Title: Colonic TRPV4 overexpression is related to constipation severity
Version: 2 Date: 24 Aug 2022
Reviewer's report:
Dr. Mihara and colleagues have examined regulatory mechanisms of TRPV4 expression by
co-culturing colon epithelial cell lines with intestinal bacteria and their derivatives. The topic
is interesting especially with regard to the relationship between chronic constipation and TRP
channels. This is an interesting area of research however I have a number of concerns as
described below.
1, The authors investigated the effects of intestinal bacteria at various time points. However,
the time pints is unclear in Fig.1A, C, Fig.2B, C, D, and Fig.4. Add each time points in results
and figure legends.
2, “In this study, we evaluated 12 representative clinical isolates of enterobacteria, followed by
10 standard isolates of enterobacteria that are known to alter TRPV4 expression (Table S1).
We did not intentionally select enteric bacteria that have been suggested to be associated with
constipation.”
The authors  should show the reason "not intentionally select enteric bacteria" because this study
focuse on the choronic constipation.
3, In supplementary Fig.1, the authors investigated only three bacteria in methylation. I think it
is better to investigate other bacteria such as E.coli. Furthermore, data of TRPV4 methylation
is important. This data should be shown as Fig.3.
4, Although, the authors discussed the effects of bacterial metabolites (Fig.2A and 4) and the
effects of TNF-alpha (Fig2C and 4), these results are complicated. Therefore, the authors should
add more information to discuss these points.
Title: Colonic TRPV4 overexpression is related to constipation severity
Version: 2 Date: 24 Aug 2022
Reviewer's report:
Dr. Mihara and colleagues have examined regulatory mechanisms of TRPV4 expression by
co-culturing colon epithelial cell lines with intestinal bacteria and their derivatives. The topic
is interesting especially with regard to the relationship between chronic constipation and TRP
channels. This is an interesting area of research however I have a number of concerns as
described below.
1, The authors investigated the effects of intestinal bacteria at various time points. However,
the time pints is unclear in Fig.1A, C, Fig.2B, C, D, and Fig.4. Add each time points in results
and figure legends.
2, “In this study, we evaluated 12 representative clinical isolates of enterobacteria, followed by
10 standard isolates of enterobacteria that are known to alter TRPV4 expression (Table S1).
We did not intentionally select enteric bacteria that have been suggested to be associated with
constipation.”
The authors  should show the reason "not intentionally select enteric bacteria" because this study
focuse on the choronic constipation.
3, In supplementary Fig.1, the authors investigated only three bacteria in methylation. I think it
is better to investigate other bacteria such as E.coli. Furthermore, data of TRPV4 methylation
is important. This data should be shown as Fig.3.
4, Although, the authors discussed the effects of bacterial metabolites (Fig.2A and 4) and the
effects of TNF-alpha (Fig2C and 4), these results are complicated. Therefore, the authors should
add more information to discuss these points.
Please review this paper.
user
Title: Colonic TRPV4 overexpression is related to constipation severity
Version: 2 Date: 24 Aug 2022
Reviewer's report:
Dr. Mihara and colleagues have examined regulatory mechanisms of TRPV4 expression by
co-culturing colon epithelial cell lines with intestinal bacteria and their derivatives. The topic
is interesting especially with regard to the relationship between chronic constipation and TRP
channels. This is an interesting area of research however I have a number of concerns as
described below.
1, The authors investigated the effects of intestinal bacteria at various time points. However,
the time pints is unclear in Fig.1A, C, Fig.2B, C, D, and Fig.4. Add each time points in results
and figure legends.
2, “In this study, we evaluated 12 representative clinical isolates of enterobacteria, followed by
10 standard isolates of enterobacteria that are known to alter TRPV4 expression (Table S1).
We did not intentionally select enteric bacteria that have been suggested to be associated with
constipation.”
The authors  should show the reason "not intentionally select enteric bacteria" because this study
focuse on the choronic constipation.
3, In supplementary Fig.1, the authors investigated only three bacteria in methylation. I think it
is better to investigate other bacteria such as E.coli. Furthermore, data of TRPV4 methylation
is important. This data should be shown as Fig.3.
4, Although, the authors discussed the effects of bacterial metabolites (Fig.2A and 4) and the
effects of TNF-alpha (Fig2C and 4), these results are complicated. Therefore, the authors should
add more information to discuss these points.
assistant