Reviewer's report
Title:Small intestinal mucosa expression of putative chaperone fls485
Version: 2Date:5 December 2009
Reviewer: Michael George
Reviewer's report:
Major Compulsory Revisions:
1. The authors have attempted to address the lack of functional analysis in their
characterization of fls485 in the original manuscript by utilizing a Caco2 in vitro
intestinal epithelial model system. Unfortunately, simply showing a slight increase
in fls485 transcript levels between high and low density Caco2 cultures did not
provide any functional insights. This set of experiments could have been
designed better. The authors should assay expression of known biomarkers to
demonstrate differentiation since Caco2 is a well-established model system, and
attempted to determine if alterations in fls485 expression are associated with
changes in Caco2 maturation/differentiation characteristics.
2. In situ mRNA analyses (Figure 2A/B) suggests the fls485 transcript is
widespread but more concentrated in the crypt regions and, intracellularly,
accumulating towards the luminal region of the plasma membrane. On the other
hand, immunohistochemistry suggests fls485 protein is concentrated toward the
upper regions of the villus and not in the crypts. Furthermore, the protein signal is
not accumulated at the plasma membrane as the mRNA is. Because multiple
products are recognized in Western blots by the antibodies used and differential
staining patterns are observed, depending on the Ab clone and/or cell line used,
the localization studies of tissue sections are difficult to interpret and
inconclusive. The authors devote part of the Discussion to address this issue, yet
in the Discussion, they also state that “immunostainings of sectioned
paraffin-embedded normal human small intestinal mucosa with #7, #10 or
subclones revealed an essential overlap with fls485 mRNA in situ hybridization
pattern. The authors also state in the Discussion that “… colocalization of EGFP
and fls485 immunosignaling was found in EGFP-fls485158 3T3 transfectants”.
However, there was no “co-localization” data presented, only Western blots
confirming an EGFP-fls485158 chimeric protein was expressed. The inaccurate
statements should be revised in the Discussion and the discrepancy between
tissue localization of fls485 mRNA and protein really does need to be resolved.
The authors’ suggestion of laser capture microdissection could be very effective
in this regard.
Minor Essential Revisions:
1. The writing is difficult to read in several places and should be edited more
carefully.
Discretionary revisions:
1. It would be informative perhaps to see internal localization of the
EGFP-fls485158 chimeric protein and whether this may change as Caco2 cells
grow to confluence and differentiate.
Level of interest: An article of limited interest
Quality of written English: Needs some language corrections before being
published
Statistical review: Yes, but I do not feel adequately qualified to assess the
statistics.
Declaration of competing interests:
I declare that I have no competing interests
Reviewer's report
Title:Small intestinal mucosa expression of putative chaperone fls485
Version: 2Date:5 December 2009
Reviewer: Michael George
Reviewer's report:
Major Compulsory Revisions:
1. The authors have attempted to address the lack of functional analysis in their
characterization of fls485 in the original manuscript by utilizing a Caco2 in vitro
intestinal epithelial model system. Unfortunately, simply showing a slight increase
in fls485 transcript levels between high and low density Caco2 cultures did not
provide any functional insights. This set of experiments could have been
designed better. The authors should assay expression of known biomarkers to
demonstrate differentiation since Caco2 is a well-established model system, and
attempted to determine if alterations in fls485 expression are associated with
changes in Caco2 maturation/differentiation characteristics.
2. In situ mRNA analyses (Figure 2A/B) suggests the fls485 transcript is
widespread but more concentrated in the crypt regions and, intracellularly,
accumulating towards the luminal region of the plasma membrane. On the other
hand, immunohistochemistry suggests fls485 protein is concentrated toward the
upper regions of the villus and not in the crypts. Furthermore, the protein signal is
not accumulated at the plasma membrane as the mRNA is. Because multiple
products are recognized in Western blots by the antibodies used and differential
staining patterns are observed, depending on the Ab clone and/or cell line used,
the localization studies of tissue sections are difficult to interpret and
inconclusive. The authors devote part of the Discussion to address this issue, yet
in the Discussion, they also state that “immunostainings of sectioned
paraffin-embedded normal human small intestinal mucosa with #7, #10 or
subclones revealed an essential overlap with fls485 mRNA in situ hybridization
pattern. The authors also state in the Discussion that “… colocalization of EGFP
and fls485 immunosignaling was found in EGFP-fls485158 3T3 transfectants”.
