.
Reviewer’s comment 1
Title needs to be revised and made more intriguing to the readers
Answer:
Thank you for your suggestion. We changed the title as follows: line 1 - 2
“Uptake of microRNAs from Exosome-like Nanovesicles of Edible Plant Juice by Rat Enterocytes.”
Reviewer’s comment 2
I think introduction can be improved by exiting pool of knowledge through recent published papers.
Answer:
Thank you for your comments. As suggested, we added recent publications to the Introduction section as follows: line 50 - 57
" However, several lines of evidence suggest that plants also release EVs having various functions including symbiosis and defense against pathogens [13]. EVs from different eukaryotic and prokaryotic organisms are involved in many processes such as host-pathogen interactions, resistance transmission, and plant diseases, which may help in the prevention and treatment of pests and pathogens [14]. Interestingly, a growing body of evidence indicates that autophagy-related proteins are involved in exosome biogenesis, which indicates that there are pathways for the interaction between autophagy and EVs that may be important in the context of cancer and neurodegenerative diseases [15]."
In line 377 - 381
[13] Cui Y, Gao J, He Y, Jiang L. Plant extracellular vesicles. Protoplasma. 2020, 257, 3-12. DOI: 10.1007/s00709-019-01435-6.
[14] Woith E, Fuhrmann G, Melzig MF. Extracellular Vesicles-Connecting Kingdoms. Int J Mol Sci. 2019, 20, 5695. DOI: 10.3390/ijms20225695.
[15] Xu J, Camfield R, Gorski SM. The interplay between exosomes and autophagy - partners in crime. J Cell Sci. 2018, 131, jcs215210. DOI: 10.1242/jcs.215210.
Reviewer’s comment 3
In abstract section, line #21 please corrects the form of verb after sentence “subcellular localization”
Answer:
Thank you for your comments. As suggested, we revised the sentence as follows:
in line 21
“EV morphological features and subcellular localization were analyzed…..”
Reviewer’s comment 4
Terms like " edible plant; exosome and MicroRNAs" are already mentioned in tiltle. Authors may delete these and replace with more appropriate ones.
Answer:
Thank you for your comments. Do you recommend using abbreviations for these terms?
Reviewer’s comment 5
In line 161, please remove “in” after the words “and” in table 2
Answer:
Thank you for your comments. We revised the text as suggested (line 171).
Reviewer’s comment 6
In line 173, Please make sure the verb used after “which”
Answer:
Thank you for your comments. As suggested, we replaced “are” with “were” (line 175).
Reviewer’s comment 7
In line 74, please add the verb after word “ EVs”
Answer:
Thank you for your comments. We changed the text (line 82) from “Representative particle size distributions are shown for EVs “ to “Representative particle size distributions for EVs are as follows:"
Reviewer’s comment 8
In line 177, please add “the” before the word “Previous”
Answer:
Thank you for your comments. We added “the” before “previous” (line 187).
Reviewer’s comment 9
In line 188, please make sure the sentence is/are before “analogous”
Answer:
Thank you for your comments. We replaced “is” with “are” (line 190).
Reviewer’s comment 10
In line 190, Please add the “are” before the word “frequently”
Answer:
Thank you for your comments. We replaced “which are among the 26 most frequently observed miRNAs” with “which are frequently observed miRNAs” (line 202).
Thank you for your careful evaluation and kind comments.
Reviewer 4
The manuscript entitled “Edible Plant Juice Contains Exosome-like Nanovesicles that Harbor MicroRNAs and Are Taken up by Rat Enterocytes” an original research article is focussed on Plant miRNAs regulate the expression of certain mammalian gene and its biological functions.The exogenous plant miRNAs carried by EVs can be absorbed via the gastrointestinal tract was dealt in details. The manuscript is prepared in well mannered. Materials and Methods are described in details and in comprehensive way. The results are well elaborate. Though an Manuscript is well presented, may some minor mistake need to be addressed.
Answer:
We thank the reviewer for the valuable comments and suggestions. Our point-by-point responses to all the comments are presented below.
Reviewer’s comment 1
Title needs to be revised and made more intriguing to the readers
Answer:
Thank you for your suggestion. We changed the title as follows: line 1 - 2
“Uptake of microRNAs from Exosome-like Nanovesicles of Edible Plant Juice by Rat Enterocytes.”
Reviewer’s comment 2
I think introduction can be improved by exiting pool of knowledge through recent published papers.
Answer:
Thank you for your comments. As suggested, we added recent publications to the Introduction section as follows: line 50 - 57
" However, several lines of evidence suggest that plants also release EVs having various functions including symbiosis and defense against pathogens [13]. EVs from different eukaryotic and prokaryotic organisms are involved in many processes such as host-pathogen interactions, resistance transmission, and plant diseases, which may help in the prevention and treatment of pests and pathogens [14]. Interestingly, a growing body of evidence indicates that autophagy-related proteins are involved in exosome biogenesis, which indicates that there are pathways for the interaction between autophagy and EVs that may be important in the context of cancer and neurodegenerative diseases [15]."
