Cytidine and dCMP Deaminases—Current Methods of Activity Analysis

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The review covers the methods for determination of the activity of cytidine deaminase and deoxycytidine monophosphate deaminase, with particular emphasis on their practical aspects, advantages, and limitations. These enzymes are known to play important roles in pyrimidine metabolism. Of the approaches discussed are spectrophotometric, fluorimetric, liquid chromatography-based, radiometric, and cell-based assays. This is a potentially useful and valuable review. Subject to the following changes, the manuscript is acceptable for publication in the journal:

 

1. By figures 1 and 2 the authors show simplified schemes of the reactions catalyzed by the enzymes, that is reasonable. For the general reader, however, it would also be useful to include a figure showing the overall structure of these enzymes and the organization of their active sites. In the caption to such a figure it would make sense to give a brief description of the functionally important elements that make up the active sites and their roles in the catalysis.

 

2. For each of the methods reviewed in Chapter 2 it would be useful to add a figure from the literature, exemplifying and illustrating its practical application.

 

3. The part ‘Concluding remarks’ on further prospects for using existing methods and developing new approaches to measuring the activity of cytidine deaminase and deoxycytidine monophosphate deaminase has to be added.

 

4. “Among the well-characterized and clinically relevant nonsynonymous SNPs in the CDA gene belongs A79C (Lys27Gln) [36] and G208A (Ala70Thr) [37,38].” (lines 117-119).

Unclear sentence, please rephrase.

 

5. Line 227

NAD+

‘+’ should be superscript

 

6. Line 318

Delete the extra dot

 

7. Line 699

Give the reference number

 

Author Response

We thank you for your thorough review and constructive comments, which have helped us improve the quality of our manuscript. In addition to addressing your specific points below, the entire manuscript has now been professionally proofread by a native-speaking editor to improve overall clarity and style. All pages referring to corrections and changes correspond to the pages in the manuscript with the tracked changes. We have addressed the comments point by point as follows:

1, By figures 1 and 2 the authors show simplified schemes of the reactions catalyzed by the enzymes, that is reasonable. For the general reader, however, it would also be useful to include a figure showing the overall structure of these enzymes and the organization of their active sites. In the caption to such a figure it would make sense to give a brief description of the functionally important elements that make up the active sites and their roles in the catalysis.

We have added a new Figures 1b (page 2) and 2b (page 3) to the manuscript, which illustrate the overall structure of human CDA (1b) and DCTD (2b). In addition, we have also expanded the text in the Introduction section to describe the organization of the active sites and the catalytic mechanism for both enzymes (pages 2, 3).

2, For each of the methods reviewed in Chapter 2 it would be useful to add a figure from the literature, exemplifying and illustrating its practical application.

We have added new figures to Chapter 2 to illustrate the practical application of the discussed methods (Figures 5-8).

3, The part ‘Concluding remarks’ on further prospects for using existing methods and developing new approaches to measuring the activity of cytidine deaminase and deoxycytidine monophosphate deaminase has to be added.

We have added a new section 7, „Concluding Remarks and Future Prospects", at the end of the manuscript (pages 23-24) to address this point. This section discusses the current trends and future directions in the field.

4, “Among the well-characterized and clinically relevant nonsynonymous SNPs in the CDA gene belongs A79C (Lys27Gln) [36] and G208A (Ala70Thr) [37,38].” (lines 117-119).

The sentence has been rephrased for clarity (page 5).

5, Line 227 - NAD+ ‘+’ should be superscript

We have corrected it (page 8).

6, Line 318 - Delete the extra dot.

We have corrected it (page 12).

7, Line 699 - Give the reference number.

The appropriate reference has been added (page 20).

Reviewer 2 Report

Comments and Suggestions for Authors

The authors delved into detailing and comparing the various methodologies for studying CDA and DCTD. Each of the technique has been described in good detail with citations from the literature. Few comments for the authors:
1.  The limitations of each technique can be build upon using examples/citations from the literature.

2. The Table 1 summarizing the techniques could be more detailed. A column pertaining to sensitivity of each technique (molar concentrations) can be added. 

3. Since the authors provide this review as a resource to select the appropriate technique, Table 1 can also include a column pertaining to the feasibility/cost of each technique and ranking them on low/medium/high scale.
4. The authors could also compare and summarize in a paragraph the advantage of different methods over one another based on the assay conditions. 

Author Response

We thank you for the constructive comments. All pages referring to corrections and changes correspond to the pages in the manuscript with the tracked changes. We have addressed the points as follows:

1, The limitations of each technique can be built upon using examples/citations from the literature.

We have revised the manuscript to include specific, cited examples from the literature to better illustrate the limitations of each technique, as requested.

2, The Table 1 summarizing the techniques could be more detailed. A column pertaining to sensitivity of each technique (molar concentrations) can be added.

To address this point, we have added a new column, "Sensitivity (LOD)," to Table 1 (pages 22-23), which provides quantitative sensitivity data for each method where applicable.

3, Table 1 can also include a column pertaining to the feasibility/cost of each technique and ranking them on low/medium/high scale.

