Use of DiO-C5-3 to improve Hoechst 33342 uptake, resolution of DNA content, and survival of CHO cells

Chinese hamster cells (line CHO) stained with either 9 microM Hoechst 33342 (HO) alone or in combination with the membrane potential fluorochrome DiO-C5-3 (DiO) were analyzed using uv laser powers between 25 and 500 mW and sorted for determination of survival by a colony formation assay. The combination of HO-DiO increased fluorescence twofold and provided coefficients of variation (CVs) as low as 3.0% under conditions where viability of cells, even at 500 mW excitation, was unaffected. HO-stained cells yielded CVs of about 8.5% and survivals of approximately 90% under similar analytical conditions. At laser powers of 25 mW, CV values for HO-DiO-stained populations were 4.0% compared to 9.4% for HO-stained cells. Results with another membrane potential dye, rhodamine 123 (R 123), in combination with HO showed no improvement compared to HO-stained cells. No preferential, cell cycle phase-specific killing was observed in either the HO- or HO-DiO-stained populations. CVs of human skin diploid fibroblasts stained with HO or with HO-DiO were comparable over the entire laser power range; however, percentage survival was slightly higher for the HO-DiO-stained populations when analyzed and sorted at the higher power (400-500 mW) range. Long-term cultures of sorted CHO-K1 subpopulations, differing in DNA ploidy, were established from HO-DiO-stained cells. Advantages of this new staining procedure include improved DNA content resolution (low CV values) and the potential use of less expensive FCM uv laser systems coupled with less perturbing excitation powers.     

Quantification of riboflavin in human urine using high performance liquid chromatography-tandem mass spectrometry

We developed a selective method to measure riboflavin in human urine. Sample preparation involved solid phase extraction and concentration of the target analyte in urine. The urine concentrate was analyzed using high performance liquid chromatography-tandem mass spectrometry. Riboflavin concentrations were quantified using an isotopically labeled internal standard. The limit of detection was 11 ng/mL, and the linear range was 4.4-20,000 ng/mL. The relative standard deviation at 100, 1000, and 5000 ng/mL was 17%, 17%, and 12%, respectively. The accuracy was 90%. On average, 100 samples, including calibration standards and quality control samples, were prepared per day. Using our method, we measured concentrations of riboflavin in human urine samples that were collected from participants in a study where riboflavin was used as a surrogate chemical to simulate exposure to an environmental toxicant.     

Manipulating substrate and pH in zymography protocols selectively distinguishes cathepsins K, L, S, and V activity in cells and tissues

Cathepsins K, L, S, and V are cysteine proteases that have been implicated in tissue-destructive diseases such as atherosclerosis, tumor metastasis, and osteoporosis. Among these four cathepsins are the most powerful human collagenases and elastases, and they share 60% sequence homology. Proper quantification of mature, active cathepsins has been confounded by inhibitor and reporter substrate cross-reactivity, but is necessary to develop properly dosed therapeutic applications. Here, we detail a method of multiplex cathepsin zymography to detect and distinguish the activity of mature cathepsins K, L, S, and V by exploiting differences in individual cathepsin substrate preferences, pH effects, and electrophoretic mobility under non-reducing conditions. Specific identification of cathepsins K, L, S, and V in one cell/tissue extract was obtained with cathepsin K (37 kDa), V (35 kDa), S (25 kDa), and L (20 kDa) under non-reducing conditions. Cathepsin K activity disappeared and V remained when incubated at pH 4 instead of 6. Application of this antibody free, species independent, and medium-throughput method was demonstrated with primary human monocyte-derived macrophages and osteoclasts, endothelial cells stimulated with inflammatory cytokines, and normal and cancer lung tissues, which identified elevated cathepsin V in lung cancer.     

Inferring human population sizes, divergence times and rates of gene flow from mitochondrial, X and Y chromosome resequencing data

We estimate parameters of a general isolation-with-migration model using resequence data from mitochondrial DNA (mtDNA), the Y chromosome, and two loci on the X chromosome in samples of 25-50 individuals from each of 10 human populations. Application of a coalescent-based Markov chain Monte Carlo technique allows simultaneous inference of divergence times, rates of gene flow, as well as changes in effective population size. Results from comparisons between sub-Saharan African and Eurasian populations estimate that 1500 individuals founded the ancestral Eurasian population approximately 40 thousand years ago (KYA). Furthermore, these small Eurasian founding populations appear to have grown much more dramatically than either African or Oceanian populations. Analyses of sub-Saharan African populations provide little evidence for a history of population bottlenecks and suggest that they began diverging from one another upward of 50 KYA. We surmise that ancestral African populations had already been geographically structured prior to the founding of ancestral Eurasian populations. African populations are shown to experience low levels of mitochondrial DNA gene flow, but high levels of Y chromosome gene flow. In particular, Y chromosome gene flow appears to be asymmetric, i.e., from the Bantu-speaking population into other African populations. Conversely, mitochondrial gene flow is more extensive between non-African populations, but appears to be absent between European and Asian populations.