Simple transporter trafficking model for amphetamine-induced dopamine efflux

Amphetamine and its derivatives are important drugs of abuse causing both short-term excitatory and long-term addictive effects. The short-term excitatory effects are linked to amphetamine's ability to maintain high levels of dopamine (DA) outside the cell both by inhibiting DA reuptake after synaptic transmission and by enhancing the efflux of DA from the dopaminergic cells. The molecular mechanisms by which amphetamine elicits the efflux of DA and similar monoamines are still unclear. Recent literature data suggest that trafficking of the monoamine transporters is a phenomenon that underlies observed changes in amphetamine-induced monoamine reuptake and efflux. We develop an ordinary differential equation model incorporating the diverse mechanistic details behind amphetamine-induced DA efflux and demonstrate its utility in describing our experimental data. We also demonstrate an experimental method to track the time-varying concentration of membrane-bound transporter molecules from the DA efflux data. The good fit between our model and the experimental data supports the hypothesis that amphetamine-induced transporter trafficking is necessary to produce extended efflux of DA. This model can explain the relative significance of different processes associated with DA efflux at different times and at different concentration ranges of amphetamine and DA. Synapse 61:500–514, 2007. © 2007 Wiley-Liss, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/56075/1/20390_ftp.pd

Droplet-Based Pyrosequencing Using Digital Microfluidics

The feasibility of implementing pyrosequencing chemistry within droplets using electrowetting-based digital microfluidics is reported. An array of electrodes patterned on a printed-circuit board was used to control the formation, transportation, merging, mixing, and splitting of submicroliter-sized droplets contained within an oil-filled chamber. A three-enzyme pyrosequencing protocol was implemented in which individual droplets contained enzymes, deoxyribonucleotide triphosphates (dNTPs), and DNA templates. The DNA templates were anchored to magnetic beads which enabled them to be thoroughly washed between nucleotide additions. Reagents and protocols were optimized to maximize signal over background, linearity of response, cycle efficiency, and wash efficiency. As an initial demonstration of feasibility, a portion of a 229 bp Candida parapsilosis template was sequenced using both a de novo protocol and a resequencing protocol. The resequencing protocol generated over 60 bp of sequence with 100% sequence accuracy based on raw pyrogram levels. Excellent linearity was observed for all of the homopolymers (two, three, or four nucleotides) contained in the C. parapsilosis sequence. With improvements in microfluidic design it is expected that longer reads, higher throughput, and improved process integration (i.e., “sample-to-sequence” capability) could eventually be achieved using this low-cost platform