Reviewer’s report
Title: 
Gene Expression Profiling of Inflammatory Cytokines in Esophageal
Biopsies of Different Phenotypes of Gastroesophageal Reflux Disease: a cross-
sectional study
Version: 
2 
Date: 
11 Jan 2021
Reviewer's report:
PEER REVIEWER ASSESSMENTS: 
OBJECTIVE - Full research articles: is there a clear objective that addresses one
or several testable research questions? (Brief or other article types: is there a
clear objective?)
Yes - there is a clear objective
DESIGN - Is the current approach (including controls and analysis protocols)
appropriate for the objective?
No - there are minor issues
EXECUTION - Are the experiments and analyses performed with sufficient
technical rigor to allow confidence in the results?
Yes - experiments and analyses were performed appropriately
STATISTICS - Is the use of statistics in the manuscript appropriate?
Yes - appropriate statistical analyses have been used in the study
INTERPRETATION - Is the current interpretation/discussion of the results
reasonable and not overstated?
No - there are minor issues
OVERALL MANUSCRIPT POTENTIAL - Has the author addressed your
concerns sufficiently for you to now recommend the work as a technically sound
contribution? If not, can further revisions be made to make the work technically
sound?
Probably - with minor revisions
PEER REVIEWER COMMENTS: 
GENERAL COMMENTS: The manuscript has been rewritten for clarity and
correction, and the authors have tried to improve the content by attending to the
suggestions of editor and reviewers. However, there is still a lack of clarity in the
presentation of the qPCR data, failing to provide the data in a relevant manner.
REQUESTED REVISIONS: 
The authors have provided the requested data on cytokine levels in the new table
3. However, this is still difficult to interpret, and therefore I suggest the authors
to improve the title of the table and add units, so the readers understand directly
they are showing delta-delta-Ct values. In the graphs, they might need to clarify
they are showing just delta Cts, and define those two concepts so that readers not
familiar with qPCR can understand what the table and graphs show. Besides, the
method used is RT-qPCR, not just RT-PCR as mentioned somewhere in the
paper.
The graphs are still quite confusing, since they mostly show the same data
(contained already in figure 1) in different manners (else I fail to see the
difference). For instance, in figure 1 the relative levels of MMP3 for BE and
control are shown in a type of graph. The same data is repeated in figure 2, in
another graph with a different scale, in a different comparison (same Y axis,
different groups in X). The significance levels can be added to figure 1, or
summarized in Table 3 or other, if that was the point of all the other graphs. 
In the graphs, the Y axes basically report the ratio between the level of the gene
of interest and GAPDH. There are graphs where the authors show levels at 0.5%
the signal of the GAPDH, and even in figure 4b there are bars below 0.1% the
signal of the GAPDH. I wonder if the authors have revised the raw data and the
reactions were well adjusted. I have never seen such extremely low ratios
reported as a result of a qPCR analysis. I am not sure of the relevance of the
detection of the target genes at such extremely low Cts to yield such a low delta-
Ct.
Publons Reviewer Recognition
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registered for such service or help editors to identify candidate reviewers. Please
find the details of processing in Publons’ privacy policy
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No
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 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 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.
This reviewer has been recruited by a partner organization, Research Square.
Reviewers with declared or apparent competing interests are not utilized for
these reviews. This reviewer has agreed to publication of their comments online
under a Creative Commons Attribution License attributed to Research Square
and was paid a small honorarium for completing the review within a specified
timeframe. Honoraria for reviews such as this are paid regardless of the reviewer
recommendation.
Reviewer Publication Consent.
 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
) if this manuscript is accepted for
publication. Any comments that I do not wish to be included in the published
report have been included as confidential comments to the editor, which will not
be published.
If you are not happy for us to publish this report, please contact the editorial
office before completing the review.
If you wish, you can include your name in your published report. Please note
you must decide whether to include your name at the start of the process and
confirm this decision whenever prompted. If you change your decision later, we
will not be able to publish your name.
I agree to the terms of the CC-BY 4.0 license; please 
do not
 publish my name
with my report. (default)
Is the study design appropriate to answer the research question (including the use
of appropriate controls), and are the conclusions supported by the evidence
presented?
No
Are the methods sufficiently described to allow the study to be repeated?
No
Is the use of statistics and treatment of uncertainties appropriate?
Yes
Is the presentation of the work clear?
No
Are the images in this manuscript (including electrophoretic gels and blots) free
from apparent manipulation?
Yes
Reviewer’s report
Title: 
Gene Expression Profiling of Inflammatory Cytokines in Esophageal
Biopsies of Different Phenotypes of Gastroesophageal Reflux Disease: a cross-
sectional study
Version: 
2 
Date: 
11 Jan 2021
Reviewer's report:
PEER REVIEWER ASSESSMENTS: 
OBJECTIVE - Full research articles: is there a clear objective that addresses one
or several testable research questions? (Brief or other article types: is there a
clear objective?)
Yes - there is a clear objective
DESIGN - Is the current approach (including controls and analysis protocols)
appropriate for the objective?
