Reviewer ’s report  
 
Title: Helicobacter -induced Gastric Inflammation Alte rs the Properties of Gastric Tissue 
Stem/Progenitor Cells  
 
Version: 0 Date: 18 May 2017  
 
Reviewer: Carrie Duckworth  
 
Reviewer's report:  
 
 Shibata et al present a manuscript entitled, "Helicobacter -induced gastric inflammation alters the 
properties of gastric stem/progenitor cells" which attempts to characterise mechanisms of 
Helicobac ter infection -associated gastric carcinogenesis using gastric organoid cultures.  The 
manuscript in its current form is difficult to read due to grammatical errors in places.  Whilst I 
note that the text has been examined by Text check (ref: 15042704), I s uggest that further proof 
reading is carried out by a native English speaker.  For instance, some connecting words such as 
'the', 'a' and 'an' are missing in places and the use of pluralisation can be confusing.  There are 
also several whole sentences that  I do not fully understand.   
The overall aim of the study is exciting in the attempt to study the effects of Helicobacter felis 
infection on the stem cell compartment of gastric epithelial cells without the compound effects of 
other cell systems found in  vivo.  However, I feel that the presented data fall short to satisfy the 
authors' conclusions.  Please address the following major and minor concerns:  
 
Major  
1. Please describe in the text why gastric corpus organoids were generated from H. felis 
infected mice whereas antral organoids were generated from MNU treated mice when the 
MNU model was used as a positive control (page 7 line 3).  These regions are very different 
in terms of histology, function and response to H. felis infection and putative stem cel l 
markers are also known to have different abundances in these different regions.  For 
instance, villin is thought to label a small subset of stem cells and these are predominantly 
observed in the gastric antrum.    
2. Please include the sex of C57BL/6 mic e used for H. felis infection/organoid studies.  There 
are known and well documented sex and background strain differences in susceptibility to 
pathology with this model.  
3. In the mouse model section, it is not clear as to whether the MNU study was firs t conducted 
in C57BL/6 mice to generate organoids prior to subcutaneous injection into NOD/SCID 
mice or whether tumours were generated in NOD/SCID mice. Please clarify in text.  Page 6 
line 16 suggests male mice were used and page 10 line 14 suggests femal e mice were used.  
Were organoids generated in males and grafted into females?  Was this the same for H. felis 
studies?  
4. The methods of organoid analyses have not been fully described.  Please include 
descriptions of how organoid size was measured; eg fr om how many fields of view, original 
magnification, how many replicates, software used.  
5. It is not clear from this manuscript whether organoids were passaged or used immediately 
following their establishment.  A clear indication of the experimental desi gn is needed.  
There is suggestion from the literature that parietal cell loss occurs following passage of 
gastric organoids unless they are co -cultured with a myofibroblast feeder layer (Schumacher 
et al J Physiol. 2015 Apr 15; 593(Pt 8): 1809 -1827).  Par ietal cell atrophy is also well 
characterised following H. felis infection in vivo.  
6. Were antibiotics used in organoid growth medium?  Have the authors determined whether 
extracted gastric glands from H. felis infected mice remain infected during gastric  organoid 
culture or whether the isolation and growth conditions ex vivo eliminate these bacteria?  In 
the latter case, how are these organoids different to those generated from H. felis infected 
but later eradicated mice?  Some indication of infection sta tus of these organoids is needed.  
7. Figure 2B shows that the number of organoids generated from H. felis infected mice is 
greater than that from untreated mice.  Please indicate how this experiment was 
standardised.  There is great variation in number of glands extracted from stomachs and how 
they are fragmented regardless of treatment.  The numbers presented per stomach seem 
remarkably low.   
8. The authors describe an increased organoid size and increased Ki67 staining (page 13, lines 
8-10) however, this  data is presented as 'data not shown' with no indication of quantification 
or justification for this statement.  Please include the data and indicate in the methods how 
this data was generated.  
9. The authors suggest that single organoids are derived from a single stem/progenitor cell 
(page 14 line 2) however, this depends on culture method used.  The authors have not 
generated single cell gastric epithelial extracts but have generated organoids f rom individual 
glands, each of which contain several stem cells all of which could continue cycling. Please 
amend this paragraph.   
10. The authors measured cytokines in organoids generated from infected mice and those post 
eradication however, on page 16  line 2 they suggest that this was conducted in organoids 
infected and post eradication which is not strictly true.  Please adjust to read accurately.  
11. Please add DCLK1 data presented in supplemental figure 1 to the main body of the 
manuscript includin g how many organoids were assessed and how this was analysed.  
12. Fig 4D shows gastric organoid size 3 days post cytokine stimulation vs control.  I find it 
hard to believe that these organoids are only 6 -8μm presumably (clarification in methods 
needed) in  diameter.  Single epithelial cells would be roughly this size and organoids contain 
several 100 to several 1000 cells.  Please check size (diameter?) calculations.  
13. Have the authors compared the phenotypic changes identified in organoids generated fro m 
H. felis infected and H. felis eradicated mice with organoid infected with H. felis in vitro?  
Are similar phenotypic changes observed in both models?  
 
