epidermidis >100 cfu 47 22             12 mixed coagulase-negative Staphylococci 90 39 61 69 0.81 3.20 0.07   13 S. epidermidis >100 cfu 24 16             14 P. aeruginosa >100 cfu 48 19             Total 239 51           Ethics approval for this study was granted by the Royal Brisbane and Women’s Hospital Human Ethics Board (Protocol 2008/059) and Griffith University Human Ethics Board. Semi-quantitative method The removal ACs were

examined using the semi-quantitative method [12]. This method is based on rolling a segment, usually the tip, of the removed catheter back and forth on 5% sheep blood agar plates (Oxoid, Australia) after removal. The plates were incubated at 35°C under aerobic conditions for 2-4 days. Microorganisms were then isolated see more and identified according to standard hospital protocol. Semi-quantitative tip culture was considered colonised if the YM155 mw plate grew ≥15 colony forming unit (cfu). If <15 cfu were grown, the catheter tip was considered to be uncolonised. Detailed molecular methods DNA extraction and PCR amplification Catheter tips were suspended in 200 μl of lysis buffer, which contained 20 mg/ml lysozyme, 20 mM Tris-HCl (pH 8.0), 2 mM EDTA, 1.2% Triton, and Proteinase K at 37°C overnight. After that, catheter

tips were taken out and bacterial DNA was extracted using the QIAamp DNA mini kit (Qiagen, Australia). For each catheter, a control (unused) AC was taken from the original packaging and rolled back and forth on blood Janus kinase (JAK) agar plates, with bacterial DNA extracted as above. Sixteen S rRNA genes were amplified from PRI-724 mw purified genomic DNA using the primers 8F and 1490R [20]. For each 25

μl reaction, conditions were as follows: 3 μl of DNA template (concentration ranged from neat to 1:103), 2.5 μl of 10 × reaction buffer containing 20 mM MgCl2, 2 μl of 25 mM dNTPs, 1 μl of each primer (10 μM), 0.1 U of Taq DNA polymerase (Qiagen, Australia), 5 μl of 5 × BSA and 10.4 μl of sterile deionised water (sdH2O). Each PCR run contained a negative control (sdH2O instead of template DNA) and a positive control (E. coli instead of template DNA). For each DNA sample, three replicate PCRs were performed. Thermocycling was as follows: initial denaturation at 95°C for 5 min, followed by 30 cycles of a 1-min denaturation, 1-min annealing at 55°C and 2-min elongation at 72°C, all followed by a final extension of 10 min at 72°C. Cloning and sequencing of 16S rDNA PCR products After purification using the Qiaquick PCR Purification kit (Qiagen, Australia), the PCR amplified 16S rRNA gene fragment were ligated into TOPO TA vector Cloning® system (Invitrogen, Ausralia) according to the manufacturer’s instructions. Two microliters of the ligation mixture was transferred to 1.5 ml sterile tube which was with competent Escherichia coli TOP10 cells provided by the manufacturer. The mixture was chilled on ice for 20 min before heat shocking for 45 seconds at 42°C.

Apoptosis was determinate LOXO-101 by staining cells with annexin V-FITC and propidium-iodide (PI) labeling, because annexin V can identify the externalization of phosphatidylserine during the apoptotic progression and therefore detect early apoptotic cells [29]. Cells were transduced with TG 9344 vector, on 12-well plates and treated after 24 hr by 20 μM GCV. Control cells were no transduced or untreated. After 72 hr of treatment, cells were harvested, and washed twice in PBS. The pellet was resuspended in 1 ml of 100 mM HEPES/NaOH, pH 7.5.

Then 500 μl of the cell suspension were incubated in presence of 2 μg/ml annexin V-FITC, and 10 μl of PI (100 μg/ml) for 10 min. Samples were MLN2238 cost immediately analyzed by flow cytometry on a bi-parametric histogram giving the level of annexin V-FITC and PI fluorescence. buy BI 6727 Apoptosis was assessed by DNA fragmentation assay. Samples of 5.105 pTG 9344 transduced cells with or without synchronization were treated 96 hr with 20 μM GCV. Cells then were centrifuged at 800 g for 5 min at 4°C. The pellet was resuspended in 20 μl of lysis buffer (EDTA 20 mM, Tris 100 mM, SDS 0,8%,

pH 8). Then 10 μl of 500 UI/ml RNAse (Sigma) were added for 60 min at 37°C. The mix was incubated 90 min at 50°C with 10 μl of 20 mg/ml proteinase K. Migration was achieved on 1.8% agarose gel containing 0.5 μg/ml ethidium bromide at 35 V during 4 hr. MSP I digested PBR 322 was used as a size marker. Non-transduced cells treated with MTX or GCV constituted control groups. Statistical analysis Comparisons were made using the Student’s t test. P < .05 was considered as significant. Results

Altered progression in the cell cycle by methotrexate, ara-C or aphidicolin Lepirudin We first assessed the effect of drugs on DHDK12 and HT29 cell cycles to delineate the time for which a maximum of cells were in S phase after drug removal. The effects of the three drugs, i.e. MTX, ara-C and aphidicolin, on the cell cycle were preliminary assessed in DHDK12 cells. After a 24 hr treatment with MTX, ara-C or aphidicolin, cells were analyzed between 0 and 72 hr after drug removal for DNA content by flow cytometry. In the DHDK12 cell line, 20% of cells were in S phase in the absence of drug and this rate was constant over time (Figure 1A). When DHDK12 cells were treated with ara-C or aphidicolin, 25% and 35% of cells were in S phase 10 hr after ara-C or aphidicolin removal, respectively (Additional file 1). By contrast, treatment with MTX resulted in 51% of the cells to be in S phase, while 28% were in G0-G1 phase, 10 hr after drug removal (Figure 1A). The ratio of cells in S phase remained higher than that in G1 phase up to 30 hr following MTX removal.