Title: Colonic TRPV4 overexpression is related to constipation severity
Version: 2 Date: 23 Sep 2022
Reviewer:  Suhasini Rajan
Reviewer's report:
This interesting manuscript takes a translational approach to link TRPV4 channel overexpression
to the pathology of constipation including cell culture studies, sequencing technology that
corroborate with the clinical findings. The manuscript is very well structured with a coherent
results section.
Major issues
1. Although functional assays were conducted for TRPV4 in Mihara et. al [22], a further
validation of the functionality of TRPV4, co-cultured with bacteria and an exposure to its
supernatants, should be considered.
2. The figures need to be rearranged to show as many important results in the main manuscript as
possible. Supplementary figures 2,3 and 4 which provide considerable support to the manuscript
concerning relevant pathways should be included in the main figures. Figure 3 is not clear with
a small font. It should be moved to supplementary figures. Instead of showing the pathways per
se, it will be interesting to see a table with the list of significantly altered pathways with highly
expressed genes.
3. Supplemental figure 3. Band intensity values with statistical significance should be added to
warrant the claim that there is overexpression of TRPV4 protein. TNF#R1 expression should
also be  investigated corroborating  microarray and RT-PCR results. The statistical analysis for
the same should be included in the methods section.
4. In figures 1 and 2, the controls are not satisfactory. A single control does not correspond to
the timeline of the experiment.
5. Since exposure to E. faecalis also increases TRPV4 expression in CCD841 cells (figure 1C),
it is only sensible to test the effect of supernatant versus bacterial components of E. faecalis as
in figure 2A.
Minor issues
Methods section
1. A short description of the preparation of  bacterial components (BC) and  supernatant (SN)
should be included in the methods section.
2. RT-PCR analysis was normalized to GAPDH or #-actin. There is no further justification for
why only #-actin was considered.
3. GEO accession number should be included in the methods section making it easy for the
readers.
4. The tool used to analyze microarray network with KEGG database is missing in the
manuscript.
Results section
1. Co-culture studies - The results from figure 1B needs to be restructured aiming for specific
description of the findings.
2. TRPV4 overexpression in constipated patients - Samples were acquired from 40 patients.
But the statistics are shown only for males. Although the absence of menopausal information is
mentioned, the results in women should also be included for more insight.
Figure legends
1. Figure 1 – Information about the days in which TRPV4 expression was measured is missing.
2. Figure 4 – E. coli supernatant is missing. There is no legend for p38I and p38I*10.
3. Figure S2 – TNF#R1 and R2 should be mentioned in the same way in both figure and in
it’s legend.
General issues
1. Maintain the same abbreviations throughout the manuscript. Eg. CSS sub 1 or CSS1.
2. Butyrate and butyric acid are used interchangeably. It is better to use one of the two
consistently.
Title: Colonic TRPV4 overexpression is related to constipation severity
Version: 2 Date: 23 Sep 2022
Reviewer:  Suhasini Rajan
Reviewer's report:
This interesting manuscript takes a translational approach to link TRPV4 channel overexpression
to the pathology of constipation including cell culture studies, sequencing technology that
corroborate with the clinical findings. The manuscript is very well structured with a coherent
results section.
Major issues
1. Although functional assays were conducted for TRPV4 in Mihara et. al [22], a further
validation of the functionality of TRPV4, co-cultured with bacteria and an exposure to its
supernatants, should be considered.
2. The figures need to be rearranged to show as many important results in the main manuscript as
possible. Supplementary figures 2,3 and 4 which provide considerable support to the manuscript
concerning relevant pathways should be included in the main figures. Figure 3 is not clear with
a small font. It should be moved to supplementary figures. Instead of showing the pathways per
se, it will be interesting to see a table with the list of significantly altered pathways with highly
expressed genes.
3. Supplemental figure 3. Band intensity values with statistical significance should be added to
warrant the claim that there is overexpression of TRPV4 protein. TNF#R1 expression should
also be  investigated corroborating  microarray and RT-PCR results. The statistical analysis for
the same should be included in the methods section.
4. In figures 1 and 2, the controls are not satisfactory. A single control does not correspond to
the timeline of the experiment.