In line 377 - 381
[13] Cui Y, Gao J, He Y, Jiang L. Plant extracellular vesicles. Protoplasma. 2020, 257, 3-12. DOI: 10.1007/s00709-019-01435-6.
[14] Woith E, Fuhrmann G, Melzig MF. Extracellular Vesicles-Connecting Kingdoms. Int J Mol Sci. 2019, 20, 5695. DOI: 10.3390/ijms20225695.
[15] Xu J, Camfield R, Gorski SM. The interplay between exosomes and autophagy - partners in crime. J Cell Sci. 2018, 131, jcs215210. DOI: 10.1242/jcs.215210.
Reviewer’s comment 3
In abstract section, line #21 please corrects the form of verb after sentence “subcellular localization”
Answer:
Thank you for your comments. As suggested, we revised the sentence as follows:
in line 21
“EV morphological features and subcellular localization were analyzed…..”
Reviewer’s comment 4
Terms like " edible plant; exosome and MicroRNAs" are already mentioned in tiltle. Authors may delete these and replace with more appropriate ones.
Answer:
Thank you for your comments. Do you recommend using abbreviations for these terms?
Reviewer’s comment 5
In line 161, please remove “in” after the words “and” in table 2
Answer:
Thank you for your comments. We revised the text as suggested (line 171).
Reviewer’s comment 6
In line 173, Please make sure the verb used after “which”
Answer:
Thank you for your comments. As suggested, we replaced “are” with “were” (line 175).
Reviewer’s comment 7
In line 74, please add the verb after word “ EVs”
Answer:
Thank you for your comments. We changed the text (line 82) from “Representative particle size distributions are shown for EVs “ to “Representative particle size distributions for EVs are as follows:"
Reviewer’s comment 8
In line 177, please add “the” before the word “Previous”
Answer:
Thank you for your comments. We added “the” before “previous” (line 187).
Reviewer’s comment 9
In line 188, please make sure the sentence is/are before “analogous”
Answer:
Thank you for your comments. We replaced “is” with “are” (line 190).
Reviewer’s comment 10
In line 190, Please add the “are” before the word “frequently”
Answer:
Thank you for your comments. We replaced “which are among the 26 most frequently observed miRNAs” with “which are frequently observed miRNAs” (line 202).
Thank you for your careful evaluation and kind comments.
Reviewer 4
The manuscript entitled “Edible Plant Juice Contains Exosome-like Nanovesicles that Harbor MicroRNAs and Are Taken up by Rat Enterocytes” an original research article is focussed on Plant miRNAs regulate the expression of certain mammalian gene and its biological functions.The exogenous plant miRNAs carried by EVs can be absorbed via the gastrointestinal tract was dealt in details. The manuscript is prepared in well mannered. Materials and Methods are described in details and in comprehensive way. The results are well elaborate. Though an Manuscript is well presented, may some minor mistake need to be addressed.
Answer:
We thank the reviewer for the valuable comments and suggestions. Our point-by-point responses to all the comments are presented below.
Reviewer’s comment 1
Title needs to be revised and made more intriguing to the readers
Answer:
Thank you for your suggestion. We changed the title as follows: line 1 - 2
“Uptake of microRNAs from Exosome-like Nanovesicles of Edible Plant Juice by Rat Enterocytes.”
Reviewer’s comment 2
I think introduction can be improved by exiting pool of knowledge through recent published papers.
Answer:
Thank you for your comments. As suggested, we added recent publications to the Introduction section as follows: line 50 - 57
" However, several lines of evidence suggest that plants also release EVs having various functions including symbiosis and defense against pathogens [13]. EVs from different eukaryotic and prokaryotic organisms are involved in many processes such as host-pathogen interactions, resistance transmission, and plant diseases, which may help in the prevention and treatment of pests and pathogens [14]. Interestingly, a growing body of evidence indicates that autophagy-related proteins are involved in exosome biogenesis, which indicates that there are pathways for the interaction between autophagy and EVs that may be important in the context of cancer and neurodegenerative diseases [15]."
In line 377 - 381
[13] Cui Y, Gao J, He Y, Jiang L. Plant extracellular vesicles. Protoplasma. 2020, 257, 3-12. DOI: 10.1007/s00709-019-01435-6.
[14] Woith E, Fuhrmann G, Melzig MF. Extracellular Vesicles-Connecting Kingdoms. Int J Mol Sci. 2019, 20, 5695. DOI: 10.3390/ijms20225695.
[15] Xu J, Camfield R, Gorski SM. The interplay between exosomes and autophagy - partners in crime. J Cell Sci. 2018, 131, jcs215210. DOI: 10.1242/jcs.215210.
Reviewer’s comment 3
In abstract section, line #21 please corrects the form of verb after sentence “subcellular localization”
Answer:
Thank you for your comments. As suggested, we revised the sentence as follows:
in line 21
“EV morphological features and subcellular localization were analyzed…..”
