Folate deficiency and DNA-methyltransferase inhibition modulate G-quadruplex frequency.

G-quadruplexes (G4) are highly stable tetra-stranded DNA secondary structures known to mediate gene regulation and to trigger genomic instability events during replication. G4 structural stability can be affected by DNA methylation and oxidation modifications; thus nutrients such as folate that have the ability to alter these processes could potentially modify the genomic occurrence of G4 elements. Hela cells were cultured in a range of folate concentrations or in the presence or absence of 5-aza-2'-deoxycytidine, a DNA-methyltransferase inhibitor. G4 structures were then quantified by immunofluorescence using an automated quantitative imaging system. G4 frequency in Hela cells and nuclei area mean were increased in 20nM folate medium compared with 2000nM folate, as well as in the presence of 5-aza-2'-deoxycytidine when compared to cells non-exposed to 5-aza-2'-deoxycytidine. These changes were exacerbated when pyridostatin, a G4 stabilising ligand, was added to the culture medium. G4 intensity in Hela cells cultured in deficient folate condition with pyridostatin was highly correlated with DNA damage as measured by γH2AX immunofluorescence (r = 0.71). This study showed for the first time that cellular G4 balance is modifiable by low folate concentrations and that these changes may occur as a consequence of DNA hypomethylation. Although the exact mechanism by which these changes occur is unclear, these findings establish the possibility that nutrients could be utilised as a tool for sustaining genome integrity by modifying G4 frequency at a cellular level.

Clinical outcomes of subcutaneous vs. transvenous implantable defibrillator therapy in a polymorbid patient cohort.

Background: The subcutaneous implantable cardioverter-defibrillator (S-ICD) has been designed to overcome lead-related complications and device endocarditis. Lacking the ability for pacing or resynchronization therapy its usage is limited to selected patients at risk for sudden cardiac death (SCD). Objective: The aim of this single-center study was to assess clinical outcomes of S-ICD and single-chamber transvenous (TV)-ICD in an all-comers population. Methods: The study cohort comprised a total of 119 ICD patients who underwent either S-ICD (n = 35) or TV-ICD (n = 84) implantation at the University Hospital Frankfurt from 2009 to 2017. By applying an inverse probability-weighting (IPW) analysis based on the propensity score including the Charlson Comorbidity Index (CCI) to adjust for potential extracardiac comorbidities, we aimed for head-to-head comparison on the study composite endpoint: overall survival, hospitalization, and device-associated events (including appropriate and inappropriate shocks or system-related complications). Results: The median age of the study population was 66.0 years, 22.7% of the patients were female. The underlying heart disease was ischemic cardiomyopathy (61.4%) with a median LVEF of 30%. Only 52.9% had received an ICD for primary prevention, most of the patients (67.3%) had advanced heart failure (NYHA class II-III) and 16.8% were in atrial fibrillation. CCI was 5 points in TV-ICD patients vs. 4 points for patients with S-ICD (p = 0.209) indicating increased morbidity. The composite endpoint occurred in 38 patients (31.9 %), revealing no significant difference between patients implanted with an S-ICD or TV-ICD (unweighted HR 1.50, 95 % confidence interval (CI) 0.78-2.90; p = 0.229, weighted HR 0.94, 95% CI, 0.61-1.50, p = 0.777). Furthermore, we observed no difference in any single clinical endpoint or device-associated outcome, neither in the unweighted cohort nor following inverse probability-weighting. Conclusion: Clinical outcomes of the S-ICD and TV-ICD revealed no differences in the composite endpoint including survival, freedom of hospitalization and device-associated events, even after careful adjustment for potential confounders. Moreover, the CCI was evaluated in a S-ICD cohort demonstrating higher survival rates than predicted by the CCI in young, polymorbid (S-)ICD patients.

Folate modulates guanine-quadruplex frequency and DNA damage in Werner syndrome.

