Mitochondrial DNA Profiling by Fractal Lacunarity to Characterize the Senescent Phenotype as Normal Aging or Pathological Aging
Round 1
Reviewer 1 Report
Review of manuscript fractalfract-1638910
Based on chaos game representation (CGR) and fractal lacunarity analysis, regarding point mutations as ‘holes’ or ‘gaps’ in mtDNA sequences, the presented approach emerges as a promising analysis tool for mutated mtDNA in particular to differentiate the senescent phenotype in physiological and pathological aging. The paper is interesting. However, there are some issues and comments that should be addressed to deserve publishing.
Comments and suggestions
- In the paragraph 2.3. p.5-6
“The lacunarity function Λ(b) has a decreasing trend and its graph resembles a hyperbola. Therefore, this function can be approximated by using the model Eq. 4.”
Hence, in Eq. (4) to avoid confusion it should be Λ(b;a,β,γ) instead of L(…) or (alternatively) the sentence in the line 205 “The best-fit of lacunarity function Λ(b) with model (4) defines parameters...“ should be modified as “The best-fit of lacunarity function Λ(b) with model function L(...) (4) defines parameters...“.
Accordingly, the designation of ordinate axis in Fig. 2 should be changed to Λ(b) or Λ(b),L(...)
Also in further text L is used to denote Substrings lengths so it would be advisable to denote (hyperbolic) model function with Λ(b;a,β,γ) instead of L(…)
Also. in Eq.4 it should be stated/described how values bmin and bmax are determined/selected. It seems that bmax is determined by subsequences lengths L in the matrix construction process (2L). Hence, in the inset graph (L vs.b that should be Λ vs. b ) on Fig. 2 the dotted line (lacunarity plot) should end at b=32.
- In Section 3. Results
Fig.5. Ordinate values: true values of β should be written (e.g .1.5x10-3 instead of 1.5) or indication provided on axis title: Lacunarity, parameter β (x103).
The opening paragraph of section 3.Results states that “mDNA sequences were processed by the modified CGR method to generate matrix description of each sequence for L=5 and L=6 …”. Here, some explanation for the selection L=5 and L=6 should be provided, especially because in previous research the authors used mtDNA sequences for L=4 to L=7 to find the proper combination of coefficients for the best characterization using CGR.
Although paragraph 3.2 (p. 7 and 8) claims that lacunarity analysis of mtDNA was performed on CGR matrices 32x32 (L=5) and 64x64 (L=6) the presented and discussed results refer only to L=5. (Excepion is Fig3 b where CGR images of different matrices L=1-6 are given without any comment regarding L-dependance, or some explanation short of „resambles Seirpinski triangle“)
A short paragraph should discuss relation between fractal parameters, particularly β and size of CGR matrices (L-value) derived for the same mDNA sequence analyzed.
Generally in discussion of results there is no consideration of uncertainty (of measurement/derivation) . Namely, although the differences of mean values of nA and pA groups are significant (cf. tables 2 and 3) the corresponding standard deviations (measurement uncertainty) are so high that there is significant overlap between the results. Higher uncertainty in a result, especially β, influences the interpretation. This should be addressed in the manuscript revision.
Furthermore, there is no information on quality of fit of data with Eq.4 (e.g. regression coefficient) used to infer β-value.
- In Discussion:
The discussion is comprehensive and general but lacks broader discussion of obtained results for lacunarity and its dispersion and corresponding interpretation.
Generally, patterns having more or larger gaps generally have higher lacunarity. The higher the lacunarity, the more inhomogeneous the examined fractal area and vice versa.
Lower lacunarity values observed for pathological aging, means lesser gaps distributions in CGR image, i.e. less mutations since point mutations are regarded as ‘holes’ or ‘gaps’ in mtDNA sequences. This is somewhat in contradiction with statement (line 277) that lower β values of nA and pA mtDNA account for a high degree of mutations. This should be clarified.
Could you comment on finding that centenarians show a higher than average degree of heteroplasmy [1] while your results show that higher heteroplasmic mutations’ number is found in pA mtDNA sequences when compared with nA ones (line 251). Corresponding mean velues are sinificantly different (10 and5) but the corresponding SD are large resulting in significant overlap.
[1] Rose G, Passarino G, et al. (2007). BMC Genomics. 8: 293
Author Response
Please see the attachment
Author Response File: 
Author Response.pdf
Reviewer 2 Report
It is advised to avoid the use of a verb such as “can characterize” in the title. It leaves the impression of a full sentence. Please rewrite the title.
Please revise the English language and address the following corrections:
Line 18: Please avoid in the sentence: “MtDNA sequences from hospitalized and non-hospitalized old subjects, …”, the expression “old subjects”, it is better to use old age subjects or elder subjects.
Line 50: The word “law” should be used here, please revise. “It is known that a complex system, following the low of deterministic chaos, can…”
Line 58: It is better to use: Aging has been defined as a “progressive, …in the sentence “Aging has been defined a “progressive, generalized impairment of functions…”
Line 73: Please remove the full stop before the references in the following text: ”…only a fraction. [22,23].”
Line 79: Please remove the full stop before the references in the following text: ”…levels of energetic demand such as cerebral, cardiac, and muscular tissues. [26].”
Line 126: Please add full stop at the end of the following sentence: ”…-tion of Helsinki”
Line 193: Please correct and use « as follows » in the following sentence: “as follow:…”
Line 220: Please avoid in the sentence: “18 outpatient old subjects…”, the expression “old subjects”, it is better to use old age subjects or elder subjects.
Line 222: Add that or which before the verb served in the following sentence “served as age-matched controls in two previous studies on…” in order to better connect the text with the another verb later used.
Line 268: Please revise and use this instead of these: “…by using these set of coefficients…”
The article does not contain any new information or does not use original software.
Author Response
Please, see the attachment
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
All reviewer's comment and recommendations were successfully addressed resulting in significantly improved manuscript.
Author Response
Dearest,
We warmly wish to thank you for your precious comments and suggestions that allowed improving the quality of our manuscript.
Sincerely,
the Authors
