Reviewer's report
Title:Alkaline sphingomyelinase (NPP7) activity and isoforms in bile from
patients undergoing endoscopic retrograde cholangiopancreatography (ERCP)
Version:1Date:1 March 2014
Reviewer: Sergio Gradilone
Reviewer's report:
This group has a long track record on the study of NPP7, from discovery to
cloning to functional characterization. The present manuscript compares the
activity of NPP7 in bile of different cholangiopathies. The observation that NPP7
is decreased in cholangiocarcinoma has potentially an enormous significance. If
further confirmed, NPP7 expression in bile would be a plausible disease
biomarker with prognostic value. Even more, the suggestion that NPP7 products
could work as tumor suppressors could have therapeutic implications.
Nevertheless, since this descriptive work represents a small group of samples
(only 5 CCA), the authors need to expand both experimentally and analytically to
provide a stronger conclusion.
Major Revisions:
1- Since the authors have shown that NPP7 has several glycosylation sites and
its activity depends on them; is it possible that the decreased activity and
different molecular weight detected by western blots reflects a lack of
glycosylation as previously shown by this group?
2- Again, and somehow related to the previous comment, the western blot in
figure 4 need to be done and shown with molecular weight markers (clearly
labeled) and positive controls. For instance, as the authors previously published,
COS-7 cells expressing the 1.4 and 1.2 isoforms would be a good positive
control. Also the same cells treated with tunicamycin, in order to inhibit
glycosylation, would be other controls needed. Besides, would be interesting if
the authors can run the western with representative samples of the different
groups and identify them in the figure/figure legend.
3- Would be interesting to validate in vitro the observation that NPP7 is
decreased in CCA. Using normal and CCA cell lines, and perhaps HCC cell lines,
to address the expression of the isoforms by RT-PCR, the protein and activity
levels in cell lysates, and the protein and activity levels secreted (if any) to the
cell media, will support these observations.
Minor Revisions:
4- Figure 2 should also show the gallstone, PSC, and other groups to make the
comparison more visual.
5- Just for consistency, figure 5 x-axis should read “NPP7 activity” as used in the
previous figures and throughout the text.
Discretionary Revisions:
6- What are the potential mechanisms for the appearance of NPP7 in bile? Is it
cholangiocytes or hepatocytes derived? Is it dissociated from the apical
membrane by bile salts? Is it secreted in vesicles? Perhaps the in vitro
experiments suggested above may help to address this question.
7- Are NPP7-/- mice prone to GI tumor development?
8- To support some of the conclusions regarding the potential role of NPP7 as a
tumor suppressor, a simple in vitro experiment where the enzyme or its products
are added to CCA cell lines, and proliferation/apoptosis/colony formation
assessed, would be very helpful.
9- The staining of NPP7 by IHC in liver samples of the different disease groups,
or at least CCA and non-malignant controls, will be also interesting to see.
Level of interest: An article of importance in its field
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:Alkaline sphingomyelinase (NPP7) activity and isoforms in bile from
patients undergoing endoscopic retrograde cholangiopancreatography (ERCP)
Version:1Date:1 March 2014
Reviewer: Sergio Gradilone
Reviewer's report:
This group has a long track record on the study of NPP7, from discovery to
cloning to functional characterization. The present manuscript compares the
activity of NPP7 in bile of different cholangiopathies. The observation that NPP7
is decreased in cholangiocarcinoma has potentially an enormous significance. If
further confirmed, NPP7 expression in bile would be a plausible disease
biomarker with prognostic value. Even more, the suggestion that NPP7 products
could work as tumor suppressors could have therapeutic implications.
Nevertheless, since this descriptive work represents a small group of samples
(only 5 CCA), the authors need to expand both experimentally and analytically to
provide a stronger conclusion.
Major Revisions:
1- Since the authors have shown that NPP7 has several glycosylation sites and
its activity depends on them; is it possible that the decreased activity and
different molecular weight detected by western blots reflects a lack of
glycosylation as previously shown by this group?
2- Again, and somehow related to the previous comment, the western blot in
figure 4 need to be done and shown with molecular weight markers (clearly
labeled) and positive controls. For instance, as the authors previously published,
COS-7 cells expressing the 1.4 and 1.2 isoforms would be a good positive
control. Also the same cells treated with tunicamycin, in order to inhibit
glycosylation, would be other controls needed. Besides, would be interesting if
the authors can run the western with representative samples of the different
groups and identify them in the figure/figure legend.
