Size-controllable quartz nanostructure for signal enhancement of DNA chip.

A mask-free, cost-effective dry-etching method for the fabrication of height- and spacing-controlled, pillar-like nanostructures was established in order to detect DNA molecules. The height and spacing of the quartz nanostructure were regulated by successive O(2) and CF(4) reactive ion etching times. The height and spacing of the nanostructures were tuned between 118 and 269 nm and between 107 and 161 nm, respectively. Probe DNA was immobilized on the structure and hybridized with fluorescently-labeled target DNA. Increases in the height and spacing of the nanopillar structure positively correlated with the fluorescence intensity of bound DNA. Usage of the nanostructure increased the DNA detection limit by up to 100-fold.

Occurrence of scuticociliatosis in olive flounder Paralichthys olivaceus by Phiasterides dicentrarchi (Ciliophora: scuticociliatida).

In the course of identifying scuticociliates recently obtained from systemically infected olive flounder Paralichthys olivaceus in Korea, we found a scuticociliate species whose small subunit ribosomal RNA (SS rRNA) gene was not amplified by species-specific primers previously designed for Uronema marinum and Pseudocohnilembus persalinus. By studying morphological characteristics of wet-mounted and stained specimens, we identified the species as Philasterides dicentrarchi, which has been reported to cause systemic infection in the European sea bass Dicentrarchus labrax and turbot Scophthalmus maximus. In this study, we compared morphological characteristics of our specimens with previously reported Philasterides species, including P. dicentrarchi, and sequenced the SS rRNA gene in order to design P. dicentrarchi specific primers. This is the first report on scuticociliatosis caused by P. dicentrarchi from marine fish in Asia.

Pseudocohnilembus persalinus (Ciliophora: Scuticociitida) is an additional species causing scuticociliatosis in olive flounder Paralichthys olivaceus.

In the present study, Pseudocohnilembus persalinus was first reported as a species causing scuticociliatosis in olive flounder Paralichthys olivaceus. Based on the stained specimens, P. persalinus was clearly differentiated from Uronema marinum, which has been shown to be a cause of scuticociliatosis in farmed olive flounder in Korea from its characteristic oral infraciliature structure. The 1754 bp small subunit ribosomal RNA (SS rRNA) gene sequence of P. persalinus showed 95% homology with the partial sequence of P. hargisi SS rRNA. Moreover, multiplex PCR based on the species-specific amplification of the SS rRNA gene sequence enabled us to distinguish P. persalinus from U. marinum in a simple and rapid manner. P. persalinus was clearly differentiated from U. marinum even when the host was infected simultaneously with both species. These data suggest that the multiplex PCR procedure would make it possible to avoid the cumbersome and time-consuming procedures of morphological analysis for the definitive identification of ciliates.

Light- and electron-microscope description of Kudoa paralichthys n. sp. (Myxozoa, Myxosporea) from the brain of cultured olive flounder Paralichthys olivaceus in Korea.

A new Myxosporea, Kudoa paralichthys n. sp., is described from the brain of cultured olive flounder Paralichthys olivaceus in South Korea. Mature spores were quadrate in apical view, measuring 5.19 +/- 0.54 microm in length, 8.23 +/- 0.50 microm in width, and 6.87 +/- 0.45 microm in thickness. Four valves were equal in size, each with 1 polar capsule. Polar capsules were pyriform in shape, measuring 2.2 +/- 0.22 microm in length and 1.2 +/- 0.14 microm in breadth. The sporoplasm consisted of a larger outer cell completely surrounding a smaller inner one, and had cytoplasmic projections. The junctions of shell valves were L-shaped. The sutural planes converged at the anterior ends of the spores and were associated with 4 small apex prominences in the central meeting point of the spores.

Measurement of protease activity of live Uronema marinun (Ciliata: Scuticociliatida) by fluorescence polarization.

The proteolytic activity of live Uronema marinum was analyzed by a fluorescence polarization (FP) technique. Protease activity was measured as a decrease in the FP value using fluorescein isothiocynate (FITC)-casein as a protein substrate. A time-dependent decrease in FP occurred in plate wells containing live U. marinum. Supplementation with the cysteine protease inhibitor E-64 had no significant inhibitory effect on the decrease in FP at any of the concentrations used. In contrast, supplementation with 1,10-phenanthroline resulted in complete inhibition of proteolysis for 30 min at 1 mM and for 1 h at 2 and 5 mM. Effective inhibition of the proteolytic activity of live U. marinum by 1,10-phenanthroline indicated that metalloproteases are the main proteases excreted by U. marinum. As U. marinum has a high potential for systemically invading and destroying fish tissues, the metalloproteases excreted by live U. marinum are likely to be involved in the invasion of host tissues and the pathogenicity of the parasite.