Reviewer ’s report  
 
Title: In search for interplay between stool microRNAs, microbiota  and short chain fatty acids in 
Crohn’s disease. A preliminary study.  
 
Version: 0 Date: 22 Mar 2020  
 
Reviewer's report:   
 
Ambrozkiewicz, Karczmarski &amp;  colleagues present a study intended to explore the levels of 
faecal bacteria, SCFA and host mi -RNAs in the context of IBD. Multi -omics datasets are really 
interesting and provide a good opportunity to discover pot ential mechanisms involved in the 
pathology of IBD. Due to the heterogeneity of the disease as well as heterogeneity of the 
faecal/gut microbiota and the combination of these two, a large number of samples or 
longitudinal sampling is needed in order to rev eal potential biological mechanisms. Therefore, I 
am concerned about the conclusions that are drawn from the dataset presented in this study 
(n=24, 15 cases, which are split in two groups vs 9 Controls).  
 
In addition, the authors provide limited informati on in the main text on the cohort, the statistical 
methods and basic descriptive statistics of the results. Providing more information will greatly 
facilitate the interpretation and the review of the results presented in the manuscript.  
 
Major comments:  
 
1. Could the authors provide more information on the patients with IBD characteristics? 
How many of them were using any (and which) kind of medication at the time of sampling? 
How many patients (in both the active and inactive groups) have ileocolonic, il eal or colonic 
inflammation. How are age and sex distributed between the groups used for analyses?  
  
2. It looks like the authors only adjusted the p -values in the 16S data for multiple testing. 
What is the reason for not doing the same for the miRNA and SCFA data?  
 
3. Could the authors provide summary statistics for each of the tested features/subgroups? 
Means, medians, standard deviations, number of zeros and non -zeros per each of the 
metabolites/taxa/miRNA in each of the tested groups (active/non -activ e/controls).  
 
4. Did the authors adjust or perform any multi -variate analysis to consider the effects of 
potential confounding factors (for example, time that the sample was stored, inflammation 
location in the gut or disease duration)? Did the authors re cord the Bristol Stool scale of each 
sample (or any other measurement of the intestinal transit time)?  
  
5. Although the authors used standard approaches and tools, it is not clear which decision 
the authors took in terms of statistical analyses. It would  be useful if the authors provide the 
codes or a more detailed information of the statistical analyses. For example, how did the authors 
take the differences in library sizes between samples into consideration, did the authors rarefy 
16S counts or did they  regress out any potential read -depth effect? Did the authors perform any 
data transformation of the read counts/concentrations (i.e. to relative abundances, center -log 
values)? Moreover, it is not clear how the authors correlated different multi -omic laye rs.  
 
6. It is not clear if the authors have performed any kind of validation on their 
discriminatory model, I assume that, because of the total sample size this task is complicated. 
Therefore, I ask myself if there is any value in this discriminatory anal ysis.  
 
7. In the discussion section the authors do not acknowledge the limitations of their study. 
They have to add this. In addition, I suggest to reduce the description of other studies and 
provide a direct comparison of their findings with the latest l iterature on the topic. It would be 
interesting if the authors could speculate why they see more differences between patients with 
inactive disease than between patients with active disease, this seems counterintuitive. In the 
discussion section the author s also mention changes in the abundance of F.prausnitzii, however 
in the supplementary tables this bacteria does not pass the significance threshold (p -
adjust &lt;0.05), could the authors clarify this point?  
 
8. The figures could, in general, be more infor mative. The figure legends do not help to 
interpret the figure. A more extensive description to help the interpretation is needed. I suggest to 
change all boxplots to violin plots with individual values represented as dots. Figure 1 uses two 
different and overlapping color scales, making it very difficult to read. Figure 5 is difficult to 
understand if no explanation is provided. I recommend to switch to a network plot.   
 
 
Other comments:  
 
- In the abstract the authors conclude: "indicating complex relationships among all these 
factors in response to intestinal inflammation.". This is a rather generic statement. I suggest to 
replace this with a conclusion that refers directly to the outcomes o f their study.  
 
