Reviewer ’s report  
 
Title: Cobalt Chloride Compromises Transepithelial Barrier Properties of CaCo -2 BBe Human 
Gastrointestinal Epithelial Cell Layers  
 
Version: 0 Date: 09 Apr 2017  
 
Reviewer: Susanne M.  Krug  
 
Reviewer's report:  
 
Although the article of DiGuilio et al. deals with a very interesting topic, there are unfortunately 
many major concerns that I have with this study.  
 
 
Besides being a pure descriptive a nd somehow only preliminary study, an adequate discussion of 
the result is missing. Also, the style of presentation and writing in general is not appropriate. I 
have doubts that a very well -established researcher like James Mullin was the one preparing the  
manuscript.  
 
 
Often unusual descriptions are used and sometimes these may lead to confusion.  
 
 
To give a few examples, "barrier friendly" and "unfriendly", or "both cell surfaces" when 
meaning apical and basolateral side. What is "physical scraping", or what  would be non -physical 
scraping? Rt is less common than Rt or TER (or TEER). "mw" is no common unit, use Da.  
 
 
The order of presentation is also a bit confusing. Before doing the experiments with one dose, the 
dose-finding data should be shown. Also the dat a finding the application -side should be one of 
the first data.  
 
 
The descriptions of the methods is very detailed one on hand, but misses important information 
on the other.  
 
 
 
- The cell line used is not named correctly. It is CaCo -2 BBe, not CACO -2. The ad dition 
BBe as mentioned in the methods section is essential, as CaCo -2 and CaCo -2 BBe cells 
are not identical.  
 
 
- How were the electrophysiological measurements performed? Were Ussing chambers 
used? Which kind or brand? Which solutions were used, Ringer so lutions? Please also 
add the composition.  
 
 
- What do the authors mean with "various nutraneuticals … at the concentration that 
provided maximal barrier enhancement"?  
 
 
- Although analyzing some tight junction proteins, there is no adequate discussion of the 
results. What do the respective changes mean for the paracellular barrier? Is each 
analyzed tight junction protein of similar impact? Why were those tight junction proteins 
chosen and other not? In addition, a short introduction into the tight junction fie ld would 
be helpful for readers being not a specialist in this.  
 
 
 
Furthermore, there are several tight junction proteins mentioned to be analyzed in the methods, 
but no data are shown. By the way, it very untypical to incubate with antibodies for only two 
minutes - might this explain why there were no data shown?  
 
 
- For analysis of the tight junction expression the authors treated cells cultured in flasks. 
As they show that cobalt only is effective from the basolateral side, how can they expect 
any effects in flask -culture? The cells grow as monolayer on the plastic bottom, so that 
there is no realistic chance to be reached from basolateral side. The HIF -beta expression 
should be shown as control, too.  
 
 
- How did the authors ensure that same protein concentratio ns were analyzed on the 
Western blots? There are no loading controls shown, e.g. beta -actin. Densitometric 
results should be corrected for that and images of blots should be shown, too.  
 
 
- The authors mention the Cytotox96 cell viability assay, but do not show the data. "Data 
not shown" is a bad habit. If not very important for the whole study, one could always 
add such data as supplementary information.  
 
 
- Does "seven -day post -confluent" m ean that cells were grown until being confluent (when 
are they confluent?) and were analyzed 7 days later? Why do the authors not use the 
usual descrition of days after seeding?  
 
 
As CaCo -2 cells in general (not only BBe) are very variable in their differen tiation there needs to 
be an explanation how the authors assured similar quality and properties in their experiments. 
Some studies use two -week old cells other three -weeks etc. A good choice would be the regular 
control of the TER by chopstick -electrode sy stems to check the differentiation easily.  
 
 
- Most of the figures could be reduced or even combined. They also need huge improvement. I 
suggest the authors to have a look on different other publications. Bars in the diagrams are too 
broad and labeling is to o small if one takes in account that in a paper the figures do not cover a 
whole page. Also labeling of the axes should be improved.  
 
 
Fig. 1: "Percent of normalized control resistance" would be "Resistance (or TER) in % of 
controls". In this context, I al so would like the authors to unify the type of presentation. 
Sometimes they show normalization, sometimes concrete values in other figures. Why did they 
chose one or the other? The "inset" is a sub -figure and should be properly labeled. What is 
shown here should be obvious without having to read the complete figure legend first.  
 