Minor  
1. Abstract: please use consistency when describing Helicobacter felis infection and use 
Helicob acter felis in full at first use.  
2. Please add the concentration and volume of H. felis administered to mice (page 7 line 11)  
3. Please describe the route of administration of antibiotics (page 7, line 17)  
4. Please add concentration of N -acetylcysteine ( page 8, line 15)  
5. Fig 1B: I am not convinced by the images that there is an upregulation of Tff2 and Muc2 
over the course of infection.  Parietal cells look positively stained particularly in the Tff2 
control tissues.   Additionally, please check the mag nification of 3M DCLK1 image and 
include scale bars on figures.  
6. I am unclear as to how E -cadherin IHC allows the confirmation of a single layer of epithelial 
cells (page 13, line 4).  Please include data not shown data.  
7. Fig 2D: The authors suggest t hat the Muc4 expressing region is expanded in gastric corpus 
glands between 3 months, 6 months and 12 months of H. felis infection, it is difficult to 
appreciate this from the 3 month image as this largely contains lymphoid cells.  Please show 
a region con taining mainly epithelium as for 6 moths and 12 months. How was this 
quantified?  
8. How were organoids prepared and quantified for subcutaneous grafts?  
9. Why is one paragraph in the discussion underlined? - the last sentence of this paragraph is 
particul arly not well phrased.  
10. Isx is discussed in the discussion section however, the only previous mention of this gene 
was from the initial cRNA microarray.  Have the authors conducted follow up studies by 
qRT-PCR as they have for other genes?  
11. I am not clear as to how the top paragraph on page 21 is relevant to the data presented in the 
manuscript.  Ie how are Sall4, Klf5, Cdx1, NFκB  related to any of the genes identified in 
presented data?  
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 adequately 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 would you recommend an  
additional statistical review?  
If an additional statistical review is recommended, please specify what aspects require further  
assessment in your comments to the editors.  
 
I am able to assess the statistics  
 
Quality of written English  
Please indicate the quality of language in the manuscript:  
 
Needs some language corrections before being published  
 
Declaration of competing interests  
Please complete a declaration of competing inter ests, 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  
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 c ontent 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 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 interests' 
below.  If your reply is yes to any, please give details below.  
 
I declare that I have no com peting 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 t hat 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: Helicobacter -induced Gastric Inflammation Alte rs the Properties of Gastric Tissue 
Stem/Progenitor Cells  
 
Version: 0 Date: 18 May 2017  
 
Reviewer: Carrie Duckworth  
 
Reviewer's report:  
 
 Shibata et al present a manuscript entitled, "Helicobacter -induced gastric inflammation alters the 
properties of gastric stem/progenitor cells" which attempts to characterise mechanisms of 
Helicobac ter infection -associated gastric carcinogenesis using gastric organoid cultures.  The 
manuscript in its current form is difficult to read due to grammatical errors in places.  Whilst I 
note that the text has been examined by Text check (ref: 15042704), I s uggest that further proof 
reading is carried out by a native English speaker.  For instance, some connecting words such as 
'the', 'a' and 'an' are missing in places and the use of pluralisation can be confusing.  There are 
also several whole sentences that  I do not fully understand.   
The overall aim of the study is exciting in the attempt to study the effects of Helicobacter felis 
infection on the stem cell compartment of gastric epithelial cells without the compound effects of 
other cell systems found in  vivo.  However, I feel that the presented data fall short to satisfy the 
authors' conclusions.  Please address the following major and minor concerns:  
 