No - there are minor issues
EXECUTION - Are the experiments and analyses performed with sufficient
technical rigor to allow confidence in the results?
Yes - experiments and analyses were performed appropriately
STATISTICS - Is the use of statistics in the manuscript appropriate?
Yes - appropriate statistical analyses have been used in the study
INTERPRETATION - Is the current interpretation/discussion of the results
reasonable and not overstated?
No - there are minor issues
OVERALL MANUSCRIPT POTENTIAL - Has the author addressed your
concerns sufficiently for you to now recommend the work as a technically sound
contribution? If not, can further revisions be made to make the work technically
sound?
Probably - with minor revisions
PEER REVIEWER COMMENTS: 
GENERAL COMMENTS: The manuscript has been rewritten for clarity and
correction, and the authors have tried to improve the content by attending to the
suggestions of editor and reviewers. However, there is still a lack of clarity in the
presentation of the qPCR data, failing to provide the data in a relevant manner.
REQUESTED REVISIONS: 
The authors have provided the requested data on cytokine levels in the new table
3. However, this is still difficult to interpret, and therefore I suggest the authors
to improve the title of the table and add units, so the readers understand directly
they are showing delta-delta-Ct values. In the graphs, they might need to clarify
they are showing just delta Cts, and define those two concepts so that readers not
familiar with qPCR can understand what the table and graphs show. Besides, the
method used is RT-qPCR, not just RT-PCR as mentioned somewhere in the
paper.
The graphs are still quite confusing, since they mostly show the same data
(contained already in figure 1) in different manners (else I fail to see the
difference). For instance, in figure 1 the relative levels of MMP3 for BE and
control are shown in a type of graph. The same data is repeated in figure 2, in
another graph with a different scale, in a different comparison (same Y axis,
different groups in X). The significance levels can be added to figure 1, or
summarized in Table 3 or other, if that was the point of all the other graphs. 
In the graphs, the Y axes basically report the ratio between the level of the gene
of interest and GAPDH. There are graphs where the authors show levels at 0.5%
the signal of the GAPDH, and even in figure 4b there are bars below 0.1% the
signal of the GAPDH. I wonder if the authors have revised the raw data and the
reactions were well adjusted. I have never seen such extremely low ratios
reported as a result of a qPCR analysis. I am not sure of the relevance of the
detection of the target genes at such extremely low Cts to yield such a low delta-
Ct.
Publons Reviewer Recognition
. Springer Nature can send verification of this
review directly to Publons (a subsidiary of Clarivate Analytics). If you would
like to take advantage of this service, please click on the “Yes” option below.
Your name, email address, title of the reviewed manuscript, name of the journal,
and date of your review submission (the “Review Data”) will then be transmitted
to Publons after the final decision on the manuscript has been made. If you have
already registered at Publons, they will notify you of the receipt of this review
and update your profile 
as per your settings and their policy. If you are not
registered with Publons, you will receive an email from them asking you to
register in order for them to be able to recognize your review on your new
profile page. Publons may use the Review Data to generate derivative metadata
for the benefit of Publons and you as a reviewer, carefully considering the
sensitivity of such information. For example, Publons may verify your record as
a reviewer by updating your profile published on its webservice if you have
registered for such service or help editors to identify candidate reviewers. Please
find the details of processing in Publons’ privacy policy
https://publons.com/about/terms
No
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 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 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.
This reviewer has been recruited by a partner organization, Research Square.
Reviewers with declared or apparent competing interests are not utilized for
these reviews. This reviewer has agreed to publication of their comments online
under a Creative Commons Attribution License attributed to Research Square
and was paid a small honorarium for completing the review within a specified
timeframe. Honoraria for reviews such as this are paid regardless of the reviewer
recommendation.
Reviewer Publication Consent.
 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
) if this manuscript is accepted for
publication. Any comments that I do not wish to be included in the published
report have been included as confidential comments to the editor, which will not
be published.
If you are not happy for us to publish this report, please contact the editorial
office before completing the review.
If you wish, you can include your name in your published report. Please note
you must decide whether to include your name at the start of the process and
confirm this decision whenever prompted. If you change your decision later, we
will not be able to publish your name.
I agree to the terms of the CC-BY 4.0 license; please 
do not
 publish my name
with my report. (default)
Is the study design appropriate to answer the research question (including the use
of appropriate controls), and are the conclusions supported by the evidence
presented?
No
Are the methods sufficiently described to allow the study to be repeated?
No
Is the use of statistics and treatment of uncertainties appropriate?
Yes
Is the presentation of the work clear?
No
Are the images in this manuscript (including electrophoretic gels and blots) free
from apparent manipulation?
Yes
Please review this paper.
user
Reviewer’s report
Title: 
Gene Expression Profiling of Inflammatory Cytokines in Esophageal
Biopsies of Different Phenotypes of Gastroesophageal Reflux Disease: a cross-
sectional study
Version: 
2 
Date: 
11 Jan 2021
Reviewer's report:
PEER REVIEWER ASSESSMENTS: 
OBJECTIVE - Full research articles: is there a clear objective that addresses one
or several testable research questions? (Brief or other article types: is there a
clear objective?)
