Development and evaluation of an indirect enzyme-linked immunosorbent assay for detection of foot-and-mouth disease virus nonstructural protein antibody using a chemically synthesized 2B peptide as antigen.

Forty peptides were synthesized corresponding to hydrophilic clusters of amino acids within the sequences of foot-and-mouth disease virus (FMDV) nonstructural proteins (NSP). Six peptides were studied in more detail and the most promising, a 2B peptide, was evaluated in enzyme-linked immunosorbent assay (ELISA) using sera from naive, vaccinated, and vaccinated-and-challenged cattle as well as bovine sera from field outbreaks. The performance of the new NSP peptide ELISA was compared to that of 4 commercial NSP ELISA kits. Antibody to 2B was detectable from the end of the first week to the second week after infection in most of the nonvaccinated animals and by the second to third week in vaccinated-and-challenged animals. The sensitivity of the 2B peptide ELISA was comparable to the 3ABC Ceditest (Ceditest FMDV-NS, Cedi Diagnostics B.V.; Chung et al., 2002). With some modification and further validation, this 2B test could be useful as a screening or conformational NSP test in postvaccination surveillance for FMD.     

Guided bone regeneration with beta-tricalcium phosphate and poly L-lactide-co-glycolide-co-epsilon-caprolactone membrane in partial defects of canine humerus

This study was performed to evaluate the effect of beta-tricalcium phosphate and poly L-lactide-co-glycolide-co-epsilon-caprolactone (TCP/PLGC) membrane in the repair of partial bone defects in canine proximal humerus. Three adult mixed-breed dogs were used during the experimental period. The length of the defect was quarter of the full length of humerus, and width of the defect was quarter of middle diameter of the lateral aspect of humerus. The humeri of each dog were divided into treatment (TCP/PLGC) and control groups. The defect was covered with TCP/PLGC membrane in treatment group. To evaluate regeneration of the bone, computerized tomography (CT) and histopathologic examination were performed. The radiopaque lines were appeared at the original defect sites in TCP/PLGC group but below the original site in control at 4th week. Radiopacity and thickness of the defect sites, and radiopaque lines were more increased at 8th week than those of 4th week. Histopathologic findings revealed fibrous connective tissue migration into the defect and the migration inhibited the structure of new cortex to be placed in the original level in control whereas new cortex growth was found in the level of original line in TCP/PLGC group. However, the new cortical bone in the TCP/PLGC group was thinner and less organized than the adjacent intact cortex, and the amount of new cancellous bones were also scanty. The result suggested that TCP/PLGC membrane is a good guided bone regeneration material to restore the original morphology of humerus in partial defect.     

Dye-resist properties of hetero-multifunctional dye-resist agents in acid dyeing of wool

The dye-resist effect and leveling properties of hetero-mulifunctional dye-resist agents in acid dyeing of wool were investigated. The dye-resist agent with dichlorotriazinyl group achieved better resist effectiveness than those with monochlorotriazinyl group. The resist effectiveness was improved by increasing the number of sulfonate group in dye-resist agents. Also, the resist agents with more sulfonate groups showed better dye-assist effectivness, attributable to the increased electrostatic attraction between dye-resist agents and the cationic dye. However, the leveling properties of dye-resist agents decreased with the number of sulfonate groups in the molecule.     

MET amplification leads to gefitinib resistance in lung cancer by activating ERBB3 signaling

The epidermal growth factor receptor (EGFR) kinase inhibitors gefitinib and erlotinib are effective treatments for lung cancers with EGFR activating mutations, but these tumors invariably develop drug resistance. Here, we describe a gefitinib-sensitive lung cancer cell line that developed resistance to gefitinib as a result of focal amplification of the MET proto-oncogene. inhibition of MET signaling in these cells restored their sensitivity to gefitinib. MET amplification was detected in 4 of 18 (22%) lung cancer specimens that had developed resistance to gefitinib or erlotinib. We find that amplification of MET causes gefitinib resistance by driving ERBB3 (HER3)-dependent activation of PI3K, a pathway thought to be specific to EGFR/ERBB family receptors. Thus, we propose that MET amplification may promote drug resistance in other ERBB-driven cancers as well.     

