Localization and classification of scattered nonlinear ultrasonic signatures in bio-mechanical media using time reversal approach.

This paper deals with the time reversal approach along with signal classification using ϕ-divergences in biomedical applications for localization and statistical classification of ultrasonic nonlinearities. The time reversal (TR) approach in combination with nonlinear elastic wave spectroscopy (NEWS) is used to obtain the nonlinear signature of air bubbles with different sizes and ultrasound contrast agents in a liquid. An optimized chirp-coded signal in the range of 0.6-3 MHz is used as a compression coding. The signal classification is performed using the fuzzy classification method and the divergence decision tree algorithm using specific ϕ-divergence spectral measures extracted from the received ultrasonic response containing acoustic nonlinearities. The classification results prove that different types of nonlinearities extracted with classical "pulse inversion" based coding methods can be identified. Simultaneously, the different positions of scattered sources are distinguished by ϕ-divergence methods. The potential of time reversal nonlinear elastic wave spectroscopy methods for understanding of ultrasonic wave propagation in complex media is clearly exhibited.

Guanine quadruplexes in the RNA genome of the tick-borne encephalitis virus: their role as a new antiviral target and in virus biology.

We have identified seven putative guanine quadruplexes (G4) in the RNA genome of tick-borne encephalitis virus (TBEV), a flavivirus causing thousands of human infections and numerous deaths every year. The formation of G4s was confirmed by biophysical methods on synthetic oligonucleotides derived from the predicted TBEV sequences. TBEV-5, located at the NS4b/NS5 boundary and conserved among all known flaviviruses, was tested along with its mutated variants for interactions with a panel of known G4 ligands, for the ability to affect RNA synthesis by the flaviviral RNA-dependent RNA polymerase (RdRp) and for effects on TBEV replication fitness in cells. G4-stabilizing TBEV-5 mutations strongly inhibited RdRp RNA synthesis and exhibited substantially reduced replication fitness, different plaque morphology and increased sensitivity to G4-binding ligands in cell-based systems. In contrast, strongly destabilizing TBEV-5 G4 mutations caused rapid reversion to the wild-type genotype. Our results suggest that there is a threshold of stability for G4 sequences in the TBEV genome, with any deviation resulting in either dramatic changes in viral phenotype or a rapid return to this optimal level of G4 stability. The data indicate that G4s are critical elements for efficient TBEV replication and are suitable targets to tackle TBEV infection.

Systematic investigation of sequence requirements for DNA i-motif formation.

The formation of intercalated motifs (iMs) - secondary DNA structures based on hemiprotonated C.C+ pairs in suitable cytosine-rich DNA sequences, is reflected by typical changes in CD and UV absorption spectra. By means of spectroscopic methods, electrophoresis, chemical modifications and other procedures, we characterized iM formation and stability in sequences with different cytosine block lengths interrupted by various numbers and types of nucleotides. Particular attention was paid to the formation of iMs at pH conditions close to neutral. We identified the optimal conditions and minimal requirements for iM formation in DNA sequences, and addressed gaps and inaccurate data interpretations in existing studies to specify principles of iM formation and modes of their folding.

Socioeconomic and gender inequalities in smoking. Findings from the Czech National Tobacco Surveys 2012-2015.

