Development of an attenuated smallpox vaccine candidate: The KVAC103 strain.

Smallpox, a disease caused by the variola virus, is one of the most dangerous diseases and had killed numerous people before it was eradicated in 1980. However, smallpox has emerged as the most threatening bio-terrorism agent; as the first- and second-generation smallpox vaccines have been controversial and have caused severe adverse reactions, new demands for safe smallpox vaccines have been raised and some attenuated smallpox vaccines have been developed. We have developed a cell culture-based highly attenuated third-generation smallpox vaccine candidate KVAC103 strain by 103 serial passages of the Lancy-Vaxina strain derived from the Lister in Vero cells. Several clones were selected, taking into consideration their shape, size, and growth rate in mammalian cells. The clones were then inoculated intracerebrally in suckling mice to test for neurovirulence by observing survival. Protective immune responses in adult mice were examined by measuring the levels of neutralization antibodies and IFN-γ expression. Among several clones, clone 7 was considered the best alternative candidate because there was no mortality in suckling mice against a lethal challenge. In addition, enhanced neutralizing antibodies and T-cell mediated IFN-γ production were observed in clone 7-immunized mice. Clone 7 was named "KVAC103" and was used for the skin toxicity test and full-genome analysis. KVAC103-inoculated rabbits showed reduced skin lesions compared to those inoculated with the Lister strain, Lancy-Vaxina. A whole genome analysis of KVAC103 revealed two major deleted regions that might contribute to the reduced virulence of KVAC103 compared to the Lister strain. Phylogenetic inference supported the close relationship with the Lister strain. Collectively, our data demonstrate that KVAC103 holds promise for use as a third-generation smallpox vaccine strain due to its enhanced safety and efficacy.

Outbreak of CTX-M-15-Producing Enterotoxigenic Escherichia coli O159:H20 in the Republic of Korea in 2016.

We investigated an outbreak of enterotoxigenic Escherichia coli (ETEC) O159:H20 associated with the consumption of a tossed-noodle dish in a high school in 2016. Thirty-three ETEC strains isolated from clinical and food samples were genetically indistinguishable. The outbreak strains were resistant to third-generation cephalosporins and harbored a blaCTX-M-15 gene on a 97-kb self-transferable IncK plasmid. This is the first outbreak caused by CTX-M-15-producing ETEC strains.

Functional characteristics of the natural polymorphisms of HIV-1 gp41 in HIV-1 isolates from enfuvirtide-naïve Korean patients.

HIV-1 gp41 plays a key role in viral entry. The insertion of Thr at position 4 and Met/Val/Phe substitutions at position 7 are frequently observed in the fusion peptide (FP) motif of gp41 without major enfuvirtide resistance associated with mutation in heptad repeats 1/2 (HR1/2) of HIV-1 isolates from Korean patients. Here, the influence of these mutations on their biological function was evaluated by employing HIV-1 variants with mutant FPs as shown previously and with recombinant HIV-1 using the env genes of 20 HIV-1 isolates from Korean patients. In an infectivity assay, all FP mutants showed lower infectivity than the wild-type NL4-3. In particular, the substitutions at position 7 led to much greater reductions in infectivity than the insertions at position 4. Nevertheless, the replication kinetics of most mutants were similar to those of the wild type, except that the FP mutants with an Ile insertion at position 4 and a Phe substitution at position 7 showed reduced replication. Moreover, most point mutants showed lower IC50 values for enfuvirtide than the wild type, whereas the L7M substitution resulted in a slightly increased IC50 value. The infectivity using the HIV-1 env recombinant viruses decreased in 14 cases but increased slightly in six cases compared with the wild type. Most recombinants were more susceptible to enfuvirtide than the wild type, except for three recombinants that showed slight resistance. Our findings may help to explain the potential mechanisms corresponding to the natural polymorphism of gp41 and to predict the efficiency of enfuvirtide in treatment of HIV-1-infected patients in Korea.

Quantification of recombinant human erythropoietin by amino acid analysis using isotope dilution liquid chromatography-tandem mass spectrometry.