Major  
1. Please describe in the text why gastric corpus organoids were generated from H. felis 
infected mice whereas antral organoids were generated from MNU treated mice when the 
MNU model was used as a positive control (page 7 line 3).  These regions are very different 
in terms of histology, function and response to H. felis infection and putative stem cel l 
markers are also known to have different abundances in these different regions.  For 
instance, villin is thought to label a small subset of stem cells and these are predominantly 
observed in the gastric antrum.    
2. Please include the sex of C57BL/6 mic e used for H. felis infection/organoid studies.  There 
are known and well documented sex and background strain differences in susceptibility to 
pathology with this model.  
3. In the mouse model section, it is not clear as to whether the MNU study was firs t conducted 
in C57BL/6 mice to generate organoids prior to subcutaneous injection into NOD/SCID 
mice or whether tumours were generated in NOD/SCID mice. Please clarify in text.  Page 6 
line 16 suggests male mice were used and page 10 line 14 suggests femal e mice were used.  
Were organoids generated in males and grafted into females?  Was this the same for H. felis 
studies?  
4. The methods of organoid analyses have not been fully described.  Please include 
descriptions of how organoid size was measured; eg fr om how many fields of view, original 
magnification, how many replicates, software used.  
5. It is not clear from this manuscript whether organoids were passaged or used immediately 
following their establishment.  A clear indication of the experimental desi gn is needed.  
There is suggestion from the literature that parietal cell loss occurs following passage of 
gastric organoids unless they are co -cultured with a myofibroblast feeder layer (Schumacher 
et al J Physiol. 2015 Apr 15; 593(Pt 8): 1809 -1827).  Par ietal cell atrophy is also well 
characterised following H. felis infection in vivo.  
6. Were antibiotics used in organoid growth medium?  Have the authors determined whether 
extracted gastric glands from H. felis infected mice remain infected during gastric  organoid 
culture or whether the isolation and growth conditions ex vivo eliminate these bacteria?  In 
the latter case, how are these organoids different to those generated from H. felis infected 
but later eradicated mice?  Some indication of infection sta tus of these organoids is needed.  
7. Figure 2B shows that the number of organoids generated from H. felis infected mice is 
greater than that from untreated mice.  Please indicate how this experiment was 
standardised.  There is great variation in number of glands extracted from stomachs and how 
they are fragmented regardless of treatment.  The numbers presented per stomach seem 
remarkably low.   
8. The authors describe an increased organoid size and increased Ki67 staining (page 13, lines 
8-10) however, this  data is presented as 'data not shown' with no indication of quantification 
or justification for this statement.  Please include the data and indicate in the methods how 
this data was generated.  
9. The authors suggest that single organoids are derived from a single stem/progenitor cell 
(page 14 line 2) however, this depends on culture method used.  The authors have not 
generated single cell gastric epithelial extracts but have generated organoids f rom individual 
glands, each of which contain several stem cells all of which could continue cycling. Please 
amend this paragraph.   
10. The authors measured cytokines in organoids generated from infected mice and those post 
eradication however, on page 16  line 2 they suggest that this was conducted in organoids 
infected and post eradication which is not strictly true.  Please adjust to read accurately.  
11. Please add DCLK1 data presented in supplemental figure 1 to the main body of the 
manuscript includin g how many organoids were assessed and how this was analysed.  
12. Fig 4D shows gastric organoid size 3 days post cytokine stimulation vs control.  I find it 
hard to believe that these organoids are only 6 -8μm presumably (clarification in methods 
needed) in  diameter.  Single epithelial cells would be roughly this size and organoids contain 
several 100 to several 1000 cells.  Please check size (diameter?) calculations.  
13. Have the authors compared the phenotypic changes identified in organoids generated fro m 
H. felis infected and H. felis eradicated mice with organoid infected with H. felis in vitro?  
Are similar phenotypic changes observed in both models?  
 
