Reviewer ’s report  
 
Title: Cobalt Chloride Compromises Transepithelial Barrier Properties of CaCo -2 BBe Human 
Gastrointestinal Epithelial Cell Layers  
 
Version: 0 Date: 06 Apr 2017  
 
Reviewer: Rita Rosenthal  
 
Reviewer's report:  
 
James Mullin and coworkers have submitted an original manuscript to BMC Gastroenterology 
entitled "Cobalt Chloride Compromises Transepit helial Barrier Properties of CACO -2 Human  
Gastrointestinal Epithelial Cell Layers", which describes the effect of cobalt chloride on 
transepithelial electrical resistance and transepithelial flux of paracellular flux markers with 
different molecular weight . The authors attribute the barrier -damaging effect of cobalt on 
inhibition of proyl hydroxylase enzymes and the resulting increase in HIF -1, which affects the 
expression of tight junction proteins. The involvement of cell death in the barrier breakdown w as 
excluded by cell viability assays.  
 
 
This is an interesting study on an important issue, barrier breakdown as result of hypoxic 
conditions, presenting novel data from a group with high reputation. However, there are many 
open questions and the manuscript and especially the presentation of the results and the 
discussion need major revision before final acceptance.  
 
 
 
 
Major points:  
 
As described in the Materials and Methods part, the whole cell lysates for analysis of tight 
junction protein expression (probabl y also of HIF -1 and HIF -1ß expression) are prepared from 
cells grown in culture flasks until they are confluent, before CoCl2 is applied. In Fig. 3, it is 
shown, that neither the resistance decreases nor the flux rate increases when CoCl2 is applied to 
the apical compartment of the cells. How can CoCl2 reach the basolateral membrane on 
confluent cell layers cultured in flasks?  
 
 
The authors attribute the barrier -damaging effect of CoCl2 on an increase in HIF -1 expression 
and a resulting change in the expre ssion of tight junction proteins occludin, which is 
downregulated, and claudin -5, which is upregulated. The downregulation of occludin could be 
associated with the barrier breakdown, but claudin -5 belongs to the sealing tight junction 
proteins and an upreg ulation of claudin -5 cannot account for a decrease in transepithelial 
resistance when claudin -5 is localized within the tight junction. In case of an internalization or 
redistribution of claudin -5, which is known to occur under pathologic conditions, claud in-5 can 
be upregulated, but has no barrier function. Immunofluorescence stainings in combination with 
confocal microscopy would clarify the localization of claudin -5 in CoCl2 -treated cells. Anyway, 
the role of the tight junction proteins analyzed in the s tudy should be discussed in detail.  
 
 
The experiments were performed on seven -day post -confluent cell layers (Fig. 1, 3,4,5, 6, 7) or 
three -day post -confluent cell layers (Fig. 9), or 3 -day, 7 -day, and 21 -day cell layers (Fig. 8). 
What's the difference? Ho w long is a seven -day post -confluent cell layer in culture?   
 
 
How were the electrophysiological measurements and the flux measurements performed? The 
measurement of short circuit current ISC would indicate the measurement in Ussing chambers? 
The electroph ysiological and flux measurements should be described in more detail.  
 
 
The cell viability data mentioned in the results should be shown, maybe in a supplemental figure.  
 
 
Fig. 1: Inset, Expression of HIF -1. Suggestion: Fig. 1A Expression of HIF -1 under C oCl2, 
Fig. 1B Transepithelial resistance, Fig. 1 C Flux rateI. In Fig. 1, the densitometric analysis of 
HIF-1 expression should be shown. Is there a correlation between HIF -1 increase and the 
effect on electrical resistance or flux?  
 
 
Fig. 9: The same sh ould be done in Fig. 9, and instead of numbers 1 -8, Control KD and HIF -1ß 
KD (not HIF -1B).  
 