Yes - there is a clear objective
DESIGN - Is the current approach (including controls and analysis protocols)
appropriate for the objective?
No - there are minor issues
EXECUTION - Are the experiments and analyses performed with sufficient
technical rigor to allow confidence in the results?
Yes - experiments and analyses were performed appropriately
STATISTICS - Is the use of statistics in the manuscript appropriate?
Yes - appropriate statistical analyses have been used in the study
INTERPRETATION - Is the current interpretation/discussion of the results
reasonable and not overstated?
No - there are minor issues
OVERALL MANUSCRIPT POTENTIAL - Has the author addressed your
concerns sufficiently for you to now recommend the work as a technically sound
contribution? If not, can further revisions be made to make the work technically
sound?
Probably - with minor revisions
PEER REVIEWER COMMENTS: 
GENERAL COMMENTS: The manuscript has been rewritten for clarity and
correction, and the authors have tried to improve the content by attending to the
suggestions of editor and reviewers. However, there is still a lack of clarity in the
presentation of the qPCR data, failing to provide the data in a relevant manner.
REQUESTED REVISIONS: 
The authors have provided the requested data on cytokine levels in the new table
3. However, this is still difficult to interpret, and therefore I suggest the authors
to improve the title of the table and add units, so the readers understand directly
they are showing delta-delta-Ct values. In the graphs, they might need to clarify
they are showing just delta Cts, and define those two concepts so that readers not
familiar with qPCR can understand what the table and graphs show. Besides, the
method used is RT-qPCR, not just RT-PCR as mentioned somewhere in the
paper.
The graphs are still quite confusing, since they mostly show the same data
(contained already in figure 1) in different manners (else I fail to see the
difference). For instance, in figure 1 the relative levels of MMP3 for BE and
control are shown in a type of graph. The same data is repeated in figure 2, in
another graph with a different scale, in a different comparison (same Y axis,
different groups in X). The significance levels can be added to figure 1, or
summarized in Table 3 or other, if that was the point of all the other graphs. 
In the graphs, the Y axes basically report the ratio between the level of the gene
of interest and GAPDH. There are graphs where the authors show levels at 0.5%
the signal of the GAPDH, and even in figure 4b there are bars below 0.1% the
signal of the GAPDH. I wonder if the authors have revised the raw data and the
reactions were well adjusted. I have never seen such extremely low ratios
reported as a result of a qPCR analysis. I am not sure of the relevance of the
detection of the target genes at such extremely low Cts to yield such a low delta-
Ct.
Publons Reviewer Recognition
. Springer Nature can send verification of this
review directly to Publons (a subsidiary of Clarivate Analytics). If you would
like to take advantage of this service, please click on the “Yes” option below.
Your name, email address, title of the reviewed manuscript, name of the journal,
and date of your review submission (the “Review Data”) will then be transmitted
to Publons after the final decision on the manuscript has been made. If you have
already registered at Publons, they will notify you of the receipt of this review
and update your profile 
as per your settings and their policy. If you are not
registered with Publons, you will receive an email from them asking you to
register in order for them to be able to recognize your review on your new
profile page. Publons may use the Review Data to generate derivative metadata
for the benefit of Publons and you as a reviewer, carefully considering the
sensitivity of such information. For example, Publons may verify your record as
a reviewer by updating your profile published on its webservice if you have
registered for such service or help editors to identify candidate reviewers. Please
find the details of processing in Publons’ privacy policy
https://publons.com/about/terms
No
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 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 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.
This reviewer has been recruited by a partner organization, Research Square.
Reviewers with declared or apparent competing interests are not utilized for
these reviews. This reviewer has agreed to publication of their comments online
under a Creative Commons Attribution License attributed to Research Square
and was paid a small honorarium for completing the review within a specified
timeframe. Honoraria for reviews such as this are paid regardless of the reviewer
recommendation.
Reviewer Publication Consent.
 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
) if this manuscript is accepted for
publication. Any comments that I do not wish to be included in the published
report have been included as confidential comments to the editor, which will not
be published.
If you are not happy for us to publish this report, please contact the editorial
office before completing the review.
If you wish, you can include your name in your published report. Please note
you must decide whether to include your name at the start of the process and
confirm this decision whenever prompted. If you change your decision later, we
will not be able to publish your name.
I agree to the terms of the CC-BY 4.0 license; please 
do not
 publish my name
with my report. (default)
Is the study design appropriate to answer the research question (including the use
of appropriate controls), and are the conclusions supported by the evidence
presented?
No
Are the methods sufficiently described to allow the study to be repeated?
No
Is the use of statistics and treatment of uncertainties appropriate?
Yes
Is the presentation of the work clear?
No
Are the images in this manuscript (including electrophoretic gels and blots) free
from apparent manipulation?
Yes
assistant