Glutathione content of in vivo and in vitro matured canine oocytes collected from different reproductive stages

Glutathione (GSH) concentrations of oocytes are considered as an important marker of the cytoplasmic maturation. The present study was designed to compare GSH concentrations of in vivo and in vitro matured canine oocytes. In vivo matured oocytes were collected 72 hr after ovulation by flushing fallopian tubes after laparotomy. Ovaries were collected from bitches with different reproductive stages, and collected oocytes were divided into 2 groups according to the size viz. < 120 microm and > 120 microm in diameter and cultured for 72 hr in Tissue Culture Medium-199 supplemented with 10% FBS, 2.2 mg/ml sodium bicarbonate, 2.0 microg/ml estrogen, 0.5 microg/ml FSH, 0.03 IU/ml hCG, and 1% penicillin-streptomycin solution in the presence or absence of 50 microM beta-mercaptoethanol. GSH concentrations were determined by the dithionitrobenzoic acid-glutathione disulfide (DTNB-GSSG) reductase recycling assay. GSH concentrations of immature canine oocytes were 2.9 and 3.8, 3.5 and 6.8, and 3.1 and 6.5 pM/oocyte for < 120 microm and > 120 microm in diameter oocyte groups at anestrous, follicular and luteal stage, respectively (P<0.05). In vivo matured oocytes had significantly higher GSH concentrations compared with in vitro matured oocytes. The GSH content was 19.2 pM/oocyte for in vivo matured oocytes, while 4.1 to 8.1 and 5.7 to 13.2 pM/oocyte for in vitro matured oocytes cultured in the absence or presence of beta-mercaptoethanol, respectively (P<0.05). Presence of beta-mercaptoethanol increased GSH synthesis in canine oocytes cultured in vitro, and oocytes collected from follicular and luteal stage was superior to anestrus oocytes.     

Impact of vitronectin concentration and surface properties on the stable propagation of human embryonic stem cells

The standard method for culturing human embryonic stem cells (hESC) uses supporting feeder layers of cells or an undefined substrate, Matrigel(?), which is a basement membrane extracted from murine sarcoma. For stem cell therapeutic applications, a superior alternative would be a defined, artificial surface that is based on immobilized human plasma vitronectin (VN), which is an adhesion-mediating protein. Therefore, VN adsorbed to diverse polymer surfaces was explored for the continuous propagation of hESC. Cells propagated on VN-coated tissue culture polystyrene (TCPS) are karyotypically normal after >10 passages of continuous culture, and are able to differentiate into embryoid bodies containing all three germ layers. Expansion rates and pluripotent marker expression verified that a minimal VN surface density threshold is required on TCPS. Further exploration of adsorbed VN was conducted on polymer substrates with different properties, ranging from hydrophilic to hydrophobic and including cationic and anionic polyelectrolyte coatings. Despite differing surface properties, these substrates adsorbed VN above the required surface density threshold and were capable of supporting hESC expansion for >10 passages. Correlating wettability of the VN-coated surfaces with the response of cultured hESC, higher cell expansion rates and OCT-4 expression levels were found for VN-coated TCPS, which exhibits a water contact angle close to 65°. Importantly, this simple, defined surface matches the performance of the benchmark Matrigel, which is a hydrogel with highly complex composition.     

A 3D-Printed Polycaprolactone/Marine Collagen Scaffold Reinforced with Carbonated Hydroxyapatite from Fish Bones for Bone Regeneration