 
In general, the authors should show the Western blots in combination with the corresponding 
loading controls. The kDalton of the detected proteins should be specif ied. Which protein did the 
authors use for normalization of the band densities? This should be mentioned in the Materials 
and Methods part.  
 
 
Fig. 2: The figure shows the normalized band densities of occludin, claudin -5, and claudin -7 in 
Caco -2 cells treat ed with 125 µM CoCl2 for 48 hrs. The Western blots of the tight junction 
proteins and the loading controls are missing. In this figure, the normalized band densities of the 
tight junction proteins is shown, why not the expression in comparison to the contr ol (=100 %) 
as shown for the resistance and flux rate?  
 
 
What's about the other tight junction proteins? It is indicated in the Materials and Methods part 
that claudin -1, -2, -3, -4, -5, -7, occludin, and tricellulin have been analyzed, but only occludin, 
claudin -5, and claudin -7 are mentioned in the results.   
 
Fig. 4: This figure shows the time course of changes in transepithelial electrical resistance and 
transepithelial mannitol flux after CoCl2 exposure. The values presented in this figure are 
normaliz ed to control values without exposure to CoCl2.  It would be helpful to see the time 
course of the control cells without CoCl2  treatment and in the presence of CoCl2. normalized to 
the value at t=0 hrs.  
 
 
Fig. 5: The dose -effect -relation presented in figure 5 should be shown in figure 1, either with 
normalized values as already shown in figure 1 for the concentration of 125 µM CoCl2or with 
absolute values as shown in figure 5.   
 
 
Fig. 7: Why do the authors measure the flux 72 hrs after addition of CoCl2? The other 
experiments were performed 48 hrs after addition of CoCl2.  
 
 
Fig. 8: Figure 8 shows the effect of CoCl2 on transepithelial electrical resistance and mannitol 
flux dependent on the differentiation s tate of the cell layer. The cells were treated with CoCl2 on 
day 3, 7, and 21 after seeding and measured 48 hrs after CoCl2 appication. Are the cells 
confluent 3 days after seeding? Is anything known about the effect of cobalt on proliferation? 
Concerning these experiments, it would be interesting to compare the absolute values of 
transepithelial resistance and mannitol fluxes.  
 
 
Fig. 9: Figure 9 shows the effect of CoCl2 on transepithelial electrical resistance and 
transepithelial mannitol flux of HIF -1β knockdown CACO -2 cell layers and the corresponding 
controls. The measurements were performed on three -day post confluent cells, whereas the other 
experiments were performed on seven -day post confluent cells. For better comparison of the 
effect of 125 µM CoC l2, the study on HIF -1β knockdown cells should be performed on seven -
day post confluent cells.  
 
 
 
Minor points:  
 
In the result part: cell cytoxicity as a result of 48 hr exposure to  change to 48 hrs  
 
 
In the discussion:  by three pieces of evidence: 11) the increased levels of HIF -1α after Co++ 
exposure, a typical  change to 1) the increased levels…….  
 
 
In the Statistics:  All data is expressed as the mean ± standard error of the mean (SEM), in the 
Figure Legends mean ± SE  
 
 
Are the methods appropriate and well described?  
If not,  please specify what is required in your comments to the authors.  
 
No 
 
Does the work include the necessary controls?  
If not, please specify which controls are required in your comments to the authors.  
 
Yes 
 
Are the conclusions drawn adequatel y supported by the data shown?  
If not, please explain in your comments to the authors.  
 
Yes 
 
Are you able to assess any statistics in the manuscript or wo uld you recommend an 
additional statistical review?  
If an additional statistical review is recommend ed, please specify what aspects require further 
assessment in your comments to the editors.  
 
I am able to assess t he statistics  
 
Quality of written English  
Please indicate the quality of language in the manuscript:  
 
Acceptable  
 
Are the methods appropriate and well described?  
If not, please specify what is required i n your comments to the authors.  
 
