Reviewer's report
Title:Transporter function and cyclic AMP turnover in normal colonic mucosa
from patients with and without colorectal neoplasia
Version: 1Date:27 February 2012
Reviewer: J.J. Eloranta
Reviewer's report:
In this manuscript the authors Kleberg et al have investigated the colonic
transport of cyclic nucleotides using electrophysiological measurements in
mini-Ussing air suction chamber set-up, using human colonic biopsy tissue. They
show that colorectal neoplasia seems interestingly to be associated with lower
transport of or sensitivity to the cyclic nucleotide dibuturyl-cAMP. In the second
part, the authors study the expression of selectecd ABC and OATP transporters
in colonic tissue material by real-time PCR and immunohistochemistry.
Major compulsory revisions:
1. The range of OATP transporters, the expression levels of which in CRN vs.
non-CRN tissue have been studied is not comprehensive enough. Based on the
past study by Hilgendorf et al (DMD 2007; 35:1333-1340), in which the
expression of eight SLCO genes in human colon tissue was studied, the authors
have selected OATP1C1, OATP2B1, OATP4A1, and OATP4C1 to be measured.
Please see the recent review by Obaidat et al. (Annu. Rev. Pharmacol. Toxicol.
2012; 52:135-151) for further members of the SLCO gene superfamily that would
be of interest to study in colorectal neoplasia, as well as relevant literature
references therein. For example:
• OATP2A1 has been detected in the colon.
• Normally liver-specific OATP1B1 and OATP1B3 have been found expressed in
colon cancer cell lines and colon cancer tissue.
• OATP3A1 has been detected in colon adenocarcinoma cell lines.
In fact, the best approach would be to include all 11 human OATPs in the study.
2. The authors have not sufficiently described the method of calculating relative
expression levels in real-time PCR experiments. Have they used the comparative
threshold cycle method?
Discretionary revisions:
1. It is regrettable that the authors have used the non-specifically intercalating
dye SYBR Green in real-time PCR experiments, instead of the TaqMan probe
technology, superior in its specificity. At least they have performed the
dissociation curve analysis to control this - perhaps these data could be provided
to the reviewers?
Minor essential revisions:
1. Page 8, line six from bottom: “dictoystelium” should be “Dictyostelium”.
2. Page 10, bottom line: “OAT2B1” should be “OATP2B1”.
3. Methods: The exact details of the antibodies used for immunohistochemistry
should be provided (ie. catalogue numbers, final concentrations used etc.).
Level of interest: An article whose findings are important to those with closely
related research interests
Quality of written English: Acceptable
Statistical review: No, the manuscript does not need to be seen by a
statistician.
Declaration of competing interests:
I declare that I have no competing interests.
Reviewer's report
Title:Transporter function and cyclic AMP turnover in normal colonic mucosa
from patients with and without colorectal neoplasia
Version: 1Date:27 February 2012
Reviewer: J.J. Eloranta
Reviewer's report:
In this manuscript the authors Kleberg et al have investigated the colonic
transport of cyclic nucleotides using electrophysiological measurements in
mini-Ussing air suction chamber set-up, using human colonic biopsy tissue. They
show that colorectal neoplasia seems interestingly to be associated with lower
transport of or sensitivity to the cyclic nucleotide dibuturyl-cAMP. In the second
part, the authors study the expression of selectecd ABC and OATP transporters
in colonic tissue material by real-time PCR and immunohistochemistry.
Major compulsory revisions:
1. The range of OATP transporters, the expression levels of which in CRN vs.
non-CRN tissue have been studied is not comprehensive enough. Based on the
past study by Hilgendorf et al (DMD 2007; 35:1333-1340), in which the
expression of eight SLCO genes in human colon tissue was studied, the authors
have selected OATP1C1, OATP2B1, OATP4A1, and OATP4C1 to be measured.
Please see the recent review by Obaidat et al. (Annu. Rev. Pharmacol. Toxicol.
2012; 52:135-151) for further members of the SLCO gene superfamily that would
be of interest to study in colorectal neoplasia, as well as relevant literature
references therein. For example:
• OATP2A1 has been detected in the colon.
