Automated sorting of live transgenic embryos.

The vast selection of Drosophila mutants is an extraordinary resource for exploring molecular events underlying development and disease. We have designed and constructed an instrument that automatically separates Drosophila embryos of one genotype from a larger population of embryos, based on a fluorescent protein marker. This instrument can also sort embryos from other species, such as Caenorhabditis elegans. The machine sorts 15 living Drosophila embryos per second with more than 99% accuracy. Sorting living embryos will solve longstanding problems, including (1) the need for large quantities of RNA from homozygous mutant embryos to use in DNA microarray or gene-chip experiments, (2) the need for large amounts of protein extract from homozygous mutant embryos for biochemical studies, for example to determine whether a multiprotein complex forms or localizes correctly in vivo when one component is missing, and (3) the need for rapid genetic screening for gene expression changes in living embryos using a fluorescent protein reporter.

Electrophysiological effects of myocardial stretch and mechanical determinants of stretch-activated arrhythmias.

BACKGROUND: Although the existence of myocardial mechanoelectrical feedback is well established, the mechanism of arrhythmia induction by ventricular dilatation or stretch remains insufficiently defined. In particular, controversy exists when comparing the arrhythmogenic potential of chronic versus acute myocardial stretch. Also, assessment of cellular electrophysiological effects of myocardial stretch has been incomplete. METHODS AND RESULTS: To evaluate the electrophysiological and arrhythmogenic effects of slow versus rapid ventricular wall stretch, we developed an isolated Langendorff-perfused rabbit heart model in which left ventricular (LV) volume can be changed by a computer-controlled servopump. Cellular electrophysiological effects and premature ventricular excitations (PVEs) and their origin produced by the volume increases were assessed by a multiple-site monophasic action potential (MAP) recording system and by volume-conducted ECGs obtained by immersing the entire preparation in a saline-filled tank. Volume was increased either gradually with slow volume ramps (0.1 ml/sec) or suddenly by volume pulses of varying pulse waveforms (three different amplitudes and five different rise velocities) applied randomly 250-350 times to each of eight hearts. Gradual LV volume loading caused gradual decreases in MAP resting and action potential amplitude, whereas rapid, transient volume pulses caused transient depolarizations. Despite similar membrane potential effects of stretch, gradual volume increases rarely (11%) produced PVEs, even with large volume loads, whereas rapid volume pulses of moderate amplitudes regularly triggered PVEs (45-100% of interventions). Logistic regression analysis showed that the probability of PVE occurrence increased independently with both the amplitude and the velocity of the volume increase, with the greatest sensitivity to stretch velocity exhibited at low and intermediate pulse amplitudes. Faster volume pulse rise velocities triggered PVEs at a lower instantaneous pulse amplitude than lower rise velocities, further corroborating the dependence of stretch-activated arrhythmias on the velocity of stretch. In contrast, an increase in the basic ventricular volume had no effect on the probability of PVE occurrence during the volume pulses. The MAP recordings demonstrated spatial variability in the extent of local depolarizations and site of PVE origin; transient depolarizations occurred, and PVEs originated most often in the posterolateral region of the left ventricle. CONCLUSIONS: Membrane depolarization is caused by both gradual and rapid ventricular stretch, but PVEs are more easily elicited by rapid stretch. Regions of greater myocardial compliance that experience greater relative stretch may act as "foci" for stretch-activated arrhythmias during dynamic ventricular loading. These whole-heart data corroborate the existence of stretch-activated membrane channels in ventricular myocardium and may help explain ventricular ectopy under conditions of differential ventricular loading, as in ventricular dyskinesia, or regional muscle traction, as in mitral valve prolapse syndrome.

The interaction between excimer laser energy and vascular tissue.

The effects of XeF1 excimer laser on isolated normal and atherosclerotic aorta were studied. Experiments were performed in flowing water at constant temperature, flow rate, water depth, pulse width (10 nsec), wavelength (351 nm), beam size (1 mm2) and focal length (50 cm). The number of pulses, the pulse energy, and the pulse frequency were varied, and the vascular tissue was studied histologically. The following observations were made: tissue ablation required a minimum threshold pulse energy and was nonlinearly proportional to the number of pulses and the pulse energy delivered; precise tissue ablation occurred at low pulse frequencies, but changes resembling a thermal process were seen as pulse frequency increased; calcified plaque was more photoresistant than atheroma or normal vessel; excimer laser energy was markedly attenuated by blood; and the time interval between pulses and high peak power are related to the precision of ablation by pulsed excimer laser. It is concluded that excimer laser can rapidly and precisely ablate vascular tissue by a photothermal process.

Percutaneous transluminal laser angioplasty for treatment of peripheral vascular disease. Clinical experience with 16 patients.

Percutaneous transluminal laser angioplasty of the peripheral arteries was performed in 16 patients with pain at rest, objective evidence of severe peripheral ischemia, conditions requiring amputation, and/or medical contraindications to surgery. In 14 patients the ipsilateral femoral artery was entered in an antegrade direction using the Seldinger technique, base-line angiograms taken, and laser angioplasty performed using argon ions transported to the target site by a 400-mu quartz fiber. Patency was established in 50% of cases and correlated directly with both total energy delivered and time and power per exposure: the lower the energy, the higher the patency rate. Complications included spasm, pain, and mechanical or laser perforation. The authors conclude that while percutaneous transluminal laser angioplasty of peripheral arteries using argon radiation is possible in man, its clinical value has not been established.

Selective absorption of ultraviolet laser energy by human atherosclerotic plaque treated with tetracycline.

Tetracycline is an antibiotic that absorbs ultraviolet light at 355 nm and preferentially binds to atherosclerotic plaque both in vitro and in vivo. Tetracycline-treated human cadaveric aorta was compared with untreated aorta using several techniques: absorptive spectrophotometry, which demonstrated a distinct absorptive peak at 355 nm in tetracycline-treated plaque that was absent in treated normal vessel; ultraviolet microscopy, which showed that treated atheroma acquired the characteristic fluorescence of tetracycline under ultraviolet light; and tissue uptake of radiolabeled tetracycline, which showed 4-fold greater uptake by atheroma than by normal vessel. In addition, intravenous tetracycline administered to patients undergoing vascular surgery demonstrated characteristic fluorescence in surgically excised diseased arteries. Because of tetracycline's unique properties, we exposed tetracycline-treated and untreated aorta to ultraviolet laser radiation at a wavelength of 355 nm. We found enhanced ablation of tetracycline-treated atheroma compared with untreated atheroma. The plaque ablation caused by ultraviolet laser radiation was twice as extensive in tetracycline-treated vs nontreated plaque (2.2 +/- 0.25 mm vs 1.3 +/- 0.55 mm, p less than 0.017). This study demonstrates the potential of tetracycline plaque enhancement for the selective destruction of atheroma by ultraviolet laser radiation.