Reviewer ’s report  
 
Title:  The impact of the rs8005161 polymorphism on G protein -coupled receptor GPR65 
(TDAG8) pH -associated activation in i ntestinal inflammation  
 
Version: 0 Date: 30 Oct 2018 
 
Reviewer:  Maike  Glitsch  
 
Reviewer’s report:  
 
The study by Tcymarevich et al. is a very interesting and important one that draws on pati ent as 
well as functional data.  
 
My main concern is the lack of baseline data for Figures 1 and 2 - however, the authors may 
have the data and so this may not be an issue at all.  
 
I don't think it's valid to normalise all data to healthy controls at pH7.6. F or instance, by doing 
this, the impression one gets is that, at pH7.6 GPR65 promotes cAMP formation because the 
pH6.5 value in the presence of the GPR65 antagonist is clearly lower than the pH7.6 value 
(though it's hard to tell if the difference would be s ignificant because the authors did not 
normalise the control pH7.6 data points to the average cAMP value, which they probably 
should). However, at pH7.6, GPR65 should not be active or only minimally so. Hence, the 
authors should use pH7.6 + GPR65 inhibitor  as the baseline for that value.  
 
My concern with lack of displayed baseline data is that one can't know whether basal cAMP 
differs for the different genotypes. It could hence be that an actual difference is masked by 
normalising everything to the healthy c AMP level at pH7.6.  If the authors have the data (and I 
appreciate that cell numbers would have been low and they may have decided not to include 
those controls) then I would strongly suggest that they display it (i.e. pH7.6 baseline for the 
different gen otypes and conditions) and maybe re -analyse their data, looking at differences 
between pH7.6 and pH6.5 data for each genotype (healthy CC, IBD -CC, IBD -CT, IBD -TT).  
 
The same applies to Figure 2: it would be good to see basal RhoA activity because it may co ntain 
important information/provide insight.  
 
If the authors do not have baseline values for the different genotypes then I think they need to 
tone down the interpretation of their data.  
 
 
Minor comment:  
 
In the description of the pH experiments, the authors state that pH was adjusted by adding 
HEPES and the appropriate amounts of NaOH or HCl. Have they also altered the bicarbonate 
concentration? This can help cells maintain physiological intracellular pH a s well as support the 
maintenance of the desired extracellular pH, especially over such long exposure times.  
Are the methods appropriate and well described?  
If not, please specify what is required in your comments to the authors.  
 
Yes 
 
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 adequately 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 would you recommend an 
additional statistical review?  
If an additional statistical review is recommended, please specify what aspects require further 
assessment in your commen ts to the editors.  
 
I am able to assess the statistics  
 
Quality of written English  
Please indicate the quality of language in the manuscript:  
 
Acceptable  
 
Declaration of competing interests  
Please complete a declaration of competing interests, considering the following questions:  
 
1. Have you in the past five years received reimbursements, fees, funding, or salary from an 
organisation that may in any way gain or lose financially from the publication of this 
manus cript, 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 p atents 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 content of the manuscript?  
 
5. Do you have any other financial competing i nterests?  
 
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 interests' 
below. If your reply is yes to any, please give details below.  
 
I declare that I have no competing interests.  
 
I agree to the open peer review policy of the journal. I understand that my name will be included 
on my report to the authors and, if the manuscript is accepted for 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 under an Open Access Creative Commons 
CC-BY license ( http://creativecommons.org/licenses/by/4. 0/). I understand that any 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 the journal  
Reviewer ’s report  
 
Title:  The impact of the rs8005161 polymorphism on G protein -coupled receptor GPR65 
(TDAG8) pH -associated activation in i ntestinal inflammation  
 
Version: 0 Date: 30 Oct 2018 
 
Reviewer:  Maike  Glitsch  
 
Reviewer’s report:  
 
The study by Tcymarevich et al. is a very interesting and important one that draws on pati ent as 
well as functional data.  
 
My main concern is the lack of baseline data for Figures 1 and 2 - however, the authors may 
have the data and so this may not be an issue at all.  
 
I don't think it's valid to normalise all data to healthy controls at pH7.6. F or instance, by doing 
this, the impression one gets is that, at pH7.6 GPR65 promotes cAMP formation because the 
pH6.5 value in the presence of the GPR65 antagonist is clearly lower than the pH7.6 value 
(though it's hard to tell if the difference would be s ignificant because the authors did not 
normalise the control pH7.6 data points to the average cAMP value, which they probably 
should). However, at pH7.6, GPR65 should not be active or only minimally so. Hence, the 
authors should use pH7.6 + GPR65 inhibitor  as the baseline for that value.  
 
My concern with lack of displayed baseline data is that one can't know whether basal cAMP 
differs for the different genotypes. It could hence be that an actual difference is masked by 
normalising everything to the healthy c AMP level at pH7.6.  If the authors have the data (and I 
appreciate that cell numbers would have been low and they may have decided not to include 
those controls) then I would strongly suggest that they display it (i.e. pH7.6 baseline for the 
different gen otypes and conditions) and maybe re -analyse their data, looking at differences 
between pH7.6 and pH6.5 data for each genotype (healthy CC, IBD -CC, IBD -CT, IBD -TT).  
 