OBJECTIVE: Smoking significantly affects morbidity and mortality of the population. The incidence of smoking is determined by gender and socioeconomic status (SES) of an individual. The aim of this study is to analyse the relationship between gender and SES indicators and smoking. METHODS: The analysis is based on data from the Czech National Tobacco Surveys from 2012 to 2015 (N∼1,800 per year). The prevalence of smoking, average daily consumption of cigarettes, initiation ratio and quit ratio were monitored. Smoking habits of the respondents were surveyed using the Czech version of the standard Tobacco Questions for Surveys (TQS) questionnaire. SES was measured by a composite index comprising three variables (level of education, income and job prestige); it had four categories: low, lower-middle, upper-middle, and high. RESULTS: In comparison with women, men had a higher smoking prevalence (OR=1.41, 95% CI=1.09-1.84), higher consumption of cigarettes (B=4.11, 95% CI=1.97-6.26), and higher rate of smoking initiation (OR=1.38, 95% CI=1.10-1.74), but they did not differ in the quit rate (OR=0.85, 95% CI=0.60-1.21). Persons in the low SES category had higher prevalence of smoking and higher initiation ratio compared with those in the high SES category (OR=2.59, 95% CI=1.36-4.97; OR=2.23, 95% CI=1.26-3.95). Cigarette consumption and quit ratio did not differ according to SES. The prevalence of smoking in the years 2012-2014 did not differ; in 2015, it was lower compared to the previous three years. CONCLUSIONS: Inequalities in socioeconomic status affect smoking, which significantly contributes to morbidity and mortality. Measures aimed at reducing inequalities in health must take into account both smoking as a risk factor and socioeconomic status, which affects its occurrence. Programmes to reduce tobacco use should reflect the different needs of individuals with different SES levels. It is especially necessary to seek effective approaches for smokers with low socioeconomic status.

i-Motif of cytosine-rich human telomere DNA fragments containing natural base lesions.

i-Motif (iM) is a four stranded DNA structure formed by cytosine-rich sequences, which are often present in functionally important parts of the genome such as promoters of genes and telomeres. Using electronic circular dichroism and UV absorption spectroscopies and electrophoretic methods, we examined the effect of four naturally occurring DNA base lesions on the folding and stability of the iM formed by the human telomere DNA sequence (C3TAA)3C3T. The results demonstrate that the TAA loop lesions, the apurinic site and 8-oxoadenine substituting for adenine, and the 5-hydroxymethyluracil substituting for thymine only marginally disturb the formation of iM. The presence of uracil, which is formed by enzymatic or spontaneous deamination of cytosine, shifts iM formation towards substantially more acidic pH values and simultaneously distinctly reduces iM stability. This effect depends on the position of the damage sites in the sequence. The results have enabled us to formulate additional rules for iM formation.

Spectroscopic insights into quadruplexes of five-repeat telomere DNA sequences upon G-block damage.

BACKGROUND: The DNA lesions, resulting from oxidative damage, were shown to destabilize human telomere four-repeat quadruplex and to alter its structure. Long telomere DNA, as a repetitive sequence, offers, however, other mechanisms of dealing with the lesion: extrusion of the damaged repeat into loop or shifting the quadruplex position by one repeat. METHODS: Using circular dichroism and UV absorption spectroscopy and polyacrylamide electrophoresis, we studied consequences of lesions at different positions of the model five-repeat human telomere DNA sequences on the structure and stability of their quadruplexes in sodium and in potassium. RESULTS: The repeats affected by lesion are preferentially positioned as terminal overhangs of the core quadruplex structurally similar to the four-repeat one. Forced affecting of the inner repeats leads to presence of variety of more parallel folds in potassium. In sodium the designed models form mixture of two dominant antiparallel quadruplexes whose population varies with the position of the affected repeat. The shapes of quadruplex CD spectra, namely the height of dominant peaks, significantly correlate with melting temperatures. CONCLUSION: Lesion in one guanine tract of a more than four repeats long human telomere DNA sequence may cause re-positioning of its quadruplex arrangement associated with a shift of the structure to less common quadruplex conformations. The type of the quadruplex depends on the loop position and external conditions. GENERAL SIGNIFICANCE: The telomere DNA quadruplexes are quite resistant to the effect of point mutations due to the telomere DNA repetitive nature, although their structure and, consequently, function might be altered.

Clustered abasic lesions profoundly change the structure and stability of human telomeric G-quadruplexes.