• Normally liver-specific OATP1B1 and OATP1B3 have been found expressed in
colon cancer cell lines and colon cancer tissue.
• OATP3A1 has been detected in colon adenocarcinoma cell lines.
In fact, the best approach would be to include all 11 human OATPs in the study.
2. The authors have not sufficiently described the method of calculating relative
expression levels in real-time PCR experiments. Have they used the comparative
threshold cycle method?
Discretionary revisions:
1. It is regrettable that the authors have used the non-specifically intercalating
dye SYBR Green in real-time PCR experiments, instead of the TaqMan probe
technology, superior in its specificity. At least they have performed the
dissociation curve analysis to control this - perhaps these data could be provided
to the reviewers?
Minor essential revisions:
1. Page 8, line six from bottom: “dictoystelium” should be “Dictyostelium”.
2. Page 10, bottom line: “OAT2B1” should be “OATP2B1”.
3. Methods: The exact details of the antibodies used for immunohistochemistry
should be provided (ie. catalogue numbers, final concentrations used etc.).
Level of interest: An article whose findings are important to those with closely
related research interests
Quality of written English: Acceptable
Statistical review: No, the manuscript does not need to be seen by a
statistician.
Declaration of competing interests:
I declare that I have no competing interests.
Please review this paper.
user
Reviewer's report
Title:Transporter function and cyclic AMP turnover in normal colonic mucosa
from patients with and without colorectal neoplasia
Version: 1Date:27 February 2012
Reviewer: J.J. Eloranta
Reviewer's report:
In this manuscript the authors Kleberg et al have investigated the colonic
transport of cyclic nucleotides using electrophysiological measurements in
mini-Ussing air suction chamber set-up, using human colonic biopsy tissue. They
show that colorectal neoplasia seems interestingly to be associated with lower
transport of or sensitivity to the cyclic nucleotide dibuturyl-cAMP. In the second
part, the authors study the expression of selectecd ABC and OATP transporters
in colonic tissue material by real-time PCR and immunohistochemistry.
Major compulsory revisions:
1. The range of OATP transporters, the expression levels of which in CRN vs.
non-CRN tissue have been studied is not comprehensive enough. Based on the
past study by Hilgendorf et al (DMD 2007; 35:1333-1340), in which the
expression of eight SLCO genes in human colon tissue was studied, the authors
have selected OATP1C1, OATP2B1, OATP4A1, and OATP4C1 to be measured.
Please see the recent review by Obaidat et al. (Annu. Rev. Pharmacol. Toxicol.
2012; 52:135-151) for further members of the SLCO gene superfamily that would
be of interest to study in colorectal neoplasia, as well as relevant literature
references therein. For example:
• OATP2A1 has been detected in the colon.
• Normally liver-specific OATP1B1 and OATP1B3 have been found expressed in
colon cancer cell lines and colon cancer tissue.
• OATP3A1 has been detected in colon adenocarcinoma cell lines.
In fact, the best approach would be to include all 11 human OATPs in the study.
2. The authors have not sufficiently described the method of calculating relative
expression levels in real-time PCR experiments. Have they used the comparative
threshold cycle method?
Discretionary revisions:
1. It is regrettable that the authors have used the non-specifically intercalating
dye SYBR Green in real-time PCR experiments, instead of the TaqMan probe
technology, superior in its specificity. At least they have performed the
dissociation curve analysis to control this - perhaps these data could be provided
to the reviewers?
Minor essential revisions:
1. Page 8, line six from bottom: “dictoystelium” should be “Dictyostelium”.
2. Page 10, bottom line: “OAT2B1” should be “OATP2B1”.
3. Methods: The exact details of the antibodies used for immunohistochemistry
should be provided (ie. catalogue numbers, final concentrations used etc.).
Level of interest: An article whose findings are important to those with closely
related research interests
Quality of written English: Acceptable
Statistical review: No, the manuscript does not need to be seen by a
statistician.
Declaration of competing interests:
I declare that I have no competing interests.
assistant