The same applies to Figure 2: it would be good to see basal RhoA activity because it may co ntain 
important information/provide insight.  
 
If the authors do not have baseline values for the different genotypes then I think they need to 
tone down the interpretation of their data.  
 
 
Minor comment:  
 
In the description of the pH experiments, the authors state that pH was adjusted by adding 
HEPES and the appropriate amounts of NaOH or HCl. Have they also altered the bicarbonate 
concentration? This can help cells maintain physiological intracellular pH a s well as support the 
maintenance of the desired extracellular pH, especially over such long exposure times.  
Are the methods appropriate and well described?  
If not, please specify what is required in your comments to the authors.  
 
Yes 
 
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 adequately 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 would you recommend an 
additional statistical review?  
If an additional statistical review is recommended, please specify what aspects require further 
assessment in your commen ts to the editors.  
 
I am able to assess the statistics  
 
Quality of written English  
Please indicate the quality of language in the manuscript:  
 
Acceptable  
 
Declaration of competing interests  
Please complete a declaration of competing interests, considering the following questions:  
 
1. Have you in the past five years received reimbursements, fees, funding, or salary from an 
organisation that may in any way gain or lose financially from the publication of this 
manus cript, 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 p atents 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 content of the manuscript?  
 
5. Do you have any other financial competing i nterests?  
 
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 interests' 
below. If your reply is yes to any, please give details below.  
 
I declare that I have no competing interests.  
 
I agree to the open peer review policy of the journal. I understand that my name will be included 
on my report to the authors and, if the manuscript is accepted for 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 under an Open Access Creative Commons 
CC-BY license ( http://creativecommons.org/licenses/by/4. 0/). I understand that any 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 the journal  
Please review this paper.
user
Reviewer ’s report  
 
Title:  The impact of the rs8005161 polymorphism on G protein -coupled receptor GPR65 
(TDAG8) pH -associated activation in i ntestinal inflammation  
 
Version: 0 Date: 30 Oct 2018 
 
Reviewer:  Maike  Glitsch  
 
Reviewer’s report:  
 
The study by Tcymarevich et al. is a very interesting and important one that draws on pati ent as 
well as functional data.  
 
My main concern is the lack of baseline data for Figures 1 and 2 - however, the authors may 
have the data and so this may not be an issue at all.  
 
I don't think it's valid to normalise all data to healthy controls at pH7.6. F or instance, by doing 
this, the impression one gets is that, at pH7.6 GPR65 promotes cAMP formation because the 
pH6.5 value in the presence of the GPR65 antagonist is clearly lower than the pH7.6 value 
(though it's hard to tell if the difference would be s ignificant because the authors did not 
normalise the control pH7.6 data points to the average cAMP value, which they probably 
should). However, at pH7.6, GPR65 should not be active or only minimally so. Hence, the 
authors should use pH7.6 + GPR65 inhibitor  as the baseline for that value.  
 
My concern with lack of displayed baseline data is that one can't know whether basal cAMP 
differs for the different genotypes. It could hence be that an actual difference is masked by 
normalising everything to the healthy c AMP level at pH7.6.  If the authors have the data (and I 
appreciate that cell numbers would have been low and they may have decided not to include 
those controls) then I would strongly suggest that they display it (i.e. pH7.6 baseline for the 
different gen otypes and conditions) and maybe re -analyse their data, looking at differences 
between pH7.6 and pH6.5 data for each genotype (healthy CC, IBD -CC, IBD -CT, IBD -TT).  
 
The same applies to Figure 2: it would be good to see basal RhoA activity because it may co ntain 
important information/provide insight.  
 
If the authors do not have baseline values for the different genotypes then I think they need to 
tone down the interpretation of their data.  
 
 
Minor comment:  
 
In the description of the pH experiments, the authors state that pH was adjusted by adding 
HEPES and the appropriate amounts of NaOH or HCl. Have they also altered the bicarbonate 
concentration? This can help cells maintain physiological intracellular pH a s well as support the 
maintenance of the desired extracellular pH, especially over such long exposure times.  
Are the methods appropriate and well described?  
If not, please specify what is required in your comments to the authors.  
 
Yes 
 
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 adequately 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 would you recommend an 
additional statistical review?  
If an additional statistical review is recommended, please specify what aspects require further 
assessment in your commen ts to the editors.  
 
I am able to assess the statistics  
 
Quality of written English  
Please indicate the quality of language in the manuscript:  
 
Acceptable  
 
Declaration of competing interests  
Please complete a declaration of competing interests, considering the following questions:  
 
1. Have you in the past five years received reimbursements, fees, funding, or salary from an 
organisation that may in any way gain or lose financially from the publication of this 
manus cript, 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 p atents 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 content of the manuscript?  
 
5. Do you have any other financial competing i nterests?  
 
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 interests' 
below. If your reply is yes to any, please give details below.  
 
I declare that I have no competing interests.  
 
I agree to the open peer review policy of the journal. I understand that my name will be included 
on my report to the authors and, if the manuscript is accepted for 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 under an Open Access Creative Commons 
CC-BY license ( http://creativecommons.org/licenses/by/4. 0/). I understand that any 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 the journal  
assistant