1. Have you in the past five years received reimbursements, fees, funding, or salary from an 
organisation that may in any way ga in or lose financially from the publication of this 
manuscript, either now or in the future?  
 
2. Do you hold any stocks or shares in an organisation that may in any way gain or lose 
financially from the publication of this manuscript, either now or in the  future?  
 
3. Do you hold or are you currently applying for any patents relating to the content of the 
manuscript?  
 
4. Have you received reimbursements, fees, funding, or salary from an organization that 
holds or has applied for patents relating to the co ntent of the manuscript?  
 
5. Do you have any other financial competing interests?  
 
6. Do you have any non -financial competing interests in relation to this paper?  
If you can answer no to all of the above, write 'I declare that I have no competing inter ests' 
below. If your reply is yes to any, please give details below.  
 
I declare that I have no competing interest.  
 
I agree to the open peer review policy of the journal. I understand t hat my name will be included 
on my report to the authors and, if the m anuscript is accepted f or publication, my named report 
including any attachments I upload will be posted on the website along  with the authors' 
responses. I agree for my report to be made available und er an Open Access  Creative Commons 
CC-BY license (http://creativecommons.org/licenses/by/4.0/ ). I understand that a ny comments 
which I do not wish to be included in my named report can be included as confidential  comments 
to the editors, which will not be published.  
 
I agree to the open peer review policy of t he journal  
 
Reviewer ’s report  
 
Title: Cobalt Chloride Compromises Transepithelial Barrier Properties of CaCo -2 BBe Human 
Gastrointestinal Epithelial Cell Layers  
 
Version: 0 Date: 06 Apr 2017  
 
Reviewer: Rita Rosenthal  
 
Reviewer's report:  
 
James Mullin and coworkers have submitted an original manuscript to BMC Gastroenterology 
entitled "Cobalt Chloride Compromises Transepit helial Barrier Properties of CACO -2 Human  
Gastrointestinal Epithelial Cell Layers", which describes the effect of cobalt chloride on 
transepithelial electrical resistance and transepithelial flux of paracellular flux markers with 
different molecular weight . The authors attribute the barrier -damaging effect of cobalt on 
inhibition of proyl hydroxylase enzymes and the resulting increase in HIF -1, which affects the 
expression of tight junction proteins. The involvement of cell death in the barrier breakdown w as 
excluded by cell viability assays.  
 
 
This is an interesting study on an important issue, barrier breakdown as result of hypoxic 
conditions, presenting novel data from a group with high reputation. However, there are many 
open questions and the manuscript and especially the presentation of the results and the 
discussion need major revision before final acceptance.  
 
 
 
 
Major points:  
 
As described in the Materials and Methods part, the whole cell lysates for analysis of tight 
junction protein expression (probabl y also of HIF -1 and HIF -1ß expression) are prepared from 
cells grown in culture flasks until they are confluent, before CoCl2 is applied. In Fig. 3, it is 
shown, that neither the resistance decreases nor the flux rate increases when CoCl2 is applied to 
the apical compartment of the cells. How can CoCl2 reach the basolateral membrane on 
confluent cell layers cultured in flasks?  
 
 
The authors attribute the barrier -damaging effect of CoCl2 on an increase in HIF -1 expression 
and a resulting change in the expre ssion of tight junction proteins occludin, which is 
downregulated, and claudin -5, which is upregulated. The downregulation of occludin could be 
associated with the barrier breakdown, but claudin -5 belongs to the sealing tight junction 
proteins and an upreg ulation of claudin -5 cannot account for a decrease in transepithelial 
resistance when claudin -5 is localized within the tight junction. In case of an internalization or 
redistribution of claudin -5, which is known to occur under pathologic conditions, claud in-5 can 
be upregulated, but has no barrier function. Immunofluorescence stainings in combination with 
confocal microscopy would clarify the localization of claudin -5 in CoCl2 -treated cells. Anyway, 
the role of the tight junction proteins analyzed in the s tudy should be discussed in detail.  
 
