Average beats were checked and intervals set using the first derivative of the tracing

Damage and PARP Activity in COPD Data are presented as mean 6 SEM or n. To test the equality of the data across the study groups, Kruskal-Wallis test and Pearson’s chi-square test was applied for numeric and nominal variables, respectively; in case of smoking-related variables, non-smoking controls were omitted from the analysis. doi:10.1371/journal.pone.0070333.t002 COPD exacerbation 20 13 the Comet Assay IV were used. In addition, a set of novel parameters were tested for their ability to more precisely characterise the DNA damage by the progression of COPD: tail moment length, extent tail moment, cell length, tail length/cell length ratio and tail migration/cell length ratio. Tail moment length shows the distance from the centre of the head to the centre of the tail, extent tail moment was calculated by dividing DNA % in the tail by the tail length and cell length shows the distance from the beginning of the head until the end of the tail.Detection of PARP Activity PARP activity was measured using 6-biotin-17-nicotinamideadenine-dinucleotide, as previously described. PBMC were cultured on poly-L-lysine-coated coverslips in RPMI medium, supplemented with 10% FCS. After 20 min incubation allowing cells to attach to slides, the medium was removed and replaced with PARP reaction buffer. After Aphrodine manufacturer 60-min incubation at 37uC, the cells were fixed at -20uC in 95% ethanol followed by 10% trichloroacetic acid. The coverslips were rinsed in PBS and endogenous peroxidase was blocked by 0.5% H2O2/ methanol for 15 min. After rinses with PBS, the coverslips were 19380617 blocked in 1% bovine serum albumin/PBS for 30 min, followed by rinses in PBSTriton X-100. Incorporated biotin was detected by streptavidinperoxidase for 30 min. Coverslips were washed with PBSTriton X100 and colour was developed with cobalt-enhanced nickel-3,39diaminobenzidine substrate. The coverslips were mounted with glycerol on slides and viewed under a Zeiss Axiolab microscope. The pictures were taken with a Zeiss Axiocam digital camera and analysed using AlphaView Software. Measurement of PARP-1 mRNA Expression The PBMC suspension was divided into halves with one part cultured in the presence of 500 nM UPF17 and the other without UPF17 that served as a control in RPMI-1640 medium. After 12 hours, RNA was extracted from PBMC with the Trizol method according to manufacturer’s protocol and stored at 80uC until cDNA synthesis. cDNA was synthesized with reverse transcriptase reaction, from total RNA using the SuperScript III enzyme, according to manufacturer’s instructions. The gene expression levels were detected using the TaqManqRT-PCR method using Hs00242302_m1 for PARP-1 and hypoxanthine phosphoribosyltransferase-1 as house keeper gene. All reactions were carried out in quadruplicates. The comparative Ct method was used for quantification of mRNA, where the 22634634 amount of target transcript was normalized to the level of endogenous HPRT-1. Non-obstructive smokers 34.4610.4 7 7 5 – 61.163.3 Non-smoking controls 7 27.462.0 64.063.4 25.761.6 – – 97.664.5 81.065.6 Statistical Analysis The equality of the baseline data across the study groups was evaluated with Kruskal-Wallis test in numeric variables and Pearson’s chi-square test in nominal variables. Associations of the comet assay parameters, as well as the PARP-1 expression and PARP activity with the presumably increasing degree of impairment from healthy never-smokers through non-obstructive smokers, stable COPD patients of GOLD stages I-IV to p