Biochemical characterization of a recombinant Japanese encephalitis virus RNA-dependent RNA polymerase

<p>Abstract</p> <p>Background</p> <p>Japanese encephalitis virus (JEV) NS5 is a viral nonstructural protein that carries both methyltransferase and RNA-dependent RNA polymerase (RdRp) domains. It is a key component of the viral RNA replicase complex that presumably includes other viral nonstructural and cellular proteins. The biochemical properties of JEV NS5 have not been characterized due to the lack of a robust <it>in vitro </it>RdRp assay system, and the molecular mechanisms for the initiation of RNA synthesis by JEV NS5 remain to be elucidated.</p> <p>Results</p> <p>To characterize the biochemical properties of JEV RdRp, we expressed in <it>Escherichia coli </it>and purified an enzymatically active full-length recombinant JEV NS5 protein with a hexahistidine tag at the N-terminus. The purified NS5 protein, but not the mutant NS5 protein with an Ala substitution at the first Asp of the RdRp-conserved GDD motif, exhibited template- and primer-dependent RNA synthesis activity using a poly(A) RNA template. The NS5 protein was able to use both plus- and minus-strand 3'-untranslated regions of the JEV genome as templates in the absence of a primer, with the latter RNA being a better template. Analysis of the RNA synthesis initiation site using the 3'-end 83 nucleotides of the JEV genome as a minimal RNA template revealed that the NS5 protein specifically initiates RNA synthesis from an internal site, U₈₁, at the two nucleotides upstream of the 3'-end of the template.</p> <p>Conclusion</p> <p>As a first step toward the understanding of the molecular mechanisms for JEV RNA replication and ultimately for the <it>in vitro </it>reconstitution of viral RNA replicase complex, we for the first time established an <it>in vitro </it>JEV RdRp assay system with a functional full-length recombinant JEV NS5 protein and characterized the mechanisms of RNA synthesis from nonviral and viral RNA templates. The full-length recombinant JEV NS5 will be useful for the elucidation of the structure-function relationship of this enzyme and for the development of anti-JEV agents.</p

A fully integrated battery-connected switched-capacitor 4:1 voltage regulator with 70% peak efficiency using bottom-plate charge recycling

This work presents a switched-capacitor (SC) DC-DC voltage regulator that converts a 3.7V battery voltage down to ~0.8V in order to power the `brain' SoC of a flapping-wing microrobotic bee. A cascade of two 2:1 SC converters offers high efficiency for a 4:1 conversion ratio. A charge recycling technique reduces the flying capacitor's bottom-plate parasitic loss by 50% and overall conversion efficiency reaches 70%. The output droop is less than 10% of the nominal output voltage for a worst-case 47mA load step.Engineering and Applied Science

An efficient strategy for cell-based antibody library selection using an integrated vector system

BACKGROUND: Cell panning of phage-displayed antibody library is a powerful tool for the development of therapeutic and imaging agents since disease-related cell surface proteins in native complex conformation can be directly targeted. Here, we employed a strategy taking advantage of an integrated vector system which allows rapid conversion of scFv-displaying phage into scFv-Fc format for efficient cell-based scFv library selection on a tetraspanin protein, CD9. RESULTS: A mouse scFv library constructed by using a phagemid vector, pDR-D1 was subjected to cell panning against stable CD9 transfectant, and the scFv repertoire from the enriched phage pool was directly transferred to a mammalian cassette vector, pDR-OriP-Fc1. The resulting constructs enabled transient expression of enough amounts of scFv-Fcs in HEK293E cells, and flow cytometric screening of binders for CD9 transfectant could be performed simply by using the culture supernatants. All three clones selected from the screening showed correct CD9-specificity. They could immunoprecipitate CD9 molecules out of the transfectant cell lysate and correctly stain endogenous CD9 expression on cancer cell membrane. Furthermore, competition assay with a known anti-CD9 monoclonal antibody (mAb) suggested that the binding epitopes of some of them overlap with that of the mAb which resides within the large extracellular loop of CD9. CONCLUSIONS: This study demonstrates that scFv-Fc from mammalian transient expression can be chosen as a reliable format for rapid screening and validation in cell-based scFv library selection, and the strategy described here will be applicable to efficient discovery of antibodies to diverse cell-surface targets

Measurement of the Background Activities of a 100Mo-enriched powder sample for AMoRE crystal material using a single high purity germanium detector

The Advanced Molybdenum-based Rare process Experiment (AMoRE) searches for neutrino-less double-beta (0{\nu}\b{eta}\b{eta}) decay of 100Mo in enriched molybdate crystals. The AMoRE crystals must have low levels of radioactive contamination to achieve low background signals with energies near the Q-value of the 100Mo 0{\nu}\b{eta}\b{eta} decay. To produce low-activity crystals, radioactive contaminants in the raw materials used to form the crystals must be controlled and quantified. 100EnrMoO3 powder, which is enriched in the 100Mo isotope, is of particular interest as it is the source of 100Mo in the crystals. A high-purity germanium detector having 100% relative efficiency, named CC1, is being operated in the Yangyang underground laboratory. Using CC1, we collected a gamma spectrum from a 1.6-kg 100EnrMoO3 powder sample enriched to 96.4% in 100Mo. Activities were analyzed for the isotopes 228Ac, 228Th, 226Ra, and 40K. They are long-lived naturally occurring isotopes that can produce background signals in the region of interest for AMoRE. Activities of both 228Ac and 228Th were < 1.0 mBq/kg at 90% confidence level (C.L.). The activity of 226Ra was measured to be 5.1 \pm 0.4 (stat) \pm 2.2 (syst) mBq/kg. The 40K activity was found as < 16.4 mBq/kg at 90% C.L.Comment: 20 pages, 6 figures, 5 table