Minor  
1. Abstract: please use consistency when describing Helicobacter felis infection and use 
Helicob acter felis in full at first use.  
2. Please add the concentration and volume of H. felis administered to mice (page 7 line 11)  
3. Please describe the route of administration of antibiotics (page 7, line 17)  
4. Please add concentration of N -acetylcysteine ( page 8, line 15)  
5. Fig 1B: I am not convinced by the images that there is an upregulation of Tff2 and Muc2 
over the course of infection.  Parietal cells look positively stained particularly in the Tff2 
control tissues.   Additionally, please check the mag nification of 3M DCLK1 image and 
include scale bars on figures.  
6. I am unclear as to how E -cadherin IHC allows the confirmation of a single layer of epithelial 
cells (page 13, line 4).  Please include data not shown data.  
7. Fig 2D: The authors suggest t hat the Muc4 expressing region is expanded in gastric corpus 
glands between 3 months, 6 months and 12 months of H. felis infection, it is difficult to 
appreciate this from the 3 month image as this largely contains lymphoid cells.  Please show 
a region con taining mainly epithelium as for 6 moths and 12 months. How was this 
quantified?  
8. How were organoids prepared and quantified for subcutaneous grafts?  
9. Why is one paragraph in the discussion underlined? - the last sentence of this paragraph is 
particul arly not well phrased.  
10. Isx is discussed in the discussion section however, the only previous mention of this gene 
was from the initial cRNA microarray.  Have the authors conducted follow up studies by 
qRT-PCR as they have for other genes?  
11. I am not clear as to how the top paragraph on page 21 is relevant to the data presented in the 
manuscript.  Ie how are Sall4, Klf5, Cdx1, NFκB  related to any of the genes identified in 
presented data?  
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 adequately 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 would you recommend an  
additional statistical review?  
If an additional statistical review is recommended, please specify what aspects require further  
assessment in your comments to the editors.  
 
I am able to assess the statistics  
 
Quality of written English  
Please indicate the quality of language in the manuscript:  
 
Needs some language corrections before being published  
 
Declaration of competing interests  
Please complete a declaration of competing inter ests, 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  
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 c ontent 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 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 interests' 
below.  If your reply is yes to any, please give details below.  
 
I declare that I have no com peting 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 t hat 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: Helicobacter -induced Gastric Inflammation Alte rs the Properties of Gastric Tissue 
Stem/Progenitor Cells  
 
Version: 0 Date: 18 May 2017  
 
Reviewer: Carrie Duckworth  
 
Reviewer's report:  
 
 Shibata et al present a manuscript entitled, "Helicobacter -induced gastric inflammation alters the 
properties of gastric stem/progenitor cells" which attempts to characterise mechanisms of 
Helicobac ter infection -associated gastric carcinogenesis using gastric organoid cultures.  The 
manuscript in its current form is difficult to read due to grammatical errors in places.  Whilst I 
note that the text has been examined by Text check (ref: 15042704), I s uggest that further proof 
reading is carried out by a native English speaker.  For instance, some connecting words such as 
'the', 'a' and 'an' are missing in places and the use of pluralisation can be confusing.  There are 
also several whole sentences that  I do not fully understand.   
The overall aim of the study is exciting in the attempt to study the effects of Helicobacter felis 
infection on the stem cell compartment of gastric epithelial cells without the compound effects of 
other cell systems found in  vivo.  However, I feel that the presented data fall short to satisfy the 
authors' conclusions.  Please address the following major and minor concerns:  
 