5. Since exposure to E. faecalis also increases TRPV4 expression in CCD841 cells (figure 1C),
it is only sensible to test the effect of supernatant versus bacterial components of E. faecalis as
in figure 2A.
Minor issues
Methods section
1. A short description of the preparation of  bacterial components (BC) and  supernatant (SN)
should be included in the methods section.
2. RT-PCR analysis was normalized to GAPDH or #-actin. There is no further justification for
why only #-actin was considered.
3. GEO accession number should be included in the methods section making it easy for the
readers.
4. The tool used to analyze microarray network with KEGG database is missing in the
manuscript.
Results section
1. Co-culture studies - The results from figure 1B needs to be restructured aiming for specific
description of the findings.
2. TRPV4 overexpression in constipated patients - Samples were acquired from 40 patients.
But the statistics are shown only for males. Although the absence of menopausal information is
mentioned, the results in women should also be included for more insight.
Figure legends
1. Figure 1 – Information about the days in which TRPV4 expression was measured is missing.
2. Figure 4 – E. coli supernatant is missing. There is no legend for p38I and p38I*10.
3. Figure S2 – TNF#R1 and R2 should be mentioned in the same way in both figure and in
it’s legend.
General issues
1. Maintain the same abbreviations throughout the manuscript. Eg. CSS sub 1 or CSS1.
2. Butyrate and butyric acid are used interchangeably. It is better to use one of the two
consistently.
Please review this paper.
user
Title: Colonic TRPV4 overexpression is related to constipation severity
Version: 2 Date: 23 Sep 2022
Reviewer:  Suhasini Rajan
Reviewer's report:
This interesting manuscript takes a translational approach to link TRPV4 channel overexpression
to the pathology of constipation including cell culture studies, sequencing technology that
corroborate with the clinical findings. The manuscript is very well structured with a coherent
results section.
Major issues
1. Although functional assays were conducted for TRPV4 in Mihara et. al [22], a further
validation of the functionality of TRPV4, co-cultured with bacteria and an exposure to its
supernatants, should be considered.
2. The figures need to be rearranged to show as many important results in the main manuscript as
possible. Supplementary figures 2,3 and 4 which provide considerable support to the manuscript
concerning relevant pathways should be included in the main figures. Figure 3 is not clear with
a small font. It should be moved to supplementary figures. Instead of showing the pathways per
se, it will be interesting to see a table with the list of significantly altered pathways with highly
expressed genes.
3. Supplemental figure 3. Band intensity values with statistical significance should be added to
warrant the claim that there is overexpression of TRPV4 protein. TNF#R1 expression should
also be  investigated corroborating  microarray and RT-PCR results. The statistical analysis for
the same should be included in the methods section.
4. In figures 1 and 2, the controls are not satisfactory. A single control does not correspond to
the timeline of the experiment.
5. Since exposure to E. faecalis also increases TRPV4 expression in CCD841 cells (figure 1C),
it is only sensible to test the effect of supernatant versus bacterial components of E. faecalis as
in figure 2A.
Minor issues
Methods section
1. A short description of the preparation of  bacterial components (BC) and  supernatant (SN)
should be included in the methods section.
2. RT-PCR analysis was normalized to GAPDH or #-actin. There is no further justification for
why only #-actin was considered.
3. GEO accession number should be included in the methods section making it easy for the
readers.
4. The tool used to analyze microarray network with KEGG database is missing in the
manuscript.
Results section
1. Co-culture studies - The results from figure 1B needs to be restructured aiming for specific
description of the findings.
2. TRPV4 overexpression in constipated patients - Samples were acquired from 40 patients.
But the statistics are shown only for males. Although the absence of menopausal information is
mentioned, the results in women should also be included for more insight.
Figure legends
1. Figure 1 – Information about the days in which TRPV4 expression was measured is missing.
2. Figure 4 – E. coli supernatant is missing. There is no legend for p38I and p38I*10.
3. Figure S2 – TNF#R1 and R2 should be mentioned in the same way in both figure and in
it’s legend.
General issues
1. Maintain the same abbreviations throughout the manuscript. Eg. CSS sub 1 or CSS1.
2. Butyrate and butyric acid are used interchangeably. It is better to use one of the two
consistently.
assistant