Reviewer’s comment 4
Terms like " edible plant; exosome and MicroRNAs" are already mentioned in tiltle. Authors may delete these and replace with more appropriate ones.
Answer:
Thank you for your comments. Do you recommend using abbreviations for these terms?
Reviewer’s comment 5
In line 161, please remove “in” after the words “and” in table 2
Answer:
Thank you for your comments. We revised the text as suggested (line 171).
Reviewer’s comment 6
In line 173, Please make sure the verb used after “which”
Answer:
Thank you for your comments. As suggested, we replaced “are” with “were” (line 175).
Reviewer’s comment 7
In line 74, please add the verb after word “ EVs”
Answer:
Thank you for your comments. We changed the text (line 82) from “Representative particle size distributions are shown for EVs “ to “Representative particle size distributions for EVs are as follows:"
Reviewer’s comment 8
In line 177, please add “the” before the word “Previous”
Answer:
Thank you for your comments. We added “the” before “previous” (line 187).
Reviewer’s comment 9
In line 188, please make sure the sentence is/are before “analogous”
Answer:
Thank you for your comments. We replaced “is” with “are” (line 190).
Reviewer’s comment 10
In line 190, Please add the “are” before the word “frequently”
Answer:
Thank you for your comments. We replaced “which are among the 26 most frequently observed miRNAs” with “which are frequently observed miRNAs” (line 202).
Thank you for your careful evaluation and kind comments.
Reviewer 4
The manuscript entitled “Edible Plant Juice Contains Exosome-like Nanovesicles that Harbor MicroRNAs and Are Taken up by Rat Enterocytes” an original research article is focussed on Plant miRNAs regulate the expression of certain mammalian gene and its biological functions.The exogenous plant miRNAs carried by EVs can be absorbed via the gastrointestinal tract was dealt in details. The manuscript is prepared in well mannered. Materials and Methods are described in details and in comprehensive way. The results are well elaborate. Though an Manuscript is well presented, may some minor mistake need to be addressed.
Answer:
We thank the reviewer for the valuable comments and suggestions. Our point-by-point responses to all the comments are presented below.
Reviewer’s comment 1
Title needs to be revised and made more intriguing to the readers
Answer:
Thank you for your suggestion. We changed the title as follows: line 1 - 2
“Uptake of microRNAs from Exosome-like Nanovesicles of Edible Plant Juice by Rat Enterocytes.”
Reviewer’s comment 2
I think introduction can be improved by exiting pool of knowledge through recent published papers.
Answer:
Thank you for your comments. As suggested, we added recent publications to the Introduction section as follows: line 50 - 57
" However, several lines of evidence suggest that plants also release EVs having various functions including symbiosis and defense against pathogens [13]. EVs from different eukaryotic and prokaryotic organisms are involved in many processes such as host-pathogen interactions, resistance transmission, and plant diseases, which may help in the prevention and treatment of pests and pathogens [14]. Interestingly, a growing body of evidence indicates that autophagy-related proteins are involved in exosome biogenesis, which indicates that there are pathways for the interaction between autophagy and EVs that may be important in the context of cancer and neurodegenerative diseases [15]."
In line 377 - 381
[13] Cui Y, Gao J, He Y, Jiang L. Plant extracellular vesicles. Protoplasma. 2020, 257, 3-12. DOI: 10.1007/s00709-019-01435-6.
[14] Woith E, Fuhrmann G, Melzig MF. Extracellular Vesicles-Connecting Kingdoms. Int J Mol Sci. 2019, 20, 5695. DOI: 10.3390/ijms20225695.
[15] Xu J, Camfield R, Gorski SM. The interplay between exosomes and autophagy - partners in crime. J Cell Sci. 2018, 131, jcs215210. DOI: 10.1242/jcs.215210.
Reviewer’s comment 3
In abstract section, line #21 please corrects the form of verb after sentence “subcellular localization”
Answer:
Thank you for your comments. As suggested, we revised the sentence as follows:
in line 21
“EV morphological features and subcellular localization were analyzed…..”
Reviewer’s comment 4
Terms like " edible plant; exosome and MicroRNAs" are already mentioned in tiltle. Authors may delete these and replace with more appropriate ones.
Answer:
Thank you for your comments. Do you recommend using abbreviations for these terms?
Reviewer’s comment 5
In line 161, please remove “in” after the words “and” in table 2
Answer:
Thank you for your comments. We revised the text as suggested (line 171).
Reviewer’s comment 6
In line 173, Please make sure the verb used after “which”
Answer:
Thank you for your comments. As suggested, we replaced “are” with “were” (line 175).
Reviewer’s comment 7
In line 74, please add the verb after word “ EVs”
Answer:
Thank you for your comments. We changed the text (line 82) from “Representative particle size distributions are shown for EVs “ to “Representative particle size distributions for EVs are as follows:"
Reviewer’s comment 8
In line 177, please add “the” before the word “Previous”
Answer:
Thank you for your comments. We added “the” before “previous” (line 187).
