Title: Plasma and fecal zonulin are not altered by a high green leafy vegetable dietary
intervention: secondary analysis of a randomized control crossover trial.
Version: 2 Date: 14 Nov 2021
Reviewer's report:
With interest, I read the paper by Riviere et al. describing the trajectory of zonulin, inflammatory
markers, and gut microbiota in a subset of 20 participants from a randomized crossover study
in which participants were directed to consume green leafy vegetable consumption or placebo
for four weeks each (with a four week washout period between them).
While some of the results reported in the manuscript are interesting, there are some substantial
concerns and gaps that should be addressed thoroughly before further consideration could be
made:
1. The main problem of the paper is that it lacks a scientific focus.  What is the primary outcome
of this secondary analysis, and what is the STORY that the authors would like to tell??  In
this reviewer's view, the paper should focus on plasma and fecal zonulin trajectory and their
associations with other biomarkers, dietary reporting, and inflammatory state.  Comparison
between sexes is optional based on direct comparisons and not stratification according to sex. If
there is a difference between the sexes, this could be part of the report's central message.
2. How were the participants who were included in this analysis chosen?  Completely randomly?
Was randomization stratified by sex? (since an equal number of men and women are included).
3. Statistical analysis - the use of the Bonferroni method to address multiple comparisons is
appropriate.  Yet, the authors chose to perform T-tests to compare groups with a sample size of
10-20.  The proper way to reach such small groups is by using non-parametric tests.
4. Differences in dietary reportings after the diet should be reported in supplementary material
and not in the main text.
5. The figures are too heavy on the eyes, and all present correlation matrixes. It would be better
to present color figures of heat maps without numbers but with a legend that states the number
range and direction according to color and color strength.
6. How do the authors explain the elevation in inflammatory markers under the LGV diet? Was
this also observed in the main trial?
7. Please consider performing partial-linear correlations to adjust for difference confounders to
assess the relationship between zonulin as a marker of intestinal integrity and other biomarkers.
8. Please remove "further research is warranted" from the abstract's conclusions.
9. Figures 3 and 4 are very general - how do they relate to the hypothesis or aims of the paper??
It is better to thoroughly cover a specific mechanism/biomarker/phenomenon than to describe
a very general concept
10. How were the biomarkers included chosen?  Are other biomarkers relevant and available?
(for example, insulin resistance markers)
Title: Plasma and fecal zonulin are not altered by a high green leafy vegetable dietary
intervention: secondary analysis of a randomized control crossover trial.
Version: 2 Date: 14 Nov 2021
Reviewer's report:
With interest, I read the paper by Riviere et al. describing the trajectory of zonulin, inflammatory
markers, and gut microbiota in a subset of 20 participants from a randomized crossover study
in which participants were directed to consume green leafy vegetable consumption or placebo
for four weeks each (with a four week washout period between them).
While some of the results reported in the manuscript are interesting, there are some substantial
concerns and gaps that should be addressed thoroughly before further consideration could be
made:
1. The main problem of the paper is that it lacks a scientific focus.  What is the primary outcome
of this secondary analysis, and what is the STORY that the authors would like to tell??  In
this reviewer's view, the paper should focus on plasma and fecal zonulin trajectory and their
associations with other biomarkers, dietary reporting, and inflammatory state.  Comparison
between sexes is optional based on direct comparisons and not stratification according to sex. If
there is a difference between the sexes, this could be part of the report's central message.
2. How were the participants who were included in this analysis chosen?  Completely randomly?
Was randomization stratified by sex? (since an equal number of men and women are included).
3. Statistical analysis - the use of the Bonferroni method to address multiple comparisons is
appropriate.  Yet, the authors chose to perform T-tests to compare groups with a sample size of
10-20.  The proper way to reach such small groups is by using non-parametric tests.
4. Differences in dietary reportings after the diet should be reported in supplementary material
and not in the main text.
5. The figures are too heavy on the eyes, and all present correlation matrixes. It would be better
to present color figures of heat maps without numbers but with a legend that states the number
range and direction according to color and color strength.
6. How do the authors explain the elevation in inflammatory markers under the LGV diet? Was
this also observed in the main trial?
7. Please consider performing partial-linear correlations to adjust for difference confounders to
assess the relationship between zonulin as a marker of intestinal integrity and other biomarkers.
8. Please remove "further research is warranted" from the abstract's conclusions.
9. Figures 3 and 4 are very general - how do they relate to the hypothesis or aims of the paper??
It is better to thoroughly cover a specific mechanism/biomarker/phenomenon than to describe
a very general concept
10. How were the biomarkers included chosen?  Are other biomarkers relevant and available?
(for example, insulin resistance markers)
Please review this paper.
user
Title: Plasma and fecal zonulin are not altered by a high green leafy vegetable dietary
intervention: secondary analysis of a randomized control crossover trial.
Version: 2 Date: 14 Nov 2021
Reviewer's report:
With interest, I read the paper by Riviere et al. describing the trajectory of zonulin, inflammatory
markers, and gut microbiota in a subset of 20 participants from a randomized crossover study
in which participants were directed to consume green leafy vegetable consumption or placebo
for four weeks each (with a four week washout period between them).
While some of the results reported in the manuscript are interesting, there are some substantial
concerns and gaps that should be addressed thoroughly before further consideration could be
made:
1. The main problem of the paper is that it lacks a scientific focus.  What is the primary outcome
of this secondary analysis, and what is the STORY that the authors would like to tell??  In
this reviewer's view, the paper should focus on plasma and fecal zonulin trajectory and their
associations with other biomarkers, dietary reporting, and inflammatory state.  Comparison
between sexes is optional based on direct comparisons and not stratification according to sex. If
there is a difference between the sexes, this could be part of the report's central message.
2. How were the participants who were included in this analysis chosen?  Completely randomly?
Was randomization stratified by sex? (since an equal number of men and women are included).
3. Statistical analysis - the use of the Bonferroni method to address multiple comparisons is
appropriate.  Yet, the authors chose to perform T-tests to compare groups with a sample size of
10-20.  The proper way to reach such small groups is by using non-parametric tests.
4. Differences in dietary reportings after the diet should be reported in supplementary material
and not in the main text.
5. The figures are too heavy on the eyes, and all present correlation matrixes. It would be better
to present color figures of heat maps without numbers but with a legend that states the number
range and direction according to color and color strength.
6. How do the authors explain the elevation in inflammatory markers under the LGV diet? Was
this also observed in the main trial?
7. Please consider performing partial-linear correlations to adjust for difference confounders to
assess the relationship between zonulin as a marker of intestinal integrity and other biomarkers.
8. Please remove "further research is warranted" from the abstract's conclusions.
9. Figures 3 and 4 are very general - how do they relate to the hypothesis or aims of the paper??
It is better to thoroughly cover a specific mechanism/biomarker/phenomenon than to describe
a very general concept
10. How were the biomarkers included chosen?  Are other biomarkers relevant and available?
(for example, insulin resistance markers)
assistant