Guanine-quadruplexes (G4) are stable tetra-stranded DNA structures that may cause DNA replication stress and inhibit gene expression. Defects in unwinding these structures by DNA helicases may result in telomere shortening and DNA damage. Furthermore, due to mutations in WRN helicase genes in Werner syndrome, G4 motifs are likely to be key elements in the expression of premature aging phenotypes. The methylation of DNA plays a significant role in the stability and occurrence of G4. Thus, G4 frequency and DNA methylation mechanisms may be affected by excesses or deficiencies in methyl donors such as folate. B-Lymphocytes from Werner patients (n = 5) and healthy individuals (n = 5) were cultured in RPMI medium under condition of folate deficiency (20 nM) or sufficiency (200 nM) for 14 days. Cells were fixed on microscope slides for immunofluorescent staining to measure G4 frequency and γH2AX (a marker of DNA strand breaks) intensity, using automated quantitative imaging fluorescent microscopy. There was a significant increase (p < 0.05) in G4 levels in Werner syndrome patients compared to healthy controls. Werner and control cells grown in 20 nM folate media also showed significant increases in G4 (p < 0.001) and γH2AX (p < 0.01) signals compared with the same cells grown in 200 nM folate. Control cells grown in 20 nM folate also showed a significant reduction in DNA methylation levels (P < 0.05). The results of this study suggest that the occurrence of DNA G4 structures can be modulated in vitro via nutrients with important roles in methylation.

Guanine-quadruplexes are increased in mild cognitive impairment and correlate with cognitive function and chromosomal DNA damage.

Guanine-quadruplexes (G4) are highly stable DNA secondary structures known to mediate gene regulation and to trigger genomic instability events during replication. G4 are known to be associated with DNA damage and we propose that G4 are involved in the ageing disorder mild cognitive impairment (MCI). Lymphocytes were obtained from healthy controls and individuals with MCI. The intensity and frequency of G4 foci as well as γH2AX (a marker of DNA damage) intensity were measured by quantitative immunofluorescence and were correlated with cognitive function and cytokinesis-block micronucleus cytome markers of DNA damage. γH2AX intensity as well as G4 frequency and intensity were significantly elevated in MCI lymphocytes compared to controls. The combined biomarker panel was tested in a predictive statistical model, which improved the demarcation of MCI from controls with 80.3% accuracy. The results obtained from this pilot study showed for the first time that G4 levels are increased with cognitive impairment and thus, may be involved in the early development of Alzheimer's disease possibly via an association with chromosomal DNA damage and DNA double strand breaks.

Automation of the cytokinesis-block micronucleus cytome assay by laser scanning cytometry and its potential application in radiation biodosimetry.

Here we describe the adaptation of laser scanning cytometry (LSC) to measure micronuclei (MN) automatically in lymphocytes. MN frequencies were determined in irradiated human lymphocytes using the cytokinesis-block technique, and the results from LSC were compared with visual scoring results obtained from slides of cells stained using Fast Green and 4',6-diamidino-2-phenylindole (DAPI). This fluorescent approach allowed clear identification of binucleated cells and detection of MN. The dose responses measured visually and by LSC showed similar trends and correlated positively (r = 0.9689; P < 0.0001). High-content analysis was developed to further automatically score MN within mono-, tri- and tetra-nucleated cells and to determine the nuclear division index and nuclear circularity values. The high-throughput nature of LSC can provide unique advantages in future DNA damage diagnostics in experimental and epidemiological studies. Importantly, it allows for co-detection of other biomarkers of interest within a single lymphocyte, and further development of this capability is anticipated.

Eletrodos com superfície fractal: princípios, tecnologia e resultados clínicos / Fractally-coated leads: principles, technology and clinical results

Este artigo aborda os fatos e achados clínicos relacionados ao revestimento fractal. A introdução, enfocando sucintamente o princípio do revestimento fractal, é seguida pela revisão das propriedades da estrutura. São feitas considerações sobre publicações de trabalhos clínicos que mencionam tal tecnologia. A principal característica do revestimento fractal é sua superfície ativa ser aproximadamente 1.000 vezes maior que eletrodos de área equivalente, do que decorrem as três vantagens desta tecnologia: captação dos sinais intracardíacos sem distorção (potencial de ação monofásico / "Monophasic Action Potential - MAP " / potencial evocado), ausência do efeito de polarização e transferência de carga otimizada. Conseqüentemente, os limiares de estimulação agudo e crônico desses eletrodos são comparáveis àqueles observados em eletrodos com liberação de esteróides. Este artigo também trata das novas tendências em eletrodos cardíacos implantáveis, apresentando o que vem sendo pesquisado atualmente na área