Ionizing radiation produces clustered damage to DNA which is difficult to repair and thus more harmful than single lesions. Clustered lesions have only been investigated in dsDNA models. Introducing the term 'clustered damage to G-quadruplexes' we report here on the structural effects of multiple tetrahydrofuranyl abasic sites replacing loop adenines (A/AP) and tetrad guanines (G/AP) in quadruplexes formed by the human telomere d[AG3(TTAG3)3] (htel-22) and d[TAG3(TTAG3)3TT] (htel-25) in K+ solutions. Single to triple A/APs increased the population of parallel strands in their structures by stabilizing propeller type loops, shifting the antiparallel htel-22 into hybrid or parallel quadruplexes. In htel-25, the G/APs inhibited the formation of parallel strands and these adopted antiparallel topologies. Clustered G/AP and A/APs reduced the thermal stability of the wild-type htel-25. Depending on position, A/APs diminished or intensified the damaging effect of the G/APs. Taken together, clustered lesions can disrupt the topology and stability of the htel quadruplexes and restrict their conformational space. These in vitro results suggest that formation of clustered lesions in the chromosome capping structure can result in the unfolding of existing G-quadruplexes which can lead to telomere shortening.

Wild-type p53 binds to MYC promoter G-quadruplex.

G-quadruplexes are four-stranded nucleic acid structures that are implicated in the regulation of transcription, translation and replication. Genome regions enriched in putative G-quadruplex motifs include telomeres and gene promoters. Tumour suppressor p53 plays a critical role in regulatory pathways leading to cell cycle arrest, DNA repair and apoptosis. In addition to transcriptional regulation mediated via sequence-specific DNA binding, p53 can selectively bind various non-B DNA structures. In the present study, wild-type p53 (wtp53) binding to G-quadruplex formed by MYC promoter nuclease hypersensitive element (NHE) III1 region was investigated. Wtp53 binding to MYC G-quadruplex is comparable to interaction with specific p53 consensus sequence (p53CON). Apart from the full-length wtp53, its isolated C-terminal region (aa 320-393) as well, is capable of high-affinity MYC G-quadruplex binding, suggesting its critical role in this type of interaction. Moreover, wtp53 binds to MYC promoter region containing putative G-quadruplex motif in two wtp53-expressing cell lines. The results suggest that wtp53 binding to G-quadruplexes can take part in transcriptional regulation of its target genes.

Time reversal transfer: Exploring the robustness of time reversed acoustics in media with geometry perturbations.

In this letter, fundamentals of transferring a time reversal experiment between similar objects are discussed. The time reversal experiment consists of two steps: forward propagation, when a source excites the medium and a complex wave field is created, and back propagation, resulting in time reversal focusing. Here the procedure of performing the first step on one specimen and the second step on another is investigated. The theory of time reversal transfer is explained on an example of object shape variations. However, conclusions of the theoretical analysis are applicable universally. The feasibility of the proposed procedure is validated in experiments modeling conditions in practice.

Diverse effects of naturally occurring base lesions on the structure and stability of the human telomere DNA quadruplex.

Various base lesions continuously form in cellular nucleic acids and the unrepaired lesions are promutagenic and procarcinogenic. Though natural base lesions have been extensively studied in double-stranded DNA models, these studies are only less than a decade old for non-canonical DNA models, such as quadruplexes. Here we present a report on the effects of three frequently occurring natural lesions that can form in the TTA loops on the structure of the human telomere quadruplex d[AG3(TTAG3)3]. We compared the effect of the abasic site and 8-oxoadenine replacing adenine and 5-hydroxymethyluracil substituting for thymine. The results showed that the three lesions impacted the stability and quadruplex folding in markedly different ways. The effects depended on the type of lesion and the position in the sequence. Analogous lesions of guanine in the G-tetrads extensively destabilized the quadruplex and the effects depended more on the position than on the type of lesion. The distinct effects of the loop substitutions as well as comparison of the modifications of the loops and the quadruplex tetrads are discussed in this communication.