 
Fig. 2: "Normalized band density": If normalized than one should normalized to percentage of 
the controls again. Please also show blots - and the loading control (as explained abov e). 
 
 
Fig. 3: It is common to present fluxes. But these are not suitable when comparing different 
markers (as the authors do in Fig. 7), as they may vary a lot depending on the respective 
concentrations used. It is better to use permeability, which is a con centration -independent value.  
 
 
Fig. 4: The used presentation of the time -curves is very unusual. The curve of the controls should 
be also shown as these also develop with time. So instead matching to the controls one should 
match to the initial start t=0.  
 
How is HIF1 -beta developing during the time -courses? And the tight junction proteins?  
 
 
Fig. 7: Please use permeability as fluxes are concentration -dependent and it is not clear if for all 
markers same concentrations and experimental conditions were used (p lease add in methods).  
 
 
Fig. 8: The abbreviation for days is not capitalized. Use "d". Do CaCo -2 BBe already form 
confluent monolayers three days after seeding? How are the non -normalized TER values? The 
reasons for the different reaction of different ages /differentiation states should be discussed.  
 
 
Fig. 9: Again, the "inset" is a sub -figure and should be properly presented and labeled. There is 
no loading control.  
 
 
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.  
 
Unable to assess  
 
Are the conclusions drawn adequatel y supported by the data shown?  
If not, please explain in your comments to the authors.  
 
No 
 
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:  
 
Needs some language corrections before being published  
 
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 ha ve no competing interests.  
 
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: 09 Apr 2017  
 
Reviewer: Susanne M.  Krug  
 
Reviewer's report:  
 
Although the article of DiGuilio et al. deals with a very interesting topic, there are unfortunately 
many major concerns that I have with this study.  
 
 
Besides being a pure descriptive a nd somehow only preliminary study, an adequate discussion of 
the result is missing. Also, the style of presentation and writing in general is not appropriate. I 
have doubts that a very well -established researcher like James Mullin was the one preparing the  
manuscript.  
 
 
Often unusual descriptions are used and sometimes these may lead to confusion.  
 
 
To give a few examples, "barrier friendly" and "unfriendly", or "both cell surfaces" when 
meaning apical and basolateral side. What is "physical scraping", or what  would be non -physical 
scraping? Rt is less common than Rt or TER (or TEER). "mw" is no common unit, use Da.  
 
 
The order of presentation is also a bit confusing. Before doing the experiments with one dose, the 
dose-finding data should be shown. Also the dat a finding the application -side should be one of 
the first data.  
 
 
The descriptions of the methods is very detailed one on hand, but misses important information 
on the other.  
 
 
 
- The cell line used is not named correctly. It is CaCo -2 BBe, not CACO -2. The ad dition 
BBe as mentioned in the methods section is essential, as CaCo -2 and CaCo -2 BBe cells 
are not identical.  
 
 
- How were the electrophysiological measurements performed? Were Ussing chambers 
used? Which kind or brand? Which solutions were used, Ringer so lutions? Please also 
add the composition.  
 
 
- What do the authors mean with "various nutraneuticals … at the concentration that 
provided maximal barrier enhancement"?  
 
 
- Although analyzing some tight junction proteins, there is no adequate discussion of the 
results. What do the respective changes mean for the paracellular barrier? Is each 
analyzed tight junction protein of similar impact? Why were those tight junction proteins 
chosen and other not? In addition, a short introduction into the tight junction fie ld would 
be helpful for readers being not a specialist in this.  
 
 
 
Furthermore, there are several tight junction proteins mentioned to be analyzed in the methods, 
but no data are shown. By the way, it very untypical to incubate with antibodies for only two 
minutes - might this explain why there were no data shown?  
 
 
- For analysis of the tight junction expression the authors treated cells cultured in flasks. 
As they show that cobalt only is effective from the basolateral side, how can they expect 
any effects in flask -culture? The cells grow as monolayer on the plastic bottom, so that 
there is no realistic chance to be reached from basolateral side. The HIF -beta expression 
should be shown as control, too.  
 
 
- How did the authors ensure that same protein concentratio ns were analyzed on the 
Western blots? There are no loading controls shown, e.g. beta -actin. Densitometric 
results should be corrected for that and images of blots should be shown, too.  
 