In bone tissue regeneration, extracellular matrix (ECM) and bioceramics are important factors, because of their osteogenic potential and cell–matrix interactions. Surface modifications with hydrophilic material including proteins show significant potential in tissue engineering applications, because scaffolds are generally fabricated using synthetic polymers and bioceramics. In the present study, carbonated hydroxyapatite (CHA) and marine atelocollagen (MC) were extracted from the bones and skins, respectively, of Paralichthys olivaceus. The extracted CHA was characterized using Fourier transform infrared (FTIR) spectroscopy and X-ray diffraction (XRD) analysis, while MC was characterized using FTIR spectroscopy and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The scaffolds consisting of polycaprolactone (PCL), and different compositions of CHA (2.5%, 5%, and 10%) were fabricated using a three-axis plotting system and coated with 2% MC. Then, the MC3T3-E1 cells were seeded on the scaffolds to evaluate the osteogenic differentiation in vitro, and in vivo calvarial implantation of the scaffolds was performed to study bone tissue regeneration. The results of mineralization confirmed that the MC/PCL, 2.5% CHA/MC/PCL, 5% CHA/MC/PCL, and 10% CHA/MC/PCL scaffolds increased osteogenic differentiation by 302%, 858%, 970%, and 1044%, respectively, compared with pure PCL scaffolds. Consequently, these results suggest that CHA and MC obtained from byproducts of P. olivaceus are superior alternatives for land animal-derived substances.     

Molecular epidemiology of rabies virus circulating in South Korea, 1998-2010

We analyzed the nucleotide sequences of the G-L (glycoprotein-large protein) intergenic non-coding region of 33 strains of the rabies virus (RABV) isolated in South Korea in 1998-2010 and compared the sequences with those of previously reported non-Korean strains. The similarities of the nucleotide sequences of the G-L region among all Korean RABV isolates ranged from 97.1 to 100%. Based on the phylogenetic analysis of the G-L region, the Korean RABV isolates were classified into three distinct subgroups with high similarity and were most closely related to the non-Korean NeiMeng1025C isolate, which was isolated from a rabid raccoon dog in eastern China, suggesting that the Korean RABV isolates originate from a rabid raccoon dog in northeastern Asia. Our results indicated that G-L region, as a useful phylogenetic indicator, is equivalent to the nucleoprotein (N) or glycoprotein (G) gene for study of RABV molecular epidemiology and that the Korean RABV isolates showing a few substitutions in the G-L region are continuously circulating in South Korea.     

Effect of conformation on the properties of uniaxially drawn polylacide films upon drawing temperature

Polylactide(PLA) films were drawn at various drawing temperature of 65, 90 and 120 °C. The effects of drawing temperature on structural conformation and properties of PLA films were investigated. It was confirmed that the PLA films at drawing temperature of 65 and 90 °C were composed of α′ phase crystal form. The strain-induced crystallization and molecular orientation increased with increasing the draw ratio, which result in improving the mechanical and thermal properties of α′ phase PLA films. However, at drawing temperature of 120 °C, the strain-induced crystallization and molecular orientation of PLA films were not distinctly detected. It was supposed that the rate of the chain relaxation was faster than chain orientation and strain-induced crystallization during uniaxial drawing process.     

Liquid Chromatography-Mass Spectrometry-Based Rapid Secondary-Metabolite Profiling of Marine Pseudoalteromonas sp. M2

The ocean is a rich resource of flora, fauna, and food. A wild-type bacterial strain showing confluent growth on marine agar with antibacterial activity was isolated from marine water, identified using 16S rDNA sequence analysis as Pseudoalteromonas sp., and designated as strain M2. This strain was found to produce various secondary metabolites including quinolone alkaloids. Using high-resolution mass spectrometry (MS) and nuclear magnetic resonance (NMR) analysis, we identified nine secondary metabolites of 4-hydroxy-2-alkylquinoline (pseudane-III, IV, V, VI, VII, VIII, IX, X, and XI). Additionally, this strain produced two novel, closely related compounds, 2-isopentylqunoline-4-one and 2-(2,3-dimetylbutyl)qunoline-4-(1H)-one, which have not been previously reported from marine bacteria. From the metabolites produced by Pseudoalteromonas sp. M2, 2-(2,3-dimethylbutyl)quinolin-4-one, pseudane-VI, and pseudane-VII inhibited melanin synthesis in Melan-A cells by 23.0%, 28.2%, and 42.7%, respectively, wherein pseudane-VII showed the highest inhibition at 8 µg/mL. The results of this study suggest that liquid chromatography (LC)-MS/MS-based metabolite screening effectively improves the efficiency of novel metabolite discovery. Additionally, these compounds are promising candidates for further bioactivity development.