Herein, we describe an accurate method for protein quantification based on conventional acid hydrolysis and an isotope dilution-ultra performance liquid chromatography-tandem mass spectrometry method. The analyte protein, recombinant human erythropoietin (rhEPO), was effectively hydrolyzed by incubation with 8 mol/L hydrochloric acid at 130 °C for 48 h, in which at least 1 µmol/kg of rhEPO was treated to avoid possible degradation of released amino acids during hydrolysis. Prior to hydrolysis, sample solution was subjected to ultrafiltration to eliminate potential interfering substances. In a reversed-phase column, the analytes (phenylalanine, proline, and valine) were separated within 3 min using gradient elution comprising 20 % (v/v) acetonitrile and 10 mmol/L ammonium acetate, both containing 0.3 % (v/v) trifluoroacetic acid. The optimized hydrolysis and analytical conditions in our study were strictly validated in terms of accuracy and precision, and were suitable for the accurate quantification of rhEPO. Certified rhEPO was analyzed using a conventional biochemical assay kit as an additional working calibrant for the quantification of EPO and improved the accuracy. The optimized protocol is suitable for the accurate quantification of rhEPO and satisfactorily serves as a reference analytical procedure for the certification of rhEPO and similar proteins.

X-ray structure of prephenate dehydratase from Streptococcus mutans.

Prephenate dehydratase is a key enzyme of the biosynthesis of L-phenylalanine in the organisms that utilize shikimate pathway. Since this enzymatic pathway does not exist in mammals, prephenate dehydratase can provide a new drug targets for antibiotics or herbicide. Prephenate dehydratase is an allosteric enzyme regulated by its end product. The enzyme composed of two domains, catalytic PDT domain located near the N-terminal and regulatory ACT domain located near the C-terminal. The allosteric enzyme is suggested to have two different conformations. When the regulatory molecule, phenylalanine, is not bound to its ACT domain, the catalytic site of PDT domain maintain open (active) state conformation as Sa-PDT structure. And the open state of its catalytic site become closed (allosterically inhibited) state if the regulatory molecule is bound to its ACT domain as Ct-PDT structure. However, the X-ray structure of prephenate dehydratase from Streptococcus mutans (Sm-PDT) shows that the catalytic site of Sm-PDT has closed state conformation without phenylalanine molecule bound to its regulatory site. The structure suggests a possibility that the binding of phenylalanine in its regulatory site may not be the only prerequisite for the closed state conformation of Sm-PDT.

Determination of phosphorus impurity that directly affects quantification of microbial genomic DNA using inductively coupled plasma optical emission spectrometry.

We prepared genomic DNA from human placenta, Escherichia coli, and Bacillus subtilis using various DNA extraction methods and quantified the genomic DNA using ultraviolet (UV) spectrophotometry, capillary electrophoresis (CE), and inductively coupled plasma optical emission spectrometry (ICP-OES). Application of ICP-OES unexpectedly led to a serious overestimation of phosphorus in B. subtilis genomic DNA prepared using cetyltrimethyl ammonium bromide (CTAB). Further investigations using reversed-phase high-performance liquid chromatography (RP-HPLC), ultra-performance liquid chromatography electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS), and (31)P nuclear magnetic resonance (NMR) identified the phosphorus impurity as lipoteichoic acid (LTA).

Complete solubilization and purification of recombinant human growth hormone produced in Escherichia coli.

High-level expression of recombinant human growth hormone (hGH) in Escherichia coli (E. coli) leads to the formation of insoluble aggregates as inclusion bodies devoid of biological activity. Until recently, significant efforts have been made to improve the recovery of active hGH from inclusion bodies. Here, we developed an efficient procedure for the production of completely soluble hGH by minimizing the formation of inclusion bodies and optimizing protein purification conditions. Under the newly established conditions we were able to obtain most of the total hGH in the soluble fraction. We show that the soluble protein can be efficiently purified in high yield by a series of chromatographic procedures. We analyzed the resulting hGH using various analytical techniques such as reversed-phase high-performance liquid chromatography (RP-HPLC), size-exclusion chromatography (SEC), matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry, and circular dichroism (CD). These multiple analyses support the conclusion that we obtained highly pure hGH with the expected molecular mass and intact secondary structure. The biological activity of purified hGH was also confirmed by evaluating its growth-promoting effect using a cell proliferation assay. Taken together, we describe a straightforward strategy for the production of completely soluble and biologically active hGH in E. coli.