Fluconazole Induced Fixed Drug Eruption

We report on a rare case of fluconazole induced fixed drug eruption in a 62-year old female patient. She was referred to our department for multiple erythematous itchy maculopatches on the face, neck, both upper arms, and trunk area, which had occurred over the previous 6 months. Her attending physician prescribed fluconazole for treatment of onychomycosis. Patch test and oral provocation were performed. The patch test showed a negative result; however, the result for oral provocation was positive, confirming this as a rare case of fluconazole induced fixed drug eruption. To the best of our knowledge, this is the first reported case in Korean dermatologic literature

Improvement in the hygroscopicity of inorganic binder through a dual coating process

The use of an anti-absorbent is proposed in this work to reduce the hygroscopicity of the inorganic binder in the casting mold, in which the anti-absorbent is coated on the mold prepared with an inorganic binder. Three types of polymers were used to select material with optimal water resistance. Polystyrene (PS) and polyvinyl alcohol (PVA) were used as a water-insoluble polymer and water-soluble polymer, respectively. In addition, polyurethane (PU) prepolymer has intermediate properties between PS and PVA. PVA and PU prepolymer were used for comparative testing with PS. For this testing process, the prepared green body was dipped into a solution of inorganic binder precursor mixed with tetraethyl orthosilicate (TEOS, SiO2 precursor) and sodium methoxide (NaOMe, Na2O precursor), and then dipped into a solution of coating reagent after a drying process. Thus, these series of coating processes in a green body is called a dual coating process. Finally the sample was heat-treated at 1000 °C to generate a glass phase by an organic–inorganic conversion process. In the sample prepared with PS, the highest contact angle and a high firing strength were exhibited, independent of polymer concentration, while the sample coated with PVA showed lower green and firing strengths. When prepolymer, PU, was applied, the green strength was remarkably improved, showing lower firing strength compared with that of PS. The green and firing strengths were optimized through the dual coating process with PS. Moreover, the moisture-proof effect of the dual coating process was verified through the moisture steam test

Fracture behavior and thermal durability of lanthanum zirconate-based thermal barrier coatings with buffer layer in thermally graded mechanical fatigue environments

The effects of buffer layer on the fracture behavior and lifetime performance of lanthanum zirconate (La2Zr2O7; LZO)-based thermal barrier coatings (TBCs) were investigated through thermally graded mechanical fatigue (TGMF) tests, which are designed to simulate the operating conditions of rotating parts in gas turbines. To improve the thermal durability of LZO-based TBCs, composite coats consisting of two feedstock powders of LZO and 8 wt% yttria-doped stabilized zirconia (8YSZ) were prepared by mixing different volume ratios (50:50 and 25:75, respectively). The composite coat of 50:50 volume ratio was employed as the top coat, and two types of buffer layers were introduced (25:75 volume ratio in LZO and 8YSZ, and 8YSZ only). These TBC systems were compared with a reference TBC system of 8YSZ. The TGMF tests with a tensile load of 60 MPa were performed for 1000 cycles, at a surface temperature of 1100 °C and a dwell time of 10 min, and then the samples were cooled at room temperature for 10 min in each cycle. For the single-layer TBCs, the composite top coat showed similar results as for the reference TBC system. The triple-layer coating (TLC) showed the best thermal cycle performance among all samples, suggesting that the buffer layer was efficient in improving lifetime performance. Failure modes were different for the TBC systems. Delamination and/or cracks were created at the interface between the bond and top coats or above the interface in the single-layer TBCs, but the TBCs with the buffer layer were delaminated and/or cracked at the interface between the buffer layer and the top coat, independent of buffer layer species. This study allows further understanding of the LZO-based TBC failure mechanisms in operating conditions, especially in combined thermal and mechanical environments, in order to design reliable TBC systems

Prognosis of Stage IV Gastric Cancer Patients

Aim. This study evaluated the survival of gastric cancer patients with metastasis to the hepatoduodenal, retropancreatic, mesenteric, and para-aortic lymph nodes. Materials and methods. We analyzed the survival rate of 435 gastric cancer patients who underwent operation from 2001 to 2010 at the Department of Surgery, Chonnam National University Hospital. There were 43, 25, 16, and 55 patients with metastasis to the hepatoduodenal, retropancreatic, mesenteric, and para-aortic nodes, respectively. Results. Based on tumor location, metastasis to the para-aortic lymph nodes was more common in upper-third cancer, and that to the hepatoduodenal lymph nodes was more common in lower-third cancer. The survival rate of patients with non-regional lymph node metastasis was better than that of patients with hepatic metastasis or peritoneal dissemination (p < 0.05). Conclusion. We recommend performing a more extended lymphadenectomy than a D2 lymphadenectomy in patients with advanced gastric cancer those having metastasis to the hepatoduodenal nodes