Real-time detection of DNA hybridization and melting on oligonucleotide arrays by using optical wave guides.

The challenge of the Human Genome Project is to increase the rate of DNA sequence acquisition by two orders of magnitude to complete sequencing of the human genome by the year 2000. The present work describes a rapid detection method using a two-dimensional optical wave guide that allows measurement of real-time binding or melting of a light-scattering label on a DNA array. A particulate label on the target DNA acts as a light-scattering source when illuminated by the evanescent wave of the wave guide and only the label bound to the surface generates a signal. Imaging/visual examination of the scattered light permits interrogation of the entire array simultaneously. Hybridization specificity is equivalent to that obtained with a conventional system using autoradiography. Wave guide melting curves are consistent with those obtained in the liquid phase and single-base discrimination is facile. Dilution experiments showed an apparent lower limit of detection at 0.4 nM oligonucleotide. This performance is comparable to the best currently known fluorescence-based systems. In addition, wave guide detection allows manipulation of hybridization stringency during detection and thereby reduces DNA chip complexity. It is anticipated that this methodology will provide a powerful tool for diagnostic applications that require rapid cost-effective detection of variations from known sequences.     

Widespread cutaneous necrosis associated with antiphospholipid syndrome

Summary Widespread cutaneous necrosis as the catastrophic manifestation in antiphospholipid syndrome (APLS) is rather uncommon. Even in the few documented cases, the extensive skin necrosis lesions have been stabilized and regressed with eventual healing utilizing pulse steroid therapy alone or in conjunction with further treatment with plasmapheresis.We describe one case of APLS associated with lupus-like disease. The young female suffered from widespread cutaneous necrosis of the lower legs and gangrene change in the digital end of hands and feet. This condition necessitated amputation of major lower extremities and digital amputation of both hands.     

Myostatin inhibition prevents diabetes and hyperphagia in a mouse model of lipodystrophy

Lipodystrophies are characterized by a loss of white adipose tissue, which causes ectopic lipid deposition, peripheral insulin resistance, reduced adipokine levels, and increased food intake (hyperphagia). The growth factor myostatin (MSTN) negatively regulates skeletal muscle growth, and mice with MSTN inhibition have reduced adiposity and improved insulin sensitivity. MSTN inhibition may therefore be efficacious in ameliorating diabetes. To test this hypothesis, we inhibited MSTN signaling in a diabetic model of generalized lipodystrophy to analyze its effects on glucose metabolism separate from effects on adipose mass. A-ZIP/F1 lipodystrophic mice were crossed to mice expressing a dominant-negative MSTN receptor (activin receptor type IIB) in muscle. MSTN inhibition in A-ZIP/F1 mice reduced blood glucose, serum insulin, triglyceride levels, and the rate of triglyceride synthesis, and improved insulin sensitivity. Unexpectedly, hyperphagia was normalized by MSTN inhibition in muscle. Blood glucose and hyperphagia were reduced in double mutants independent of the adipokine leptin. These results show that the effect of MSTN inhibition on insulin sensitivity is not secondary to an effect on adipose mass and that MSTN inhibition may be an effective treatment for diabetes. These results further suggest that muscle may play a heretofore unappreciated role in regulating food intake.     

A Cadaveric and Preliminary Clinical Study of Ultrasonographically Assisted Percutaneous Carpal Tunnel Release

The aim of this study was to assess the effectiveness and safety profile of a new technique for ultrasonographically assisted percutaneous carpal tunnel release. Experiments were performed on 40 hands in 20 cadavers. We first performed a detailed ultrasonographic examination and correlation study that included surgical dissection of the transverse carpal ligament, the related neurovascular structures and the bony landmarks of the radiocarpal, midcarpal and carpometacarpal joints of the right hand. We then used the measurements we made for percutaneous carpal tunnel release of the transverse carpal ligament using intra-operative ultrasonography for guidance and a hook knife on the left-hand side. The completeness of the release and the potential risks of injury to the flexor tendon and neurovascular bundles were examined. Using real-time intra-operative ultrasonographic monitoring to clearly delineate these targets, we were able to percutaneously release the transverse carpal ligament completely in 18 (90%) of the 20 hands and partially release it in 2 without injuring any neurovascular bundles. We then performed the procedure on 91 consecutive cases of carpal tunnel syndrome and found that the sensory disturbances disappeared in 100% patients 12 mo post-operatively; only 2 hands were graded as unsatisfactory. There were no intra- or post-operative complications. Based on the results from the cadaveric studies and our successful preliminary clinical outcomes, we conclude that this method is tolerable and that its clinical application can be encouraged.     