 
The experiments were performed on seven -day post -confluent cell layers (Fig. 1, 3,4,5, 6, 7) or 
three -day post -confluent cell layers (Fig. 9), or 3 -day, 7 -day, and 21 -day cell layers (Fig. 8). 
What's the difference? Ho w long is a seven -day post -confluent cell layer in culture?   
 
 
How were the electrophysiological measurements and the flux measurements performed? The 
measurement of short circuit current ISC would indicate the measurement in Ussing chambers? 
The electroph ysiological and flux measurements should be described in more detail.  
 
 
The cell viability data mentioned in the results should be shown, maybe in a supplemental figure.  
 
 
Fig. 1: Inset, Expression of HIF -1. Suggestion: Fig. 1A Expression of HIF -1 under C oCl2, 
Fig. 1B Transepithelial resistance, Fig. 1 C Flux rateI. In Fig. 1, the densitometric analysis of 
HIF-1 expression should be shown. Is there a correlation between HIF -1 increase and the 
effect on electrical resistance or flux?  
 
 
Fig. 9: The same sh ould be done in Fig. 9, and instead of numbers 1 -8, Control KD and HIF -1ß 
KD (not HIF -1B).  
 
 
In general, the authors should show the Western blots in combination with the corresponding 
loading controls. The kDalton of the detected proteins should be specif ied. Which protein did the 
authors use for normalization of the band densities? This should be mentioned in the Materials 
and Methods part.  
 
 
Fig. 2: The figure shows the normalized band densities of occludin, claudin -5, and claudin -7 in 
Caco -2 cells treat ed with 125 µM CoCl2 for 48 hrs. The Western blots of the tight junction 
proteins and the loading controls are missing. In this figure, the normalized band densities of the 
tight junction proteins is shown, why not the expression in comparison to the contr ol (=100 %) 
as shown for the resistance and flux rate?  
 
 
What's about the other tight junction proteins? It is indicated in the Materials and Methods part 
that claudin -1, -2, -3, -4, -5, -7, occludin, and tricellulin have been analyzed, but only occludin, 
claudin -5, and claudin -7 are mentioned in the results.   
 
Fig. 4: This figure shows the time course of changes in transepithelial electrical resistance and 
transepithelial mannitol flux after CoCl2 exposure. The values presented in this figure are 
normaliz ed to control values without exposure to CoCl2.  It would be helpful to see the time 
course of the control cells without CoCl2  treatment and in the presence of CoCl2. normalized to 
the value at t=0 hrs.  
 
 
Fig. 5: The dose -effect -relation presented in figure 5 should be shown in figure 1, either with 
normalized values as already shown in figure 1 for the concentration of 125 µM CoCl2or with 
absolute values as shown in figure 5.   
 
 
Fig. 7: Why do the authors measure the flux 72 hrs after addition of CoCl2? The other 
experiments were performed 48 hrs after addition of CoCl2.  
 
 
Fig. 8: Figure 8 shows the effect of CoCl2 on transepithelial electrical resistance and mannitol 
flux dependent on the differentiation s tate of the cell layer. The cells were treated with CoCl2 on 
day 3, 7, and 21 after seeding and measured 48 hrs after CoCl2 appication. Are the cells 
confluent 3 days after seeding? Is anything known about the effect of cobalt on proliferation? 
Concerning these experiments, it would be interesting to compare the absolute values of 
transepithelial resistance and mannitol fluxes.  
 
 
Fig. 9: Figure 9 shows the effect of CoCl2 on transepithelial electrical resistance and 
transepithelial mannitol flux of HIF -1β knockdown CACO -2 cell layers and the corresponding 
controls. The measurements were performed on three -day post confluent cells, whereas the other 
experiments were performed on seven -day post confluent cells. For better comparison of the 
effect of 125 µM CoC l2, the study on HIF -1β knockdown cells should be performed on seven -
day post confluent cells.  
 
