26 research outputs found
A high-throughput method to detect Plasmodium falciparum clones in limiting dilution microplates
<p>Abstract</p> <p>Background</p> <p>Molecular and cellular studies of <it>Plasmodium falciparum </it>require cloning of parasites by limiting dilution cultivation, typically performed in microplates. The parasite's slow replication rate combined with laborious methods for identification of positive wells has limited these studies. A new high-throughput method for detecting growth without compromising parasite viability is reported.</p> <p>Methods</p> <p><it>In vitro </it>parasite cultivation is associated with extracellular acidification. A survey of fluorescent pH indicators identified 5-(and-6)-carboxy SNARF-1 as a membrane-impermeant dye with a suitable <it>pK<sub>a </sub></it>value. Conditions for facile detection of viable parasites in 96-well microplates were optimized and used for limiting dilution cloning of genetic cross progeny and transfected parasites.</p> <p>Results</p> <p>5-(and-6)-carboxy SNARF-1 is a two-emission wavelength dye that accurately reported extracellular pH in parasite cultures. It readily detected parasite growth in microplate wells and yielded results comparable to labour-intensive examination of Giemsa-stained smears. The dye is non-toxic, allowing parasite detection without transfer of culture material to additional plates for separate assays. This dye was used with high-throughput limiting dilution culture to generate additional progeny clones from the HB3 × Dd2 genetic cross.</p> <p>Conclusions</p> <p>This fluorescence-based assay represents a low-cost, efficient method for detection of viable parasites in microplate wells; it can be easily expanded by automation.</p
Exercise training favors increased insulin-stimulated glucose uptake in skeletal muscle in contrast to adipose tissue: a randomized study using FDG PET imaging
Physical exercise increases peripheral insulin sensitivity, but regional differences are poorly elucidated in humans. We investigated the effect of aerobic exercise training on insulin-stimulated glucose uptake in five individual femoral muscle groups and four different adipose tissue regions, using dynamic (femoral region) and static (abdominal region) 2-deoxy-2-[18F]fluoro-d-glucose (FDG) PET/CT methodology during steady-state insulin infusion (40 mU·m−2·min−1). Body composition was measured by dual X-ray absorptiometry and MRI. Sixty-one healthy, sedentary [V̇o2max 36(5) ml·kg−1·min−1; mean(SD)], moderately overweight [BMI 28.1(1.8) kg/m2], young [age: 30(6) yr] men were randomized to sedentary living (CON; n = 17 completers) or moderate (MOD; 300 kcal/day, n = 18) or high (HIGH; 600 kcal/day, n = 18) dose physical exercise for 11 wk. At baseline, insulin-stimulated glucose uptake was highest in femoral skeletal muscle followed by intraperitoneal visceral adipose tissue (VAT), retroperitoneal VAT, abdominal (anterior + posterior) subcutaneous adipose tissue (SAT), and femoral SAT ( P < 0.0001 between tissues). Metabolic rate of glucose increased similarly (∼30%) in the two exercise groups in femoral skeletal muscle (MOD 24[9, 39] μmol·kg−1·min−1, P = 0.004; HIGH 22[9, 35] μmol·kg−1·min−1, P = 0.003) (mean[95% CI]) and in five individual femoral muscle groups but not in femoral SAT. Standardized uptake value of FDG decreased ∼24% in anterior abdominal SAT and ∼20% in posterior abdominal SAT compared with CON but not in either intra- or retroperitoneal VAT. Total adipose tissue mass decreased in both exercise groups, and the decrease was distributed equally among subcutaneous and intra-abdominal depots. In conclusion, aerobic exercise training increases insulin-stimulated glucose uptake in skeletal muscle but not in adipose tissue, which demonstrates some interregional differences. </jats:p
Trimetrexate as biochemical modulator of 5-fluorouracil/leucovorin in advanced colorectal cancer: final results of a randomised European study
Trimetrexate (TMTX) is a biochemical modulator of 5-fluorouracil (5-FU) and leucovorin (LV). Phase II trials have shown promising activity of 5-FU/LV/TMTX in patients with advanced colorectal cancer (ACC). This trial evaluated the effect of TMTX in combination with 5-FU/LV as first-line treatment in ACC. Patients with ACC were randomised to receive either intravenous LV 200 mg/m2/5-FU 600 mg/m2 or TMTX 110 mg/m2 followed 24 h later by LV 200 mg/m2/5-FU 500 mg/m2 plus oral LV rescue. Both schedules were given weekly for 6 weeks every 8 weeks. Patients were evaluated for progression-free survival (PFS), overall survival (OS), tumour response, quality of life (QoL) and toxicity. A total of 365 patients were randomised. A statistically significant prolongation of median PFS was seen in patients treated with TMTX/5-FU/LV compared with 5-FU/LV (5.4 months versus 4.1 months, respectively; P = 0.03), and a trend towards a significant benefit for OS (13.4 months versus 10.5 months, respectively; P = 0.08). Tumour response, QoL and toxicity were comparable between the two arms. Diarrhoea was the most frequently occurring grade 3 or 4 toxicity (22% and 30%, respectively). The addition of TMTX to a weekly regimen of 5-FU/LV results in a small but significant improvement in PFS without adding toxicity or worsening QoL in patients with AC