- The background section provides a good and extensive overview of the main topic of 
the paper. However, it lacks explanation of the rationale/goal of the study.  
 
- Considering that the focus of the authors' findings is in disease activit y, I would suggest 
to expand the discussion and background sections on this specific aspect of IBD.  
 
- Discussion and background sections contain repeated sentences (i.e line 339)  
 
- Check the use of italics when mentioning names of bacteria.  
 
Are the methods appropriate and well described?  
If not, please specify what is r equired 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.  
 
Unable to assess  
 
Are you able to assess any statistics in the manus cript or would you recommend an 
additional statistical review?  
If an additional statistical review is recommended, please speci fy 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 manuscr ipt: 
 
Needs some language corrections before being published  
 
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, fund ing, or salary 
from an organisation that may in any way gain or lose financial ly from the 
publication of this manuscript, either now or in the future?  
 
2.  Do you hold any stocks or shares in an organisation t hat 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 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, w rite 'I declare that I have  no competing interests' 
below. If your reply is yes to any, please give details below.  
 
I declare th at 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 f or publication, my named report 
including any attachments I up load will be posted on the website alon g 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 no t wish to be included in my named report can be in cluded 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: In search for interplay between stool microRNAs, microbiota  and short chain fatty acids in 
Crohn’s disease. A preliminary study.  
 
Version: 0 Date: 22 Mar 2020  
 
Reviewer's report:   
 
Ambrozkiewicz, Karczmarski &amp;  colleagues present a study intended to explore the levels of 
faecal bacteria, SCFA and host mi -RNAs in the context of IBD. Multi -omics datasets are really 
interesting and provide a good opportunity to discover pot ential mechanisms involved in the 
pathology of IBD. Due to the heterogeneity of the disease as well as heterogeneity of the 
faecal/gut microbiota and the combination of these two, a large number of samples or 
longitudinal sampling is needed in order to rev eal potential biological mechanisms. Therefore, I 
am concerned about the conclusions that are drawn from the dataset presented in this study 
(n=24, 15 cases, which are split in two groups vs 9 Controls).  
 
In addition, the authors provide limited informati on in the main text on the cohort, the statistical 
methods and basic descriptive statistics of the results. Providing more information will greatly 
facilitate the interpretation and the review of the results presented in the manuscript.  
 
Major comments:  
 
1. Could the authors provide more information on the patients with IBD characteristics? 
How many of them were using any (and which) kind of medication at the time of sampling? 
How many patients (in both the active and inactive groups) have ileocolonic, il eal or colonic 
inflammation. How are age and sex distributed between the groups used for analyses?  
  
2. It looks like the authors only adjusted the p -values in the 16S data for multiple testing. 
What is the reason for not doing the same for the miRNA and SCFA data?  
 
3. Could the authors provide summary statistics for each of the tested features/subgroups? 
Means, medians, standard deviations, number of zeros and non -zeros per each of the 
metabolites/taxa/miRNA in each of the tested groups (active/non -activ e/controls).  
 
4. Did the authors adjust or perform any multi -variate analysis to consider the effects of 
potential confounding factors (for example, time that the sample was stored, inflammation 
location in the gut or disease duration)? Did the authors re cord the Bristol Stool scale of each 
sample (or any other measurement of the intestinal transit time)?  
  
5. Although the authors used standard approaches and tools, it is not clear which decision 
the authors took in terms of statistical analyses. It would  be useful if the authors provide the 
codes or a more detailed information of the statistical analyses. For example, how did the authors 
take the differences in library sizes between samples into consideration, did the authors rarefy 
16S counts or did they  regress out any potential read -depth effect? Did the authors perform any 
data transformation of the read counts/concentrations (i.e. to relative abundances, center -log 
values)? Moreover, it is not clear how the authors correlated different multi -omic laye rs.  
 