 
- The authors mention the Cytotox96 cell viability assay, but do not show the data. "Data 
not shown" is a bad habit. If not very important for the whole study, one could always 
add such data as supplementary information.  
 
 
- Does "seven -day post -confluent" m ean that cells were grown until being confluent (when 
are they confluent?) and were analyzed 7 days later? Why do the authors not use the 
usual descrition of days after seeding?  
 
 
As CaCo -2 cells in general (not only BBe) are very variable in their differen tiation there needs to 
be an explanation how the authors assured similar quality and properties in their experiments. 
Some studies use two -week old cells other three -weeks etc. A good choice would be the regular 
control of the TER by chopstick -electrode sy stems to check the differentiation easily.  
 
 
- Most of the figures could be reduced or even combined. They also need huge improvement. I 
suggest the authors to have a look on different other publications. Bars in the diagrams are too 
broad and labeling is to o small if one takes in account that in a paper the figures do not cover a 
whole page. Also labeling of the axes should be improved.  
 
 
Fig. 1: "Percent of normalized control resistance" would be "Resistance (or TER) in % of 
controls". In this context, I al so would like the authors to unify the type of presentation. 
Sometimes they show normalization, sometimes concrete values in other figures. Why did they 
chose one or the other? The "inset" is a sub -figure and should be properly labeled. What is 
shown here should be obvious without having to read the complete figure legend first.  
 
 
Fig. 2: "Normalized band density": If normalized than one should normalized to percentage of 
the controls again. Please also show blots - and the loading control (as explained abov e). 
 
 
Fig. 3: It is common to present fluxes. But these are not suitable when comparing different 
markers (as the authors do in Fig. 7), as they may vary a lot depending on the respective 
concentrations used. It is better to use permeability, which is a con centration -independent value.  
 
 
Fig. 4: The used presentation of the time -curves is very unusual. The curve of the controls should 
be also shown as these also develop with time. So instead matching to the controls one should 
match to the initial start t=0.  
 
How is HIF1 -beta developing during the time -courses? And the tight junction proteins?  
 
 
Fig. 7: Please use permeability as fluxes are concentration -dependent and it is not clear if for all 
markers same concentrations and experimental conditions were used (p lease add in methods).  
 
 
Fig. 8: The abbreviation for days is not capitalized. Use "d". Do CaCo -2 BBe already form 
confluent monolayers three days after seeding? How are the non -normalized TER values? The 
reasons for the different reaction of different ages /differentiation states should be discussed.  
 
 
Fig. 9: Again, the "inset" is a sub -figure and should be properly presented and labeled. There is 
no loading control.  
 
 
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.  
 
Unable to assess  
 
Are the conclusions drawn adequatel y supported by the data shown?  
If not, please explain in your comments to the authors.  
 
No 
 
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:  
 
Needs some language corrections before being published  
 
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 ha ve no competing interests.  
 
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: 09 Apr 2017  
 
Reviewer: Susanne M.  Krug  
 
Reviewer's report:  
 
Although the article of DiGuilio et al. deals with a very interesting topic, there are unfortunately 
many major concerns that I have with this study.  
 
 
Besides being a pure descriptive a nd somehow only preliminary study, an adequate discussion of 
the result is missing. Also, the style of presentation and writing in general is not appropriate. I 
have doubts that a very well -established researcher like James Mullin was the one preparing the  
manuscript.  
 
 
Often unusual descriptions are used and sometimes these may lead to confusion.  
 
 
To give a few examples, "barrier friendly" and "unfriendly", or "both cell surfaces" when 
meaning apical and basolateral side. What is "physical scraping", or what  would be non -physical 
scraping? Rt is less common than Rt or TER (or TEER). "mw" is no common unit, use Da.  
 
 
The order of presentation is also a bit confusing. Before doing the experiments with one dose, the 
dose-finding data should be shown. Also the dat a finding the application -side should be one of 
the first data.  
 
 
The descriptions of the methods is very detailed one on hand, but misses important information 
on the other.  
 
 
 
- The cell line used is not named correctly. It is CaCo -2 BBe, not CACO -2. The ad dition 
BBe as mentioned in the methods section is essential, as CaCo -2 and CaCo -2 BBe cells 
are not identical.  
 