Rapid and slow release phenytoin in epileptic patients at steady state: Assessment of relative bioavailability utilizing Michaelis-Menten parameters

The bioavailability of phenytoin from rapid release capsule and oral solution formulations relative to that of a slow release capsule formulation was assessed in five patients who had participated in a three-way crossover study performed at steady state. The subjects then underwent dosage adjustment utilizing the slow release formulation, and estimates of their Michaelis-Menten parameters thus obtained were utilized in calculating the relative bioavailabilities. In addition, expected changes in steady-state plasma phenytoin concentrations were calculated assuming initial levels of 15 mg/liter, with increases and decreases in bioavailability of 10%. The consequences of such alterations in the extent of phenytoin absorption or average content of the dosage form may be clinically significant, particularly where the initial phenytoin level is equal to or greater than the patient's operative K_m.The authors gratefully acknowledge the support of FDA Contract #223-76-3019 and NINCDS Contract #N01-NS-5-2327 in the performance of this research.     

Rapid and slow release phenytoin in epileptic patients at steady state: Comparative plasma levels and toxicity

Phenytoin plasma level and toxicity data were compared in a three-way crossover study performed in 18 patients at steady state. Formulations compared were a rapid and a slow release capsule and an oral solution. Plasma concentration-time integrals and maximum plasma phenytoin levels were significantly greater for the rapid release capsule and solution than for the slow release capsule. The incidence of nystagmus and toxicity did not differ for the three treatments,but the occurrence of mental symptoms was more frequent for the oral solution, possibly because of the solvent used in this formulation.     

Effects of docosahexaenoic acid supplementation on blood lipids, estrogen metabolism, and in vivo oxidative stress in postmenopausal vegetarian women

BACKGROUND: Vegetarians are generally deficient in long-chain n-3 fatty acids. Long-chain n-3 fatty acids have a beneficial effect on plasma lipid levels, and some studies showed that they had breast cancer suppression effect. One of the biomarkers of breast cancer risk is the ratio of urinary 2-hydroxyestrone (2-OHE(1)) to 16alpha-hydroxyestrone (16alpha-OHE(1)). OBJECTIVE: To investigate the effect of docosahexaenoic acid (DHA, 22:6n-3) supplementation on blood lipids, estrogen metabolism and oxidative stress in vegetarians. DESIGN: Single-blind, randomized, placebo-controlled trial. INTERVENTIONS: Twenty-seven postmenopausal vegetarian women were recruited. After a 2-week run-in period with 6 g placebo corn oil, the subjects were subsequently randomized to receive either 6 g corn oil (n=13) or 6 g DHA-rich algae oil (2.14 g of DHA/day) (n=14) for 6 weeks. Two subjects in corn oil group withdrew before completion. MAIN OUTCOME MEASURES: Plasma lipids, urinary 2-OHE(1) and 16alpha-OHE(1), urinary F(2)-isoprostanes and plasma alpha-tocopherol. RESULTS: Plasma LDL-DHA and EPA level increased significantly by DHA supplementation. DHA decreased plasma cholesterol (C) levels (P=0.04), but did not influence the levels of plasma TG, LDL-C and HDL-C, alpha-tocopherol, urinary F(2)-isoprostanes, 2-OHE(1), 16alpha-OHE(1) and ratio of 2-OHE(1) to 16alpha-OHE(1) as compared to corn oil. CONCLUSION: DHA supplementation at a dose of 2.14 g/day for 42 days decreases plasma cholesterol but neither does it show beneficial effects on estrogen metabolism, nor does it induce deleterious effects on the observed in vivo antioxidant or oxidative stress marker in postmenopausal vegetarian women. SPONSORSHIP: A grant (# DOH89-TD-1062) from Department of Health, Executive Yuan, Taiwan.     

The universal influenza vaccine M2e-HBc administered intranasally in combination with the adjuvant CTA1-DD provides complete protection

Mucosal vaccination requires effective and safe adjuvants. We have evaluated the non-toxic adjuvant CTA1-DD for mucosal vaccination against influenza. CTA1-DD contains the enzymatically active CTA1 subunit of cholera toxin (CT) genetically fused to a gene encoding a dimer of the D-fragment from Staphylococcus aureus protein A. CTA1-DD only binds to Ig-receptor carrying cells of the immune system. Nasal administration of the universal influenza vaccine M2e-HBc in combination with CTA1-DD completely protected mice from a potentially lethal infection and significantly reduced morbidity. Sera of mice immunized with M2e-HBc + CTA1-DD revealed IgG subclass profiles consistent with an enhanced Th1-type immunity. When the vaccine was administered intraperitoneally, the adjuvant improved the M2e antibody titer in circulation, but did not significantly reduce the morbidity.