Reviewer's report
Title:Hyperammonia Induces Specific Liver Injury through an Intrinsic
Ca2+-independent Apoptosis Pathway
Version:2Date:29 April 2014
Reviewer: Jason Coombes
Reviewer's report:
1. Please describe the rationale for using 20 mM dose NH4Cl in the early figures.
2. Fig1. Labelling of Control and 20 mM bars in AST and ALT figures are
inconsistent and confusing. Please make them consistent. Description of ALT as
being “in a steady decline within 48 h” is not convincing. Ammonia is slightly
increased vs control at 12 h, more increased at 24 h, and relatively decreased at
48 h. Furthermore, at 48 h the difference is small and unlikely to be significant.
There is no evidence of any trends in AST and ALT based on this 48 h time
period. Additionally, presenting a dose reponse curve here may be informative.
3. Fig 2a I can’t understand the description of apoptosis provided here. Apoptosis
in primary hepatocytes appears to be increased more like 800% than the 25.97%
described. This is an error in description. It looks to be approximately 3% vs
25.97% in the graph, which does not represent a 25.97% increase. I am very
confused by this. Also, data for all cell lines is not described. In the figure legend
please briefly describe the analysis and indicate clearly which is the apoptotic
population measured.
4. Figure 3. I don’t understand what data is presented here. The Panel labelled
“figure 3” is full of FACS plots and two graphs, however it is described in the text
and in the figure legend as relating to MTT assays.
5. What does “inhibition of cells” on the y-axis of the graphs mean? What units
are this? Is it a measurement of OD, a percentage? This is very unclear.
6. The chelating drug BAPTA is used, however no justification or reasons for
selecting this drug is provided. Please provide a sensible and thoughtful
description, and also comment more on its mechanism of action.
7. Apoptosis data – mRNA is presented in panel labelled Fig5 however it is
described as being in fig 4. All of these figures are mis-labelled and as such it is
very difficult to interpret this paper. The dosages used here are not consistent
with the dosages used in previous figures. Caspase 3 mRNA and protein are not
themselves indicative of apoptosis, and a cleaved caspase detection method
should be used.
8. Cell viability, apoptosis, and mitochondrial data are presented in a strange
manner, not in any particular order. Results in figures are not presented in the
same order they are described in the text. Additionally, the order they appear in
the text is not logical and very difficult to follow.
9. Mitochondrial data – no rationale or logical reasoning is provided for
investigating mitochondria. The results are simply presented without any logical
introduction or reasons.
Summary: This paper is presented in a very strange manner. The figures are not
labelled consistently with the text, so it is almost impossible to follow. There is no
logical reasoning described for the experiments and it is not easy to understand
any logical flow.
Level of interest: An article whose findings are important to those with closely
related research interests
Quality of written English: Not suitable for publication unless extensively edited
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:Hyperammonia Induces Specific Liver Injury through an Intrinsic
Ca2+-independent Apoptosis Pathway
Version:2Date:29 April 2014
Reviewer: Jason Coombes
Reviewer's report:
1. Please describe the rationale for using 20 mM dose NH4Cl in the early figures.
2. Fig1. Labelling of Control and 20 mM bars in AST and ALT figures are
inconsistent and confusing. Please make them consistent. Description of ALT as
being “in a steady decline within 48 h” is not convincing. Ammonia is slightly
increased vs control at 12 h, more increased at 24 h, and relatively decreased at
48 h. Furthermore, at 48 h the difference is small and unlikely to be significant.
There is no evidence of any trends in AST and ALT based on this 48 h time
period. Additionally, presenting a dose reponse curve here may be informative.
3. Fig 2a I can’t understand the description of apoptosis provided here. Apoptosis
in primary hepatocytes appears to be increased more like 800% than the 25.97%
described. This is an error in description. It looks to be approximately 3% vs
25.97% in the graph, which does not represent a 25.97% increase. I am very
confused by this. Also, data for all cell lines is not described. In the figure legend
please briefly describe the analysis and indicate clearly which is the apoptotic
population measured.
4. Figure 3. I don’t understand what data is presented here. The Panel labelled
“figure 3” is full of FACS plots and two graphs, however it is described in the text
and in the figure legend as relating to MTT assays.