3- Would be interesting to validate in vitro the observation that NPP7 is
decreased in CCA. Using normal and CCA cell lines, and perhaps HCC cell lines,
to address the expression of the isoforms by RT-PCR, the protein and activity
levels in cell lysates, and the protein and activity levels secreted (if any) to the
cell media, will support these observations.
Minor Revisions:
4- Figure 2 should also show the gallstone, PSC, and other groups to make the
comparison more visual.
5- Just for consistency, figure 5 x-axis should read “NPP7 activity” as used in the
previous figures and throughout the text.
Discretionary Revisions:
6- What are the potential mechanisms for the appearance of NPP7 in bile? Is it
cholangiocytes or hepatocytes derived? Is it dissociated from the apical
membrane by bile salts? Is it secreted in vesicles? Perhaps the in vitro
experiments suggested above may help to address this question.
7- Are NPP7-/- mice prone to GI tumor development?
8- To support some of the conclusions regarding the potential role of NPP7 as a
tumor suppressor, a simple in vitro experiment where the enzyme or its products
are added to CCA cell lines, and proliferation/apoptosis/colony formation
assessed, would be very helpful.
9- The staining of NPP7 by IHC in liver samples of the different disease groups,
or at least CCA and non-malignant controls, will be also interesting to see.
Level of interest: An article of importance in its field
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:Alkaline sphingomyelinase (NPP7) activity and isoforms in bile from
patients undergoing endoscopic retrograde cholangiopancreatography (ERCP)
Version:1Date:1 March 2014
Reviewer: Sergio Gradilone
Reviewer's report:
This group has a long track record on the study of NPP7, from discovery to
cloning to functional characterization. The present manuscript compares the
activity of NPP7 in bile of different cholangiopathies. The observation that NPP7
is decreased in cholangiocarcinoma has potentially an enormous significance. If
further confirmed, NPP7 expression in bile would be a plausible disease
biomarker with prognostic value. Even more, the suggestion that NPP7 products
could work as tumor suppressors could have therapeutic implications.
Nevertheless, since this descriptive work represents a small group of samples
(only 5 CCA), the authors need to expand both experimentally and analytically to
provide a stronger conclusion.
Major Revisions:
1- Since the authors have shown that NPP7 has several glycosylation sites and
its activity depends on them; is it possible that the decreased activity and
different molecular weight detected by western blots reflects a lack of
glycosylation as previously shown by this group?
2- Again, and somehow related to the previous comment, the western blot in
figure 4 need to be done and shown with molecular weight markers (clearly
labeled) and positive controls. For instance, as the authors previously published,
COS-7 cells expressing the 1.4 and 1.2 isoforms would be a good positive
control. Also the same cells treated with tunicamycin, in order to inhibit
glycosylation, would be other controls needed. Besides, would be interesting if
the authors can run the western with representative samples of the different
groups and identify them in the figure/figure legend.
3- Would be interesting to validate in vitro the observation that NPP7 is
decreased in CCA. Using normal and CCA cell lines, and perhaps HCC cell lines,
to address the expression of the isoforms by RT-PCR, the protein and activity
levels in cell lysates, and the protein and activity levels secreted (if any) to the
cell media, will support these observations.
Minor Revisions:
4- Figure 2 should also show the gallstone, PSC, and other groups to make the
comparison more visual.
5- Just for consistency, figure 5 x-axis should read “NPP7 activity” as used in the
previous figures and throughout the text.
Discretionary Revisions:
6- What are the potential mechanisms for the appearance of NPP7 in bile? Is it
cholangiocytes or hepatocytes derived? Is it dissociated from the apical
membrane by bile salts? Is it secreted in vesicles? Perhaps the in vitro
experiments suggested above may help to address this question.
7- Are NPP7-/- mice prone to GI tumor development?
8- To support some of the conclusions regarding the potential role of NPP7 as a
tumor suppressor, a simple in vitro experiment where the enzyme or its products
are added to CCA cell lines, and proliferation/apoptosis/colony formation
assessed, would be very helpful.
9- The staining of NPP7 by IHC in liver samples of the different disease groups,
or at least CCA and non-malignant controls, will be also interesting to see.
Level of interest: An article of importance in its field
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