METHOD: Lifespan was determined by counting the number of surviving nematodes daily under a dissecting microscope after treatment with hydrogen peroxide and the tocotrienol-rich fraction. The evaluated oxidative markers included lipofuscin, which was measured using a fluorescent microscope, and protein carbonyl and 8-hydroxy-2'-deoxyguanosine, which were measured using commercially available kits.
RESULTS: Hydrogen peroxide-induced oxidative stress significantly decreased the mean lifespan of C. elegans, which was restored to that of the control by the tocotrienol-rich fraction when administered before or both before and after the hydrogen peroxide. The accumulation of the age marker lipofuscin, which increased with hydrogen peroxide exposure, was decreased with upon treatment with the tocotrienol-rich fraction (p<0.05). The level of 8-hydroxy-2'-deoxyguanosine significantly increased in the hydrogen peroxide-induced group relative to the control. Treatment with the tocotrienol-rich fraction before or after hydrogen peroxide induction also increased the level of 8-hydroxy-2'-deoxyguanosine relative to the control. However, neither hydrogen peroxide nor the tocotrienol-rich fraction treatment affected the protein carbonyl content of the nematodes.
CONCLUSION: The tocotrienol-rich fraction restored the lifespan of oxidative stress-induced C. elegans and reduced the accumulation of lipofuscin but did not affect protein damage. In addition, DNA oxidation was increased.
METHODS: We evaluated a truncating variant, p.Arg798Ter (rs137852986), and 10 missense variants of BRIP1, in 48 144 cases and 43 607 controls of European origin, drawn from 41 studies participating in the Breast Cancer Association Consortium (BCAC). Additionally, we sequenced the coding regions of BRIP1 in 13 213 cases and 5242 controls from the UK, 1313 cases and 1123 controls from three population-based studies as part of the Breast Cancer Family Registry, and 1853 familial cases and 2001 controls from Australia.
RESULTS: The rare truncating allele of rs137852986 was observed in 23 cases and 18 controls in Europeans in BCAC (OR 1.09, 95% CI 0.58 to 2.03, p=0.79). Truncating variants were found in the sequencing studies in 34 cases (0.21%) and 19 controls (0.23%) (combined OR 0.90, 95% CI 0.48 to 1.70, p=0.75).
CONCLUSIONS: These results suggest that truncating variants in BRIP1, and in particular p.Arg798Ter, are not associated with a substantial increase in breast cancer risk. Such observations have important implications for the reporting of results from breast cancer screening panels.
MATERIALS AND METHODS: We studied the samples of the pharyngeal mucosa smears taken from children aged 1-15 years with X-ray confirmed pneumonia. The selection of DNA probes for specific detection of community-acquired pneumonia pathogens (S. pneumoniae, H. influenzae, M. pneumoniae, C. pneumonia, and L. pneumophila) and development of the microarray design were carried out using the disprose program. The nucleotide sequences of pathogens were obtained from NCBI Nucleotide database. In the research we used CustomArray microarrays (USA). For a pooled sample containing S. pneumoniae and H. influenzae DNA, we performed a sequential selection of the best combinations of hybridization parameters: DNA fragment size, DNA amount, hybridization temperature. The selection criteria were: the percentage of effective probes with a standardized hybridization signal (SHS) ≥3 Z, and the excess of SHS levels of effective specific probes compared to SHS of effective nonspecific probes. We selected the probes to detect of S. pneumoniae and H. influenzae characterized by an effective hybridization signal under optimal conditions. The developed microarray was tested under the selected conditions on clinical samples containing S. pneumoniae or H. influenzae DNA. Using ROC analysis there were established threshold values for the signals of specific probes at optimal sensitivity points and the test specificity, the excess of which was interpreted as the evidence of pathogen presence in a sample.
RESULTS: A microarray design included 142 DNA probes to detect S. pneumoniae, H. influenzae, M. pneumoniae, C. pneumoniae, and L. pneumophila, the probes being synthesized onto slides. Using the example of clinical samples containing S. pneumoniae and/or H. influenza DNA, we selected optimal parameters for DNA hybridization on microarrays, which enabled to identify bacterial pathogens of community-acquired pneumonia with sufficient efficiency, specificity and reproducibility: the amount of hybridized DNA was 2 μg, the DNA fragment size: 300 nt, hybridization temperature: 47°C. There was selected a list of probes for specific detection of S. pneumoniae and H. influenzae characterized by an effective hybridization signal under the identified conditions. We determined the threshold values of standardized probe signals for specific detection of S. pneumoniae (4.5 Z) and H. influenzae (4.9 Z) in clinical samples.
CONCLUSION: A DNA microarray was developed and synthesized for parallel indication of bacterial pathogens of community-acquired pneumonia. There were selected the optimal parameters for DNA hybridization on a microarray to identify bacterial pathogens - S. pneumoniae and H. influenzae, and determined the threshold values of significant probe signals for their specific detection. The interpretation of the microarray hybridization results corresponds to those obtained by PCR. The microarray can be used to improve laboratory diagnostics of community-acquired pneumonia pathogens.