 
- How were the electrophysiological measurements performed? Were Ussing chambers 
used? Which kind or brand? Which solutions were used, Ringer so lutions? Please also 
add the composition.  
 
 
- What do the authors mean with "various nutraneuticals … at the concentration that 
provided maximal barrier enhancement"?  
 
 
- Although analyzing some tight junction proteins, there is no adequate discussion of the 
results. What do the respective changes mean for the paracellular barrier? Is each 
analyzed tight junction protein of similar impact? Why were those tight junction proteins 
chosen and other not? In addition, a short introduction into the tight junction fie ld would 
be helpful for readers being not a specialist in this.  
 
 
 
Furthermore, there are several tight junction proteins mentioned to be analyzed in the methods, 
but no data are shown. By the way, it very untypical to incubate with antibodies for only two 
minutes - might this explain why there were no data shown?  
 
 
- For analysis of the tight junction expression the authors treated cells cultured in flasks. 
As they show that cobalt only is effective from the basolateral side, how can they expect 
any effects in flask -culture? The cells grow as monolayer on the plastic bottom, so that 
there is no realistic chance to be reached from basolateral side. The HIF -beta expression 
should be shown as control, too.  
 
 
- How did the authors ensure that same protein concentratio ns were analyzed on the 
Western blots? There are no loading controls shown, e.g. beta -actin. Densitometric 
results should be corrected for that and images of blots should be shown, too.  
 
 
- The authors mention the Cytotox96 cell viability assay, but do not show the data. "Data 
not shown" is a bad habit. If not very important for the whole study, one could always 
add such data as supplementary information.  
 
 
- Does "seven -day post -confluent" m ean that cells were grown until being confluent (when 
are they confluent?) and were analyzed 7 days later? Why do the authors not use the 
usual descrition of days after seeding?  
 
 
As CaCo -2 cells in general (not only BBe) are very variable in their differen tiation there needs to 
be an explanation how the authors assured similar quality and properties in their experiments. 
Some studies use two -week old cells other three -weeks etc. A good choice would be the regular 
control of the TER by chopstick -electrode sy stems to check the differentiation easily.  
 
 
- Most of the figures could be reduced or even combined. They also need huge improvement. I 
suggest the authors to have a look on different other publications. Bars in the diagrams are too 
broad and labeling is to o small if one takes in account that in a paper the figures do not cover a 
whole page. Also labeling of the axes should be improved.  
 
 
Fig. 1: "Percent of normalized control resistance" would be "Resistance (or TER) in % of 
controls". In this context, I al so would like the authors to unify the type of presentation. 
Sometimes they show normalization, sometimes concrete values in other figures. Why did they 
chose one or the other? The "inset" is a sub -figure and should be properly labeled. What is 
shown here should be obvious without having to read the complete figure legend first.  
 
 
Fig. 2: "Normalized band density": If normalized than one should normalized to percentage of 
the controls again. Please also show blots - and the loading control (as explained abov e). 
 
 
Fig. 3: It is common to present fluxes. But these are not suitable when comparing different 
markers (as the authors do in Fig. 7), as they may vary a lot depending on the respective 
concentrations used. It is better to use permeability, which is a con centration -independent value.  
 
 
Fig. 4: The used presentation of the time -curves is very unusual. The curve of the controls should 
be also shown as these also develop with time. So instead matching to the controls one should 
match to the initial start t=0.  
 
How is HIF1 -beta developing during the time -courses? And the tight junction proteins?  
 
 
Fig. 7: Please use permeability as fluxes are concentration -dependent and it is not clear if for all 
markers same concentrations and experimental conditions were used (p lease add in methods).  
 
 
Fig. 8: The abbreviation for days is not capitalized. Use "d". Do CaCo -2 BBe already form 
confluent monolayers three days after seeding? How are the non -normalized TER values? The 
reasons for the different reaction of different ages /differentiation states should be discussed.  
 
 
Fig. 9: Again, the "inset" is a sub -figure and should be properly presented and labeled. There is 
no loading control.  
 
 
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.  
 
Unable to assess  
 
Are the conclusions drawn adequatel y supported by the data shown?  
If not, please explain in your comments to the authors.  
 
No 
 
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:  
 
Needs some language corrections before being published  
 
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 ha ve no competing interests.  
 
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