 
 
Minor points:  
 
In the result part: cell cytoxicity as a result of 48 hr exposure to  change to 48 hrs  
 
 
In the discussion:  by three pieces of evidence: 11) the increased levels of HIF -1α after Co++ 
exposure, a typical  change to 1) the increased levels…….  
 
 
In the Statistics:  All data is expressed as the mean ± standard error of the mean (SEM), in the 
Figure Legends mean ± SE  
 
 
Are the methods appropriate and well described?  
If not,  please specify what is required in your comments to the authors.  
 
No 
 
Does the work include the necessary controls?  
If not, please specify which controls are required in your comments to the authors.  
 
Yes 
 
Are the conclusions drawn adequatel y supported by the data shown?  
If not, please explain in your comments to the authors.  
 
Yes 
 
Are you able to assess any statistics in the manuscript or wo uld you recommend an 
additional statistical review?  
If an additional statistical review is recommend ed, please specify what aspects require further 
assessment in your comments to the editors.  
 
I am able to assess t he statistics  
 
Quality of written English  
Please indicate the quality of language in the manuscript:  
 
Acceptable  
 
Are the methods appropriate and well described?  
If not, please specify what is required i n your comments to the authors.  
 
1. Have you in the past five years received reimbursements, fees, funding, or salary from an 
organisation that may in any way ga in or lose financially from the publication of this 
manuscript, either now or in the future?  
 
2. Do you hold any stocks or shares in an organisation that may in any way gain or lose 
financially from the publication of this manuscript, either now or in the  future?  
 
3. Do you hold or are you currently applying for any patents relating to the content of the 
manuscript?  
 
4. Have you received reimbursements, fees, funding, or salary from an organization that 
holds or has applied for patents relating to the co ntent of the manuscript?  
 
5. Do you have any other financial competing interests?  
 
6. Do you have any non -financial competing interests in relation to this paper?  
If you can answer no to all of the above, write 'I declare that I have no competing inter ests' 
below. If your reply is yes to any, please give details below.  
 
I declare that I have no competing interest.  
 
I agree to the open peer review policy of the journal. I understand t hat my name will be included 
on my report to the authors and, if the m anuscript is accepted f or publication, my named report 
including any attachments I upload will be posted on the website along  with the authors' 
responses. I agree for my report to be made available und er an Open Access  Creative Commons 
CC-BY license (http://creativecommons.org/licenses/by/4.0/ ). I understand that a ny comments 
which I do not wish to be included in my named report can be included as confidential  comments 
to the editors, which will not be published.  
 
I agree to the open peer review policy of t he journal  
 
Please review this paper.
user
Reviewer ’s report  
 
Title: Cobalt Chloride Compromises Transepithelial Barrier Properties of CaCo -2 BBe Human 
Gastrointestinal Epithelial Cell Layers  
 
Version: 0 Date: 06 Apr 2017  
 
Reviewer: Rita Rosenthal  
 
Reviewer's report:  
 
James Mullin and coworkers have submitted an original manuscript to BMC Gastroenterology 
entitled "Cobalt Chloride Compromises Transepit helial Barrier Properties of CACO -2 Human  
Gastrointestinal Epithelial Cell Layers", which describes the effect of cobalt chloride on 
transepithelial electrical resistance and transepithelial flux of paracellular flux markers with 
different molecular weight . The authors attribute the barrier -damaging effect of cobalt on 
inhibition of proyl hydroxylase enzymes and the resulting increase in HIF -1, which affects the 
expression of tight junction proteins. The involvement of cell death in the barrier breakdown w as 
excluded by cell viability assays.  
 
 
This is an interesting study on an important issue, barrier breakdown as result of hypoxic 
conditions, presenting novel data from a group with high reputation. However, there are many 
open questions and the manuscript and especially the presentation of the results and the 
discussion need major revision before final acceptance.  
 