Efficacy of Vitamin and Antioxidant Supplements in Prevention of Esophageal Cancer: Meta-analysis of Randomized Controlled Trials.

BACKGROUND: Observational epidemiological studies have shown that higher intakes of vitamins or antioxidants were inversely associated with the risk of esophageal cancer. However, randomized controlled trials (RCTs) have reported no preventive efficacy of vitamin or antioxidant supplements on esophageal cancer. This meta-analysis aimed to investigate the efficacy of vitamin and antioxidant supplements in the prevention of esophageal cancer as reported by RCTs. METHODS: We searched PubMed, EMBASE, and the Cochrane Library in May 2013. Two authors independently reviewed and selected eligible articles based on predetermined selection criteria. RESULTS: Of 171 articles searched from three databases and relevant bibliographies, 10 RCTs were included in the final analyses. In a fixed-effect meta-analysis of 10 trials, there was no efficacy of vitamin and antioxidant supplements in the prevention of esophageal cancer (relative risk [RR], 1.04; 95% confidence interval [CI], 0.86-1.25; I(2)=0.0%). Also, subgroup meta-analyses showed that vitamin and antioxidant supplements had no preventive efficacy on esophageal cancer both in the high risk (RR, 1.04; 95% CI, 0.85-1.28; n=4) and non-high risk (RR, 1.01; 95% CI, 0.65-1.56; n=6) groups for esophageal cancer. Further, subgroup meta-analyses revealed no preventive efficacy on esophageal cancer by type of methodological quality and type of vitamin and antioxidant supplements. CONCLUSIONS: Unlike observational epidemiological studies, this meta-analysis of RCTs suggests that there is no clinical evidence to support the efficacy of vitamin and antioxidant supplements in the prevention of esophageal cancer.

Interpretation of protein quantitation using the Bradford assay: comparison with two calculation models.

The Bradford assay is a simple method for protein quantitation, but variation in the results between proteins is a matter of concern. In this study, we compared and normalized quantitative values from two models for protein quantitation, where the residues in the protein that bind to anionic Coomassie Brilliant Blue G-250 comprise either Arg and Lys (Method 1, M1) or Arg, Lys, and His (Method 2, M2). Use of the M2 model yielded much more consistent quantitation values compared with use of the M1 model, which exhibited marked overestimations against protein standards.

New genetic variants of Anaplasma phagocytophilum and Anaplasma bovis from Korean water deer (Hydropotes inermis argyropus).

Wild deer are one of the important natural reservoir hosts of Anaplasma species, which cause granulocytic anaplasmosis in equines, canines, and humans. The objective of the present study was to determine whether and what species of Anaplasma naturally infect Korean water deer (KWD) in the Republic of Korea. A total of 66 spleens from KWD carcasses were collected by the Conservation Genome Resource Bank for Korean Wildlife in Korea between March 2008 and May 2009. Polymerase chain reaction (PCR) was performed using 16S ribosomal (r)RNA, with ankA, groEL, and msp2 gene primers to amplify the genes of Anaplasma and Ehrlichia. Using 16S rRNA-based nested PCR, Anaplasma phagocytophilum and Anaplasma bovis were detected in 42 (63.6%) and 23 (34.8%) of 66 KWD spleens, respectively. The 42 A. phagocytophilum were classified into five genotypes and the 23 A. bovis were classified into two genotypes by sequence analysis. By ankA-, groEL-, and msp2-based nested PCR, A. phagocytophilum was detected in 1 (1.5%), 7 (10.6%), and 3 (4.6%) of 66 samples, respectively. These gene sequences had only one genotype. Five of seven obtained 16S rRNA gene sequences have never been identified. The ankA, groEL, and msp2 obtained gene sequences represented new genotypes. This is the first report of A. phagocytophilum and A. bovis in KWD, suggesting that they may act as reservoirs for anaplasmosis zoonotic pathogens.