6. It is not clear if the authors have performed any kind of validation on their 
discriminatory model, I assume that, because of the total sample size this task is complicated. 
Therefore, I ask myself if there is any value in this discriminatory anal ysis.  
 
7. In the discussion section the authors do not acknowledge the limitations of their study. 
They have to add this. In addition, I suggest to reduce the description of other studies and 
provide a direct comparison of their findings with the latest l iterature on the topic. It would be 
interesting if the authors could speculate why they see more differences between patients with 
inactive disease than between patients with active disease, this seems counterintuitive. In the 
discussion section the author s also mention changes in the abundance of F.prausnitzii, however 
in the supplementary tables this bacteria does not pass the significance threshold (p -
adjust &lt;0.05), could the authors clarify this point?  
 
8. The figures could, in general, be more infor mative. The figure legends do not help to 
interpret the figure. A more extensive description to help the interpretation is needed. I suggest to 
change all boxplots to violin plots with individual values represented as dots. Figure 1 uses two 
different and overlapping color scales, making it very difficult to read. Figure 5 is difficult to 
understand if no explanation is provided. I recommend to switch to a network plot.   
 
 
Other comments:  
 
- In the abstract the authors conclude: "indicating complex relationships among all these 
factors in response to intestinal inflammation.". This is a rather generic statement. I suggest to 
replace this with a conclusion that refers directly to the outcomes o f their study.  
 
- The background section provides a good and extensive overview of the main topic of 
the paper. However, it lacks explanation of the rationale/goal of the study.  
 
- Considering that the focus of the authors' findings is in disease activit y, I would suggest 
to expand the discussion and background sections on this specific aspect of IBD.  
 
- Discussion and background sections contain repeated sentences (i.e line 339)  
 
- Check the use of italics when mentioning names of bacteria.  
 
Are the methods appropriate and well described?  
If not, please specify what is r equired 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.  
 
Unable to assess  
 
Are you able to assess any statistics in the manus cript or would you recommend an 
additional statistical review?  
If an additional statistical review is recommended, please speci fy 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 manuscr ipt: 
 
Needs some language corrections before being published  
 
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, fund ing, or salary 
from an organisation that may in any way gain or lose financial ly from the 
publication of this manuscript, either now or in the future?  
 
2.  Do you hold any stocks or shares in an organisation t hat 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 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, w rite 'I declare that I have  no competing interests' 
below. If your reply is yes to any, please give details below.  
 
I declare th at 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 f or publication, my named report 
including any attachments I up load will be posted on the website alon g 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 no t wish to be included in my named report can be in cluded 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: In search for interplay between stool microRNAs, microbiota  and short chain fatty acids in 
Crohn’s disease. A preliminary study.  
 
Version: 0 Date: 22 Mar 2020  
 
Reviewer's report:   
 
Ambrozkiewicz, Karczmarski &amp;  colleagues present a study intended to explore the levels of 
faecal bacteria, SCFA and host mi -RNAs in the context of IBD. Multi -omics datasets are really 
interesting and provide a good opportunity to discover pot ential mechanisms involved in the 
pathology of IBD. Due to the heterogeneity of the disease as well as heterogeneity of the 
faecal/gut microbiota and the combination of these two, a large number of samples or 
longitudinal sampling is needed in order to rev eal potential biological mechanisms. Therefore, I 
am concerned about the conclusions that are drawn from the dataset presented in this study 
(n=24, 15 cases, which are split in two groups vs 9 Controls).  
 
In addition, the authors provide limited informati on in the main text on the cohort, the statistical 
methods and basic descriptive statistics of the results. Providing more information will greatly 
facilitate the interpretation and the review of the results presented in the manuscript.  
 
Major comments:  
 
1. Could the authors provide more information on the patients with IBD characteristics? 
How many of them were using any (and which) kind of medication at the time of sampling? 
How many patients (in both the active and inactive groups) have ileocolonic, il eal or colonic 
inflammation. How are age and sex distributed between the groups used for analyses?  
  