 
 
 
Major points:  
 
As described in the Materials and Methods part, the whole cell lysates for analysis of tight 
junction protein expression (probabl y also of HIF -1 and HIF -1ß expression) are prepared from 
cells grown in culture flasks until they are confluent, before CoCl2 is applied. In Fig. 3, it is 
shown, that neither the resistance decreases nor the flux rate increases when CoCl2 is applied to 
the apical compartment of the cells. How can CoCl2 reach the basolateral membrane on 
confluent cell layers cultured in flasks?  
 
 
The authors attribute the barrier -damaging effect of CoCl2 on an increase in HIF -1 expression 
and a resulting change in the expre ssion of tight junction proteins occludin, which is 
downregulated, and claudin -5, which is upregulated. The downregulation of occludin could be 
associated with the barrier breakdown, but claudin -5 belongs to the sealing tight junction 
proteins and an upreg ulation of claudin -5 cannot account for a decrease in transepithelial 
resistance when claudin -5 is localized within the tight junction. In case of an internalization or 
redistribution of claudin -5, which is known to occur under pathologic conditions, claud in-5 can 
be upregulated, but has no barrier function. Immunofluorescence stainings in combination with 
confocal microscopy would clarify the localization of claudin -5 in CoCl2 -treated cells. Anyway, 
the role of the tight junction proteins analyzed in the s tudy should be discussed in detail.  
 
 
The experiments were performed on seven -day post -confluent cell layers (Fig. 1, 3,4,5, 6, 7) or 
three -day post -confluent cell layers (Fig. 9), or 3 -day, 7 -day, and 21 -day cell layers (Fig. 8). 
What's the difference? Ho w long is a seven -day post -confluent cell layer in culture?   
 
 
How were the electrophysiological measurements and the flux measurements performed? The 
measurement of short circuit current ISC would indicate the measurement in Ussing chambers? 
The electroph ysiological and flux measurements should be described in more detail.  
 
 
The cell viability data mentioned in the results should be shown, maybe in a supplemental figure.  
 
 
Fig. 1: Inset, Expression of HIF -1. Suggestion: Fig. 1A Expression of HIF -1 under C oCl2, 
Fig. 1B Transepithelial resistance, Fig. 1 C Flux rateI. In Fig. 1, the densitometric analysis of 
HIF-1 expression should be shown. Is there a correlation between HIF -1 increase and the 
effect on electrical resistance or flux?  
 
 
Fig. 9: The same sh ould be done in Fig. 9, and instead of numbers 1 -8, Control KD and HIF -1ß 
KD (not HIF -1B).  
 
 
In general, the authors should show the Western blots in combination with the corresponding 
loading controls. The kDalton of the detected proteins should be specif ied. Which protein did the 
authors use for normalization of the band densities? This should be mentioned in the Materials 
and Methods part.  
 
 
Fig. 2: The figure shows the normalized band densities of occludin, claudin -5, and claudin -7 in 
Caco -2 cells treat ed with 125 µM CoCl2 for 48 hrs. The Western blots of the tight junction 
proteins and the loading controls are missing. In this figure, the normalized band densities of the 
tight junction proteins is shown, why not the expression in comparison to the contr ol (=100 %) 
as shown for the resistance and flux rate?  
 
 
What's about the other tight junction proteins? It is indicated in the Materials and Methods part 
that claudin -1, -2, -3, -4, -5, -7, occludin, and tricellulin have been analyzed, but only occludin, 
claudin -5, and claudin -7 are mentioned in the results.   
 
Fig. 4: This figure shows the time course of changes in transepithelial electrical resistance and 
transepithelial mannitol flux after CoCl2 exposure. The values presented in this figure are 
normaliz ed to control values without exposure to CoCl2.  It would be helpful to see the time 
course of the control cells without CoCl2  treatment and in the presence of CoCl2. normalized to 
the value at t=0 hrs.  
 