Major  
1. Please describe in the text why gastric corpus organoids were generated from H. felis 
infected mice whereas antral organoids were generated from MNU treated mice when the 
MNU model was used as a positive control (page 7 line 3).  These regions are very different 
in terms of histology, function and response to H. felis infection and putative stem cel l 
markers are also known to have different abundances in these different regions.  For 
instance, villin is thought to label a small subset of stem cells and these are predominantly 
observed in the gastric antrum.    
2. Please include the sex of C57BL/6 mic e used for H. felis infection/organoid studies.  There 
are known and well documented sex and background strain differences in susceptibility to 
pathology with this model.  
3. In the mouse model section, it is not clear as to whether the MNU study was firs t conducted 
in C57BL/6 mice to generate organoids prior to subcutaneous injection into NOD/SCID 
mice or whether tumours were generated in NOD/SCID mice. Please clarify in text.  Page 6 
line 16 suggests male mice were used and page 10 line 14 suggests femal e mice were used.  
Were organoids generated in males and grafted into females?  Was this the same for H. felis 
studies?  
4. The methods of organoid analyses have not been fully described.  Please include 
descriptions of how organoid size was measured; eg fr om how many fields of view, original 
magnification, how many replicates, software used.  
5. It is not clear from this manuscript whether organoids were passaged or used immediately 
following their establishment.  A clear indication of the experimental desi gn is needed.  
There is suggestion from the literature that parietal cell loss occurs following passage of 
gastric organoids unless they are co -cultured with a myofibroblast feeder layer (Schumacher 
et al J Physiol. 2015 Apr 15; 593(Pt 8): 1809 -1827).  Par ietal cell atrophy is also well 
characterised following H. felis infection in vivo.  
6. Were antibiotics used in organoid growth medium?  Have the authors determined whether 
extracted gastric glands from H. felis infected mice remain infected during gastric  organoid 
culture or whether the isolation and growth conditions ex vivo eliminate these bacteria?  In 
the latter case, how are these organoids different to those generated from H. felis infected 
but later eradicated mice?  Some indication of infection sta tus of these organoids is needed.  
7. Figure 2B shows that the number of organoids generated from H. felis infected mice is 
greater than that from untreated mice.  Please indicate how this experiment was 
standardised.  There is great variation in number of glands extracted from stomachs and how 
they are fragmented regardless of treatment.  The numbers presented per stomach seem 
remarkably low.   
8. The authors describe an increased organoid size and increased Ki67 staining (page 13, lines 
8-10) however, this  data is presented as 'data not shown' with no indication of quantification 
or justification for this statement.  Please include the data and indicate in the methods how 
this data was generated.  
9. The authors suggest that single organoids are derived from a single stem/progenitor cell 
(page 14 line 2) however, this depends on culture method used.  The authors have not 
generated single cell gastric epithelial extracts but have generated organoids f rom individual 
glands, each of which contain several stem cells all of which could continue cycling. Please 
amend this paragraph.   
10. The authors measured cytokines in organoids generated from infected mice and those post 
eradication however, on page 16  line 2 they suggest that this was conducted in organoids 
infected and post eradication which is not strictly true.  Please adjust to read accurately.  
11. Please add DCLK1 data presented in supplemental figure 1 to the main body of the 
manuscript includin g how many organoids were assessed and how this was analysed.  
12. Fig 4D shows gastric organoid size 3 days post cytokine stimulation vs control.  I find it 
hard to believe that these organoids are only 6 -8μm presumably (clarification in methods 
needed) in  diameter.  Single epithelial cells would be roughly this size and organoids contain 
several 100 to several 1000 cells.  Please check size (diameter?) calculations.  
13. Have the authors compared the phenotypic changes identified in organoids generated fro m 
H. felis infected and H. felis eradicated mice with organoid infected with H. felis in vitro?  
Are similar phenotypic changes observed in both models?  
 
Minor  
1. Abstract: please use consistency when describing Helicobacter felis infection and use 
Helicob acter felis in full at first use.  
2. Please add the concentration and volume of H. felis administered to mice (page 7 line 11)  
3. Please describe the route of administration of antibiotics (page 7, line 17)  
4. Please add concentration of N -acetylcysteine ( page 8, line 15)  
5. Fig 1B: I am not convinced by the images that there is an upregulation of Tff2 and Muc2 
over the course of infection.  Parietal cells look positively stained particularly in the Tff2 
control tissues.   Additionally, please check the mag nification of 3M DCLK1 image and 
include scale bars on figures.  
6. I am unclear as to how E -cadherin IHC allows the confirmation of a single layer of epithelial 
cells (page 13, line 4).  Please include data not shown data.  
7. Fig 2D: The authors suggest t hat the Muc4 expressing region is expanded in gastric corpus 
glands between 3 months, 6 months and 12 months of H. felis infection, it is difficult to 
appreciate this from the 3 month image as this largely contains lymphoid cells.  Please show 
a region con taining mainly epithelium as for 6 moths and 12 months. How was this 
quantified?  
8. How were organoids prepared and quantified for subcutaneous grafts?  
9. Why is one paragraph in the discussion underlined? - the last sentence of this paragraph is 
particul arly not well phrased.  
10. Isx is discussed in the discussion section however, the only previous mention of this gene 
was from the initial cRNA microarray.  Have the authors conducted follow up studies by 
qRT-PCR as they have for other genes?  
11. I am not clear as to how the top paragraph on page 21 is relevant to the data presented in the 
manuscript.  Ie how are Sall4, Klf5, Cdx1, NFκB  related to any of the genes identified in 
presented data?  
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 adequately 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 would you recommend an  
additional statistical review?  
If an additional statistical review is recommended, please specify what aspects require further  
assessment in your comments to the editors.  
 
I am able to assess the statistics  
 
Quality of written English  
Please indicate the quality of language in the manuscript:  
 
Needs some language corrections before being published  
 
Declaration of competing interests  
Please complete a declaration of competing inter ests, 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  
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 c ontent 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 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 interests' 
below.  If your reply is yes to any, please give details below.  
 
I declare that I have no com peting 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 t hat 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