We have also added a "Feasibility/Cost" column to Table 1 (pages 22-23) with a low-medium-high ranking to aid in method selection.

4, The authors could also compare and summarize in a paragraph the advantage of different methods over one another based on the assay conditions.

As suggested, we have added a new introductory paragraph to Section 6 (pages 21-22), which provides a summary and comparative overview of the advantages of the different methods based on specific applications.

Reviewer 3 Report

Comments and Suggestions for Authors

29 July 2025

Manuscript ID: ijms-377774

Title: Cytidine and dCMP Deaminases - Current Methods of Activity Analysis

Authors: Anna Ligasová, Martina Horejšová, Radana Brumarová, David

Friedecký, Karel Koberna

During my review, I provide some more detailed comments that will assist the authors with their revisions and help the Academic Editor make a decision on the manuscript.

I will answer the following questions:

 

What is the main question addressed by the research?

This study presented a review of methods of Cytidine and dCMP Deaminases activity.

Do you consider the topic original or relevant to the field? Does it address a specific gap in the field? Please also explain why this is/ is not the case.

Cytidine deaminase and deoxycytidine monophosphate deaminase (dCMP) play significant roles in pyrimidine metabolism.

In oncology, these enzymes play an important role in the metabolism of cytidine analogs. They are used in a widely used group of chemotherapeutic agents for treating various types of cancers, including myelodysplastic syndromes (MDS), acute myeloid leukemia (AML), pancreatic carcinoma, and lung cancer.

What does it add to the subject area compared with other published material?

This review examines current and proven methods for analyzing CDA and DCTD enzyme activity. It focuses on their practical aspects, advantages, and limitations. The article discusses the principles and applications of spectroscopic techniques, including traditional UV-Vis spectrophotometry and modern fluorogenic assays. It also covers chromatographic methods, methods using mass spectrometry, or combinations thereof, as well as techniques suitable for studying activity in cellular systems.

What specific improvements should the authors consider regarding the methodology?

The methodology was well implemented and presented appropriately.

Are the conclusions consistent with the evidence and arguments presented, and do they address the main question posed? Please also explain why this is/is not the case.

In Section 6, the authors did not express their opinion on the review of methods for determining the activity of CDA and DCTD.

The authors presented Table 1 and left it to the researchers to choose the method, depending on the planned experiment.

Are the references appropriate?

The references were not used appropriately.

Any additional comments on the tables and figures?

Below is a summarized analysis of comments and suggestions for improving the tables, figures, and the quality of the data and text in the manuscript:

In line 210, the abbreviations M9 and LB should be explained.

In line 227, the “NAD+” should be corrected.

In line 277, write the place of origin of the products Assay Genie and Abcam.

In line 318, delete the additional dot.

In lines 322 and 323, consider deleting the “e.g.,”

In line 623, write correctly the description of isotopes.

In lines 639 and 640, write the description of the symbol “Km” and also write “m” as a subscript.

In References, titles should be written in uppercase or lowercase letters.

In line 910, write the abbreviation of the journal (this is the same in lines 950 and 999).

Conclusion: I recommend that this manuscript be published in the International Journal of Molecular Sciences after a minor revision.

Author Response

We thank you for your thorough review and constructive comments, which have helped us improve the quality of our manuscript. All pages referring to corrections and changes correspond to the pages in the manuscript with the tracked changes. We have addressed the comments point by point as follows:

1, In Section 6, the authors did not express their opinion on the review of methods for determining the activity of CDA and DCTD. The authors presented Table 1 and left it to the researchers to choose the method, depending on the planned experiment.

We have revised the manuscript to include specific, cited examples from the literature to better illustrate the limitations of each technique. In addition, we have added three new columns to Table 1: "Sensitivity (LOD)," which provides quantitative sensitivity data for each method where applicable, "Feasibility/Cost" with a low-medium-high ranking to aid in method selection, and "Assay Type". Furthermore, we have added a new introductory paragraph to Section 6 (pages 21-22) that summarizes and compares the advantages of the different methods based on specific applications.

2, In line 210, the abbreviations M9 and LB should be explained.

We explained both abbreviations in the text (pages 7-8) and added the abbreviations to the Abbreviation list (pages24-26).

3, In line 227, the “NAD+” should be corrected.

We have corrected it (page 8).

4, In line 277, write the place of origin of the products Assay Genie and Abcam.

We added the place of origin of the products Assay Genie and Abcam (page 10).

5, In line 318, delete the additional dot.

We have corrected it (page 12).

6, In lines 322 and 323, consider deleting the “e.g.,”

We removed all „e.g.“ from these lines (page 12).

7, In line 623, write correctly the description of isotopes.

We have corrected all description of isotopes (page 19).

8, In lines 639 and 640, write the description of the symbol “Km” and also write “m” as a subscript.

We have added description what the constant Km means and we also corrected the „m“ (page 18).

9, In References, titles should be written in uppercase or lowercase letters.

We have corrected it.

10, In line 910, write the abbreviation of the journal (this is the same in lines 950 and 999).

We have corrected it.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Accept in present form