 
Fig. 5: The dose -effect -relation presented in figure 5 should be shown in figure 1, either with 
normalized values as already shown in figure 1 for the concentration of 125 µM CoCl2or with 
absolute values as shown in figure 5.   
 
 
Fig. 7: Why do the authors measure the flux 72 hrs after addition of CoCl2? The other 
experiments were performed 48 hrs after addition of CoCl2.  
 
 
Fig. 8: Figure 8 shows the effect of CoCl2 on transepithelial electrical resistance and mannitol 
flux dependent on the differentiation s tate of the cell layer. The cells were treated with CoCl2 on 
day 3, 7, and 21 after seeding and measured 48 hrs after CoCl2 appication. Are the cells 
confluent 3 days after seeding? Is anything known about the effect of cobalt on proliferation? 
Concerning these experiments, it would be interesting to compare the absolute values of 
transepithelial resistance and mannitol fluxes.  
 
 
Fig. 9: Figure 9 shows the effect of CoCl2 on transepithelial electrical resistance and 
transepithelial mannitol flux of HIF -1β knockdown CACO -2 cell layers and the corresponding 
controls. The measurements were performed on three -day post confluent cells, whereas the other 
experiments were performed on seven -day post confluent cells. For better comparison of the 
effect of 125 µM CoC l2, the study on HIF -1β knockdown cells should be performed on seven -
day post confluent cells.  
 
 
 
Minor points:  
 
In the result part: cell cytoxicity as a result of 48 hr exposure to  change to 48 hrs  
 
 
In the discussion:  by three pieces of evidence: 11) the increased levels of HIF -1α after Co++ 
exposure, a typical  change to 1) the increased levels…….  
 
 
In the Statistics:  All data is expressed as the mean ± standard error of the mean (SEM), in the 
Figure Legends mean ± SE  
 
 
Are the methods appropriate and well described?  
If not,  please specify what is required in your comments to the authors.  
 
No 
 
Does the work include the necessary controls?  
If not, please specify which controls are required in your comments to the authors.  
 
Yes 
 
Are the conclusions drawn adequatel y supported by the data shown?  
If not, please explain in your comments to the authors.  
 
Yes 
 
Are you able to assess any statistics in the manuscript or wo uld you recommend an 
additional statistical review?  
If an additional statistical review is recommend ed, please specify what aspects require further 
assessment in your comments to the editors.  
 
I am able to assess t he statistics  
 
Quality of written English  
Please indicate the quality of language in the manuscript:  
 
Acceptable  
 
Are the methods appropriate and well described?  
If not, please specify what is required i n your comments to the authors.  
 
1. Have you in the past five years received reimbursements, fees, funding, or salary from an 
organisation that may in any way ga in or lose financially from the publication of this 
manuscript, either now or in the future?  
 
2. Do you hold any stocks or shares in an organisation that may in any way gain or lose 
financially from the publication of this manuscript, either now or in the  future?  
 
3. Do you hold or are you currently applying for any patents relating to the content of the 
manuscript?  
 
4. Have you received reimbursements, fees, funding, or salary from an organization that 
holds or has applied for patents relating to the co ntent of the manuscript?  
 
5. Do you have any other financial competing interests?  
 
6. Do you have any non -financial competing interests in relation to this paper?  
If you can answer no to all of the above, write 'I declare that I have no competing inter ests' 
below. If your reply is yes to any, please give details below.  
 
I declare that I have no competing interest.  
 
I agree to the open peer review policy of the journal. I understand t hat my name will be included 
on my report to the authors and, if the m anuscript is accepted f or publication, my named report 
including any attachments I upload will be posted on the website along  with the authors' 
responses. I agree for my report to be made available und er an Open Access  Creative Commons 
CC-BY license (http://creativecommons.org/licenses/by/4.0/ ). I understand that a ny comments 
which I do not wish to be included in my named report can be included as confidential  comments 
to the editors, which will not be published.  
 
I agree to the open peer review policy of t he journal  
 
assistant