Reviewer’s comment 9
In line 188, please make sure the sentence is/are before “analogous”
Answer:
Thank you for your comments. We replaced “is” with “are” (line 190).
Reviewer’s comment 10
In line 190, Please add the “are” before the word “frequently”
Answer:
Thank you for your comments. We replaced “which are among the 26 most frequently observed miRNAs” with “which are frequently observed miRNAs” (line 202).
Thank you for your careful evaluation and kind comments.
Reviewer 4
The manuscript entitled “Edible Plant Juice Contains Exosome-like Nanovesicles that Harbor MicroRNAs and Are Taken up by Rat Enterocytes” an original research article is focussed on Plant miRNAs regulate the expression of certain mammalian gene and its biological functions.The exogenous plant miRNAs carried by EVs can be absorbed via the gastrointestinal tract was dealt in details. The manuscript is prepared in well mannered. Materials and Methods are described in details and in comprehensive way. The results are well elaborate. Though an Manuscript is well presented, may some minor mistake need to be addressed.
Answer:
We thank the reviewer for the valuable comments and suggestions. Our point-by-point responses to all the comments are presented below.
Reviewer’s comment 1
Title needs to be revised and made more intriguing to the readers
Answer:
Thank you for your suggestion. We changed the title as follows: line 1 - 2
“Uptake of microRNAs from Exosome-like Nanovesicles of Edible Plant Juice by Rat Enterocytes.”
Reviewer’s comment 2
I think introduction can be improved by exiting pool of knowledge through recent published papers.
Answer:
Thank you for your comments. As suggested, we added recent publications to the Introduction section as follows: line 50 - 57
" However, several lines of evidence suggest that plants also release EVs having various functions including symbiosis and defense against pathogens [13]. EVs from different eukaryotic and prokaryotic organisms are involved in many processes such as host-pathogen interactions, resistance transmission, and plant diseases, which may help in the prevention and treatment of pests and pathogens [14]. Interestingly, a growing body of evidence indicates that autophagy-related proteins are involved in exosome biogenesis, which indicates that there are pathways for the interaction between autophagy and EVs that may be important in the context of cancer and neurodegenerative diseases [15]."
In line 377 - 381
[13] Cui Y, Gao J, He Y, Jiang L. Plant extracellular vesicles. Protoplasma. 2020, 257, 3-12. DOI: 10.1007/s00709-019-01435-6.
[14] Woith E, Fuhrmann G, Melzig MF. Extracellular Vesicles-Connecting Kingdoms. Int J Mol Sci. 2019, 20, 5695. DOI: 10.3390/ijms20225695.
[15] Xu J, Camfield R, Gorski SM. The interplay between exosomes and autophagy - partners in crime. J Cell Sci. 2018, 131, jcs215210. DOI: 10.1242/jcs.215210.
Reviewer’s comment 3
In abstract section, line #21 please corrects the form of verb after sentence “subcellular localization”
Answer:
Thank you for your comments. As suggested, we revised the sentence as follows:
in line 21
“EV morphological features and subcellular localization were analyzed…..”
Reviewer’s comment 4
Terms like " edible plant; exosome and MicroRNAs" are already mentioned in tiltle. Authors may delete these and replace with more appropriate ones.
Answer:
Thank you for your comments. Do you recommend using abbreviations for these terms?
Reviewer’s comment 5
In line 161, please remove “in” after the words “and” in table 2
Answer:
Thank you for your comments. We revised the text as suggested (line 171).
Reviewer’s comment 6
In line 173, Please make sure the verb used after “which”
Answer:
Thank you for your comments. As suggested, we replaced “are” with “were” (line 175).
Reviewer’s comment 7
In line 74, please add the verb after word “ EVs”
Answer:
Thank you for your comments. We changed the text (line 82) from “Representative particle size distributions are shown for EVs “ to “Representative particle size distributions for EVs are as follows:"
Reviewer’s comment 8
In line 177, please add “the” before the word “Previous”
Answer:
Thank you for your comments. We added “the” before “previous” (line 187).
Reviewer’s comment 9
In line 188, please make sure the sentence is/are before “analogous”
Answer:
Thank you for your comments. We replaced “is” with “are” (line 190).
Reviewer’s comment 10
In line 190, Please add the “are” before the word “frequently”
Answer:
Thank you for your comments. We replaced “which are among the 26 most frequently observed miRNAs” with “which are frequently observed miRNAs” (line 202).
Thank you for your careful evaluation and kind comments.
Reviewer 4
The manuscript entitled “Edible Plant Juice Contains Exosome-like Nanovesicles that Harbor MicroRNAs and Are Taken up by Rat Enterocytes” an original research article is focussed on Plant miRNAs regulate the expression of certain mammalian gene and its biological functions.The exogenous plant miRNAs carried by EVs can be absorbed via the gastrointestinal tract was dealt in details. The manuscript is prepared in well mannered. Materials and Methods are described in details and in comprehensive way. The results are well elaborate. Though an Manuscript is well presented, may some minor mistake need to be addressed.
