We have described a new DNA sequencing platform based on the Sanger chemistry, in which the large-scale microfabricated channel plates and electrophoretic system result in higher-throughput DNA sequencing. Three hundred and eighty-four channels are arranged in a fan-like shape on a 25x47 cm glass plate, on which 384 oval sample holes are connected to each channel coupled to the opposite anode access holes. Two microfabricated plates are set on the sequencing apparatus, in which sequencing electrophoresis is conducted on one plate and the preparation process is on another plate. Each sample hole is loaded with 2.3 microL volume of sample and injected into separation channels electrokinetically. High-quality sequencing data were acquired using the pUC18 template, achieving an average read-length of 1001 bases with 99% accuracy and a throughput of 5 Mbases per day per instrument. To assess the performance in actual sequencing field, the bacterial artificial chromosome shotgun library of the Pseudorca crassidens genome was sequenced, using 1/80 of the quantity of Sanger reagent (0.1 microL). We believe that this is the first demonstration of the useful performance of DNA sequencing using monolithic microfabricated devices with walk-away operation.
A novel automated C-terminal fragment peptide fractionator has been constructed. Digests with lysyl endopeptidase were covalently immobilized on p-phenylene diisothiocyanate polymer beads. Only the C-terminal fragment, which contains no lysyl residue, was liberated by cleavage at the first peptide bond of the immobilized fragment peptides with trifluoroacetic acid, and it was automatically collected. The whole procedure was automatically and precisely performed under microprocessor control in a nitrogen atmosphere. The resulting fragment was sequenced without further purification. Sequences of both N- and C-terminal regions can be routinely obtained for ca. 100 pmol samples by the use of a conventional automated Edman-type protein sequencer.
Introduction
Cilostazol may be a novel therapeutic agent for Alzheimer's disease. Its metabolite, OPC‐13015, has a stronger inhibitory effect on type 3 phosphodiesterase than cilostazol.
Methods
We prospectively enrolled patients with mild cognitive impairment to whom cilostazol was newly prescribed. Patients underwent the Montreal Cognitive Assessment (MoCA) twice, at a 6‐month interval. Plasma cilostazol, OPC‐13015, OPC‐13213, and OPC‐13217 concentrations were determined using liquid chromatography‐tandem mass spectrometry.
Results
MoCA score changes from baseline to the 6‐month visit were positively correlated with ratios of OPC‐13015 to cilostazol and total metabolites (
n
= 19,
P
= .005). Patients with higher ratios of OPC‐13015 (≥0.18, median value;
n
= 10) had significantly higher MoCA scores (
P
= .036) than patients with lower ratios (the ratio <0.18,
n
= 9). The absolute value of OPC‐13015 concentration in blood was also higher in patients with preserved cognitive function (
P
= .033).
Discussion
Blood OPC‐13015 levels may be a predictive biomarker of cilostazol treatment for Alzheimer's disease.
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