However, there was no “co-localization” data presented, only Western blots
confirming an EGFP-fls485158 chimeric protein was expressed. The inaccurate
statements should be revised in the Discussion and the discrepancy between
tissue localization of fls485 mRNA and protein really does need to be resolved.
The authors’ suggestion of laser capture microdissection could be very effective
in this regard.
Minor Essential Revisions:
1. The writing is difficult to read in several places and should be edited more
carefully.
Discretionary revisions:
1. It would be informative perhaps to see internal localization of the
EGFP-fls485158 chimeric protein and whether this may change as Caco2 cells
grow to confluence and differentiate.
Level of interest: An article of limited interest
Quality of written English: Needs some language corrections before being
published
Statistical review: Yes, but I do not feel adequately qualified to assess the
statistics.
Declaration of competing interests:
I declare that I have no competing interests
Please review this paper.
user
Reviewer's report
Title:Small intestinal mucosa expression of putative chaperone fls485
Version: 2Date:5 December 2009
Reviewer: Michael George
Reviewer's report:
Major Compulsory Revisions:
1. The authors have attempted to address the lack of functional analysis in their
characterization of fls485 in the original manuscript by utilizing a Caco2 in vitro
intestinal epithelial model system. Unfortunately, simply showing a slight increase
in fls485 transcript levels between high and low density Caco2 cultures did not
provide any functional insights. This set of experiments could have been
designed better. The authors should assay expression of known biomarkers to
demonstrate differentiation since Caco2 is a well-established model system, and
attempted to determine if alterations in fls485 expression are associated with
changes in Caco2 maturation/differentiation characteristics.
2. In situ mRNA analyses (Figure 2A/B) suggests the fls485 transcript is
widespread but more concentrated in the crypt regions and, intracellularly,
accumulating towards the luminal region of the plasma membrane. On the other
hand, immunohistochemistry suggests fls485 protein is concentrated toward the
upper regions of the villus and not in the crypts. Furthermore, the protein signal is
not accumulated at the plasma membrane as the mRNA is. Because multiple
products are recognized in Western blots by the antibodies used and differential
staining patterns are observed, depending on the Ab clone and/or cell line used,
the localization studies of tissue sections are difficult to interpret and
inconclusive. The authors devote part of the Discussion to address this issue, yet
in the Discussion, they also state that “immunostainings of sectioned
paraffin-embedded normal human small intestinal mucosa with #7, #10 or
subclones revealed an essential overlap with fls485 mRNA in situ hybridization
pattern. The authors also state in the Discussion that “… colocalization of EGFP
and fls485 immunosignaling was found in EGFP-fls485158 3T3 transfectants”.
However, there was no “co-localization” data presented, only Western blots
confirming an EGFP-fls485158 chimeric protein was expressed. The inaccurate
statements should be revised in the Discussion and the discrepancy between
tissue localization of fls485 mRNA and protein really does need to be resolved.
The authors’ suggestion of laser capture microdissection could be very effective
in this regard.
Minor Essential Revisions:
1. The writing is difficult to read in several places and should be edited more
carefully.
Discretionary revisions:
1. It would be informative perhaps to see internal localization of the
EGFP-fls485158 chimeric protein and whether this may change as Caco2 cells
grow to confluence and differentiate.
Level of interest: An article of limited interest
Quality of written English: Needs some language corrections before being
published
Statistical review: Yes, but I do not feel adequately qualified to assess the
statistics.
Declaration of competing interests:
I declare that I have no competing interests
assistant