Answer:
We thank the reviewer for the valuable comments and suggestions. Our point-by-point responses to all the comments are presented below.
Reviewer’s comment 1
Title needs to be revised and made more intriguing to the readers
Answer:
Thank you for your suggestion. We changed the title as follows: line 1 - 2
“Uptake of microRNAs from Exosome-like Nanovesicles of Edible Plant Juice by Rat Enterocytes.”
Reviewer’s comment 2
I think introduction can be improved by exiting pool of knowledge through recent published papers.
Answer:
Thank you for your comments. As suggested, we added recent publications to the Introduction section as follows: line 50 - 57
" However, several lines of evidence suggest that plants also release EVs having various functions including symbiosis and defense against pathogens [13]. EVs from different eukaryotic and prokaryotic organisms are involved in many processes such as host-pathogen interactions, resistance transmission, and plant diseases, which may help in the prevention and treatment of pests and pathogens [14]. Interestingly, a growing body of evidence indicates that autophagy-related proteins are involved in exosome biogenesis, which indicates that there are pathways for the interaction between autophagy and EVs that may be important in the context of cancer and neurodegenerative diseases [15]."
In line 377 - 381
[13] Cui Y, Gao J, He Y, Jiang L. Plant extracellular vesicles. Protoplasma. 2020, 257, 3-12. DOI: 10.1007/s00709-019-01435-6.
[14] Woith E, Fuhrmann G, Melzig MF. Extracellular Vesicles-Connecting Kingdoms. Int J Mol Sci. 2019, 20, 5695. DOI: 10.3390/ijms20225695.
[15] Xu J, Camfield R, Gorski SM. The interplay between exosomes and autophagy - partners in crime. J Cell Sci. 2018, 131, jcs215210. DOI: 10.1242/jcs.215210.
Reviewer’s comment 3
In abstract section, line #21 please corrects the form of verb after sentence “subcellular localization”
Answer:
Thank you for your comments. As suggested, we revised the sentence as follows:
in line 21
“EV morphological features and subcellular localization were analyzed…..”
Reviewer’s comment 4
Terms like " edible plant; exosome and MicroRNAs" are already mentioned in tiltle. Authors may delete these and replace with more appropriate ones.
Answer:
Thank you for your comments. Do you recommend using abbreviations for these terms?
Reviewer’s comment 5
In line 161, please remove “in” after the words “and” in table 2
Answer:
Thank you for your comments. We revised the text as suggested (line 171).
Reviewer’s comment 6
In line 173, Please make sure the verb used after “which”
Answer:
Thank you for your comments. As suggested, we replaced “are” with “were” (line 175).
Reviewer’s comment 7
In line 74, please add the verb after word “ EVs”
Answer:
Thank you for your comments. We changed the text (line 82) from “Representative particle size distributions are shown for EVs “ to “Representative particle size distributions for EVs are as follows:"
Reviewer’s comment 8
In line 177, please add “the” before the word “Previous”
Answer:
Thank you for your comments. We added “the” before “previous” (line 187).
Reviewer’s comment 9
In line 188, please make sure the sentence is/are before “analogous”
Answer:
Thank you for your comments. We replaced “is” with “are” (line 190).
Reviewer’s comment 10
In line 190, Please add the “are” before the word “frequently”
Answer:
Thank you for your comments. We replaced “which are among the 26 most frequently observed miRNAs” with “which are frequently observed miRNAs” (line 202).
Thank you for your careful evaluation and kind comments.
Reviewer 4
The manuscript entitled “Edible Plant Juice Contains Exosome-like Nanovesicles that Harbor MicroRNAs and Are Taken up by Rat Enterocytes” an original research article is focussed on Plant miRNAs regulate the expression of certain mammalian gene and its biological functions.The exogenous plant miRNAs carried by EVs can be absorbed via the gastrointestinal tract was dealt in details. The manuscript is prepared in well mannered. Materials and Methods are described in details and in comprehensive way. The results are well elaborate. Though an Manuscript is well presented, may some minor mistake need to be addressed.
Answer:
We thank the reviewer for the valuable comments and suggestions. Our point-by-point responses to all the comments are presented below.
Reviewer’s comment 1
Title needs to be revised and made more intriguing to the readers
Answer:
Thank you for your suggestion. We changed the title as follows: line 1 - 2
“Uptake of microRNAs from Exosome-like Nanovesicles of Edible Plant Juice by Rat Enterocytes.”
Reviewer’s comment 2
I think introduction can be improved by exiting pool of knowledge through recent published papers.