2. It looks like the authors only adjusted the p -values in the 16S data for multiple testing. 
What is the reason for not doing the same for the miRNA and SCFA data?  
 
3. Could the authors provide summary statistics for each of the tested features/subgroups? 
Means, medians, standard deviations, number of zeros and non -zeros per each of the 
metabolites/taxa/miRNA in each of the tested groups (active/non -activ e/controls).  
 
4. Did the authors adjust or perform any multi -variate analysis to consider the effects of 
potential confounding factors (for example, time that the sample was stored, inflammation 
location in the gut or disease duration)? Did the authors re cord the Bristol Stool scale of each 
sample (or any other measurement of the intestinal transit time)?  
  
5. Although the authors used standard approaches and tools, it is not clear which decision 
the authors took in terms of statistical analyses. It would  be useful if the authors provide the 
codes or a more detailed information of the statistical analyses. For example, how did the authors 
take the differences in library sizes between samples into consideration, did the authors rarefy 
16S counts or did they  regress out any potential read -depth effect? Did the authors perform any 
data transformation of the read counts/concentrations (i.e. to relative abundances, center -log 
values)? Moreover, it is not clear how the authors correlated different multi -omic laye rs.  
 
6. It is not clear if the authors have performed any kind of validation on their 
discriminatory model, I assume that, because of the total sample size this task is complicated. 
Therefore, I ask myself if there is any value in this discriminatory anal ysis.  
 
7. In the discussion section the authors do not acknowledge the limitations of their study. 
They have to add this. In addition, I suggest to reduce the description of other studies and 
provide a direct comparison of their findings with the latest l iterature on the topic. It would be 
interesting if the authors could speculate why they see more differences between patients with 
inactive disease than between patients with active disease, this seems counterintuitive. In the 
discussion section the author s also mention changes in the abundance of F.prausnitzii, however 
in the supplementary tables this bacteria does not pass the significance threshold (p -
adjust &lt;0.05), could the authors clarify this point?  
 
8. The figures could, in general, be more infor mative. The figure legends do not help to 
interpret the figure. A more extensive description to help the interpretation is needed. I suggest to 
change all boxplots to violin plots with individual values represented as dots. Figure 1 uses two 
different and overlapping color scales, making it very difficult to read. Figure 5 is difficult to 
understand if no explanation is provided. I recommend to switch to a network plot.   
 
 
Other comments:  
 
- In the abstract the authors conclude: "indicating complex relationships among all these 
factors in response to intestinal inflammation.". This is a rather generic statement. I suggest to 
replace this with a conclusion that refers directly to the outcomes o f their study.  
 
- The background section provides a good and extensive overview of the main topic of 
the paper. However, it lacks explanation of the rationale/goal of the study.  
 
- Considering that the focus of the authors' findings is in disease activit y, I would suggest 
to expand the discussion and background sections on this specific aspect of IBD.  
 
- Discussion and background sections contain repeated sentences (i.e line 339)  
 
- Check the use of italics when mentioning names of bacteria.  
 
Are the methods appropriate and well described?  
If not, please specify what is r equired 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.  
 
Unable to assess  
 
Are you able to assess any statistics in the manus cript or would you recommend an 
additional statistical review?  
If an additional statistical review is recommended, please speci fy 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 manuscr ipt: 
 
Needs some language corrections before being published  
 
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, fund ing, or salary 
from an organisation that may in any way gain or lose financial ly from the 
publication of this manuscript, either now or in the future?  
 
2.  Do you hold any stocks or shares in an organisation t hat 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 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, w rite 'I declare that I have  no competing interests' 
below. If your reply is yes to any, please give details below.  
 
I declare th at 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 f or publication, my named report 
including any attachments I up load will be posted on the website alon g 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 no t wish to be included in my named report can be in cluded as confidential comments 
to the editors, which will not be published.  
 
I agree to the open peer review policy of the journal  
assistant