5. What does “inhibition of cells” on the y-axis of the graphs mean? What units
are this? Is it a measurement of OD, a percentage? This is very unclear.
6. The chelating drug BAPTA is used, however no justification or reasons for
selecting this drug is provided. Please provide a sensible and thoughtful
description, and also comment more on its mechanism of action.
7. Apoptosis data – mRNA is presented in panel labelled Fig5 however it is
described as being in fig 4. All of these figures are mis-labelled and as such it is
very difficult to interpret this paper. The dosages used here are not consistent
with the dosages used in previous figures. Caspase 3 mRNA and protein are not
themselves indicative of apoptosis, and a cleaved caspase detection method
should be used.
8. Cell viability, apoptosis, and mitochondrial data are presented in a strange
manner, not in any particular order. Results in figures are not presented in the
same order they are described in the text. Additionally, the order they appear in
the text is not logical and very difficult to follow.
9. Mitochondrial data – no rationale or logical reasoning is provided for
investigating mitochondria. The results are simply presented without any logical
introduction or reasons.
Summary: This paper is presented in a very strange manner. The figures are not
labelled consistently with the text, so it is almost impossible to follow. There is no
logical reasoning described for the experiments and it is not easy to understand
any logical flow.
Level of interest: An article whose findings are important to those with closely
related research interests
Quality of written English: Not suitable for publication unless extensively edited
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:Hyperammonia Induces Specific Liver Injury through an Intrinsic
Ca2+-independent Apoptosis Pathway
Version:2Date:29 April 2014
Reviewer: Jason Coombes
Reviewer's report:
1. Please describe the rationale for using 20 mM dose NH4Cl in the early figures.
2. Fig1. Labelling of Control and 20 mM bars in AST and ALT figures are
inconsistent and confusing. Please make them consistent. Description of ALT as
being “in a steady decline within 48 h” is not convincing. Ammonia is slightly
increased vs control at 12 h, more increased at 24 h, and relatively decreased at
48 h. Furthermore, at 48 h the difference is small and unlikely to be significant.
There is no evidence of any trends in AST and ALT based on this 48 h time
period. Additionally, presenting a dose reponse curve here may be informative.
3. Fig 2a I can’t understand the description of apoptosis provided here. Apoptosis
in primary hepatocytes appears to be increased more like 800% than the 25.97%
described. This is an error in description. It looks to be approximately 3% vs
25.97% in the graph, which does not represent a 25.97% increase. I am very
confused by this. Also, data for all cell lines is not described. In the figure legend
please briefly describe the analysis and indicate clearly which is the apoptotic
population measured.
4. Figure 3. I don’t understand what data is presented here. The Panel labelled
“figure 3” is full of FACS plots and two graphs, however it is described in the text
and in the figure legend as relating to MTT assays.
5. What does “inhibition of cells” on the y-axis of the graphs mean? What units
are this? Is it a measurement of OD, a percentage? This is very unclear.
6. The chelating drug BAPTA is used, however no justification or reasons for
selecting this drug is provided. Please provide a sensible and thoughtful
description, and also comment more on its mechanism of action.
7. Apoptosis data – mRNA is presented in panel labelled Fig5 however it is
described as being in fig 4. All of these figures are mis-labelled and as such it is
very difficult to interpret this paper. The dosages used here are not consistent
with the dosages used in previous figures. Caspase 3 mRNA and protein are not
themselves indicative of apoptosis, and a cleaved caspase detection method
should be used.
8. Cell viability, apoptosis, and mitochondrial data are presented in a strange
manner, not in any particular order. Results in figures are not presented in the
same order they are described in the text. Additionally, the order they appear in
the text is not logical and very difficult to follow.
9. Mitochondrial data – no rationale or logical reasoning is provided for
investigating mitochondria. The results are simply presented without any logical
introduction or reasons.
Summary: This paper is presented in a very strange manner. The figures are not
labelled consistently with the text, so it is almost impossible to follow. There is no
logical reasoning described for the experiments and it is not easy to understand
any logical flow.
Level of interest: An article whose findings are important to those with closely
related research interests
Quality of written English: Not suitable for publication unless extensively edited
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