Answer:
Thank you for your comments. As suggested, we added recent publications to the Introduction section as follows: line 50 - 57
" However, several lines of evidence suggest that plants also release EVs having various functions including symbiosis and defense against pathogens [13]. EVs from different eukaryotic and prokaryotic organisms are involved in many processes such as host-pathogen interactions, resistance transmission, and plant diseases, which may help in the prevention and treatment of pests and pathogens [14]. Interestingly, a growing body of evidence indicates that autophagy-related proteins are involved in exosome biogenesis, which indicates that there are pathways for the interaction between autophagy and EVs that may be important in the context of cancer and neurodegenerative diseases [15]."
In line 377 - 381
[13] Cui Y, Gao J, He Y, Jiang L. Plant extracellular vesicles. Protoplasma. 2020, 257, 3-12. DOI: 10.1007/s00709-019-01435-6.
[14] Woith E, Fuhrmann G, Melzig MF. Extracellular Vesicles-Connecting Kingdoms. Int J Mol Sci. 2019, 20, 5695. DOI: 10.3390/ijms20225695.
[15] Xu J, Camfield R, Gorski SM. The interplay between exosomes and autophagy - partners in crime. J Cell Sci. 2018, 131, jcs215210. DOI: 10.1242/jcs.215210.
Reviewer’s comment 3
In abstract section, line #21 please corrects the form of verb after sentence “subcellular localization”
Answer:
Thank you for your comments. As suggested, we revised the sentence as follows:
in line 21
“EV morphological features and subcellular localization were analyzed…..”
Reviewer’s comment 4
Terms like " edible plant; exosome and MicroRNAs" are already mentioned in tiltle. Authors may delete these and replace with more appropriate ones.
Answer:
Thank you for your comments. Do you recommend using abbreviations for these terms?
Reviewer’s comment 5
In line 161, please remove “in” after the words “and” in table 2
Answer:
Thank you for your comments. We revised the text as suggested (line 171).
Reviewer’s comment 6
In line 173, Please make sure the verb used after “which”
Answer:
Thank you for your comments. As suggested, we replaced “are” with “were” (line 175).
Reviewer’s comment 7
In line 74, please add the verb after word “ EVs”
Answer:
Thank you for your comments. We changed the text (line 82) from “Representative particle size distributions are shown for EVs “ to “Representative particle size distributions for EVs are as follows:"
Reviewer’s comment 8
In line 177, please add “the” before the word “Previous”
Answer:
Thank you for your comments. We added “the” before “previous” (line 187).
Reviewer’s comment 9
In line 188, please make sure the sentence is/are before “analogous”
Answer:
Thank you for your comments. We replaced “is” with “are” (line 190).
Reviewer’s comment 10
In line 190, Please add the “are” before the word “frequently”
Answer:
Thank you for your comments. We replaced “which are among the 26 most frequently observed miRNAs” with “which are frequently observed miRNAs” (line 202).
Thank you for your careful evaluation and kind comments.
Reviewer 4
The manuscript entitled “Edible Plant Juice Contains Exosome-like Nanovesicles that Harbor MicroRNAs and Are Taken up by Rat Enterocytes” an original research article is focussed on Plant miRNAs regulate the expression of certain mammalian gene and its biological functions.The exogenous plant miRNAs carried by EVs can be absorbed via the gastrointestinal tract was dealt in details. The manuscript is prepared in well mannered. Materials and Methods are described in details and in comprehensive way. The results are well elaborate. Though an Manuscript is well presented, may some minor mistake need to be addressed.
Answer:
We thank the reviewer for the valuable comments and suggestions. Our point-by-point responses to all the comments are presented below.
Reviewer’s comment 1
Title needs to be revised and made more intriguing to the readers
Answer:
Thank you for your suggestion. We changed the title as follows: line 1 - 2
“Uptake of microRNAs from Exosome-like Nanovesicles of Edible Plant Juice by Rat Enterocytes.”
Reviewer’s comment 2
I think introduction can be improved by exiting pool of knowledge through recent published papers.
Answer:
Thank you for your comments. As suggested, we added recent publications to the Introduction section as follows: line 50 - 57
" However, several lines of evidence suggest that plants also release EVs having various functions including symbiosis and defense against pathogens [13]. EVs from different eukaryotic and prokaryotic organisms are involved in many processes such as host-pathogen interactions, resistance transmission, and plant diseases, which may help in the prevention and treatment of pests and pathogens [14]. Interestingly, a growing body of evidence indicates that autophagy-related proteins are involved in exosome biogenesis, which indicates that there are pathways for the interaction between autophagy and EVs that may be important in the context of cancer and neurodegenerative diseases [15]."
In line 377 - 381
[13] Cui Y, Gao J, He Y, Jiang L. Plant extracellular vesicles. Protoplasma. 2020, 257, 3-12. DOI: 10.1007/s00709-019-01435-6.
[14] Woith E, Fuhrmann G, Melzig MF. Extracellular Vesicles-Connecting Kingdoms. Int J Mol Sci. 2019, 20, 5695. DOI: 10.3390/ijms20225695.
[15] Xu J, Camfield R, Gorski SM. The interplay between exosomes and autophagy - partners in crime. J Cell Sci. 2018, 131, jcs215210. DOI: 10.1242/jcs.215210.
Reviewer’s comment 3
In abstract section, line #21 please corrects the form of verb after sentence “subcellular localization”
Answer:
Thank you for your comments. As suggested, we revised the sentence as follows:
in line 21
“EV morphological features and subcellular localization were analyzed…..”
Reviewer’s comment 4
Terms like " edible plant; exosome and MicroRNAs" are already mentioned in tiltle. Authors may delete these and replace with more appropriate ones.
Answer:
Thank you for your comments. Do you recommend using abbreviations for these terms?
Reviewer’s comment 5
In line 161, please remove “in” after the words “and” in table 2
Answer:
Thank you for your comments. We revised the text as suggested (line 171).
Reviewer’s comment 6
In line 173, Please make sure the verb used after “which”
Answer:
Thank you for your comments. As suggested, we replaced “are” with “were” (line 175).
Reviewer’s comment 7
In line 74, please add the verb after word “ EVs”
Answer:
Thank you for your comments. We changed the text (line 82) from “Representative particle size distributions are shown for EVs “ to “Representative particle size distributions for EVs are as follows:"
Reviewer’s comment 8
In line 177, please add “the” before the word “Previous”
Answer:
Thank you for your comments. We added “the” before “previous” (line 187).
Reviewer’s comment 9
In line 188, please make sure the sentence is/are before “analogous”
Answer:
Thank you for your comments. We replaced “is” with “are” (line 190).
Reviewer’s comment 10
In line 190, Please add the “are” before the word “frequently”
Answer:
Thank you for your comments. We replaced “which are among the 26 most frequently observed miRNAs” with “which are frequently observed miRNAs” (line 202).
Thank you for your careful evaluation and kind comments.
Reviewer 4
The manuscript entitled “Edible Plant Juice Contains Exosome-like Nanovesicles that Harbor MicroRNAs and Are Taken up by Rat Enterocytes” an original research article is focussed on Plant miRNAs regulate the expression of certain mammalian gene and its biological functions.The exogenous plant miRNAs carried by EVs can be absorbed via the gastrointestinal tract was dealt in details. The manuscript is prepared in well mannered. Materials and Methods are described in details and in comprehensive way. The results are well elaborate. Though an Manuscript is well presented, may some minor mistake need to be addressed.
Answer:
We thank the reviewer for the valuable comments and suggestions. Our point-by-point responses to all the comments are presented below.
Reviewer’s comment 1
Title needs to be revised and made more intriguing to the readers
Answer:
Thank you for your suggestion. We changed the title as follows: line 1 - 2
“Uptake of microRNAs from Exosome-like Nanovesicles of Edible Plant Juice by Rat Enterocytes.”
Reviewer’s comment 2
I think introduction can be improved by exiting pool of knowledge through recent published papers.
Answer:
Thank you for your comments. As suggested, we added recent publications to the Introduction section as follows: line 50 - 57
" However, several lines of evidence suggest that plants also release EVs having various functions including symbiosis and defense against pathogens [13]. EVs from different eukaryotic and prokaryotic organisms are involved in many processes such as host-pathogen interactions, resistance transmission, and plant diseases, which may help in the prevention and treatment of pests and pathogens [14]. Interestingly, a growing body of evidence indicates that autophagy-related proteins are involved in exosome biogenesis, which indicates that there are pathways for the interaction between autophagy and EVs that may be important in the context of cancer and neurodegenerative diseases [15]."
In line 377 - 381
[13] Cui Y, Gao J, He Y, Jiang L. Plant extracellular vesicles. Protoplasma. 2020, 257, 3-12. DOI: 10.1007/s00709-019-01435-6.
[14] Woith E, Fuhrmann G, Melzig MF. Extracellular Vesicles-Connecting Kingdoms. Int J Mol Sci. 2019, 20, 5695. DOI: 10.3390/ijms20225695.
[15] Xu J, Camfield R, Gorski SM. The interplay between exosomes and autophagy - partners in crime. J Cell Sci. 2018, 131, jcs215210. DOI: 10.1242/jcs.215210.
Reviewer’s comment 3
In abstract section, line #21 please corrects the form of verb after sentence “subcellular localization”
Answer:
Thank you for your comments. As suggested, we revised the sentence as follows:
in line 21
“EV morphological features and subcellular localization were analyzed…..”
Reviewer’s comment 4
Terms like " edible plant; exosome and MicroRNAs" are already mentioned in tiltle. Authors may delete these and replace with more appropriate ones.
Answer:
Thank you for your comments. Do you recommend using abbreviations for these terms?
Reviewer’s comment 5
In line 161, please remove “in” after the words “and” in table 2
Answer:
Thank you for your comments. We revised the text as suggested (line 171).
Reviewer’s comment 6
In line 173, Please make sure the verb used after “which”
Answer:
Thank you for your comments. As suggested, we replaced “are” with “were” (line 175).
Reviewer’s comment 7
In line 74, please add the verb after word “ EVs”
Answer:
Thank you for your comments. We changed the text (line 82) from “Representative particle size distributions are shown for EVs “ to “Representative particle size distributions for EVs are as follows:"
Reviewer’s comment 8
In line 177, please add “the” before the word “Previous”
Answer:
Thank you for your comments. We added “the” before “previous” (line 187).
Reviewer’s comment 9
In line 188, please make sure the sentence is/are before “analogous”
Answer:
Thank you for your comments. We replaced “is” with “are” (line 190).
Reviewer’s comment 10
In line 190, Please add the “are” before the word “frequently”
Answer:
Thank you for your comments. We replaced “which are among the 26 most frequently observed miRNAs” with “which are frequently observed miRNAs” (line 202).
Thank you for your careful evaluation and kind comments.
Reviewer 4
The manuscript entitled “Edible Plant Juice Contains Exosome-like Nanovesicles that Harbor MicroRNAs and Are Taken up by Rat Enterocytes” an original research article is focussed on Plant miRNAs regulate the expression of certain mammalian gene and its biological functions.The exogenous plant miRNAs carried by EVs can be absorbed via the gastrointestinal tract was dealt in details. The manuscript is prepared in well mannered. Materials and Methods are described in details and in comprehensive way. The results are well elaborate. Though an Manuscript is well presented, may some minor mistake need to be addressed.
Answer:
We thank the reviewer for the valuable comments and suggestions. Our point-by-point responses to all the comments are presented below.
Reviewer’s comment 1
Title needs to be revised and made more intriguing to the readers
Answer:
Thank you for your suggestion. We changed the title as follows: line 1 - 2
“Uptake of microRNAs from Exosome-like Nanovesicles of Edible Plant Juice by Rat Enterocytes.”
Reviewer’s comment 2
I think introduction can be improved by exiting pool of knowledge through recent published papers.
Answer:
Thank you for your comments. As suggested, we added recent publications to the Introduction section as follows: line 50 - 57
" However, several lines of evidence suggest that plants also release EVs having various functions including symbiosis and defense against pathogens [13]. EVs from different eukaryotic and prokaryotic organisms are involved in many processes such as host-pathogen interactions, resistance transmission, and plant diseases, which may help in the prevention and treatment of pests and pathogens [14]. Interestingly, a growing body of evidence indicates that autophagy-related proteins are involved in exosome biogenesis, which indicates that there are pathways for the interaction between autophagy and EVs that may be important in the context of cancer and neurodegenerative diseases [15]."
In line 377 - 381
[13] Cui Y, Gao J, He Y, Jiang L. Plant extracellular vesicles. Protoplasma. 2020, 257, 3-12. DOI: 10.1007/s00709-019-01435-6.
[14] Woith E, Fuhrmann G, Melzig MF. Extracellular Vesicles-Connecting Kingdoms. Int J Mol Sci. 2019, 20, 5695. DOI: 10.3390/ijms20225695.
[15] Xu J, Camfield R, Gorski SM. The interplay between exosomes and autophagy - partners in crime. J Cell Sci. 2018, 131, jcs215210. DOI: 10.1242/jcs.215210.
Reviewer’s comment 3
In abstract section, line #21 please corrects the form of verb after sentence “subcellular localization”
Answer:
Thank you for your comments. As suggested, we revised the sentence as follows:
in line 21
“EV morphological features and subcellular localization were analyzed…..”
Reviewer’s comment 4
Terms like " edible plant; exosome and MicroRNAs" are already mentioned in tiltle. Authors may delete these and replace with more appropriate ones.
Answer:
Thank you for your comments. Do you recommend using abbreviations for these terms?
Reviewer’s comment 5
In line 161, please remove “in” after the words “and” in table 2
Answer:
Thank you for your comments. We revised the text as suggested (line 171).
Reviewer’s comment 6
In line 173, Please make sure the verb used after “which”
Answer:
Thank you for your comments. As suggested, we replaced “are” with “were” (line 175).
Reviewer’s comment 7
In line 74, please add the verb after word “ EVs”
Answer:
Thank you for your comments. We changed the text (line 82) from “Representative particle size distributions are shown for EVs “ to “Representative particle size distributions for EVs are as follows:"
Reviewer’s comment 8
In line 177, please add “the” before the word “Previous”
Answer:
Thank you for your comments. We added “the” before “previous” (line 187).
Reviewer’s comment 9
In line 188, please make sure the sentence is/are before “analogous”
Answer:
Thank you for your comments. We replaced “is” with “are” (line 190).
Reviewer’s comment 10
In line 190, Please add the “are” before the word “frequently”
Answer:
Thank you for your comments. We replaced “which are among the 26 most frequently observed miRNAs” with “which are frequently observed miRNAs” (line 202).
Thank you for your careful evaluation and kind comments.
Round 2
Reviewer 1 Report
The manuscript is now ok
Reviewer 2 Report
I have no additional comments in this manuscript.
