SNPer: An R Library for Quantitative Variant Analysis on Single Nucleotide Polymorphisms among Influenza Virus Populations

Influenza virus (IFV) can evolve rapidly leading to genetic drifts and shifts resulting in human and animal influenza epidemics and pandemics. The genetic shift that gave rise to the 2009 influenza A/H1N1 pandemic originated from a triple gene reassortment of avian, swine and human IFVs. More minor genetic alterations in genetic drift can lead to influenza drug resistance such as the H274Y mutation associated with oseltamivir resistance. Hence, a rapid tool to detect IFV mutations and the potential emergence of new virulent strains can better prepare us for seasonal influenza outbreaks as well as potential pandemics. Furthermore, identification of specific mutations by closely examining single nucleotide polymorphisms (SNPs) in IFV sequences is essential to classify potential genetic markers associated with potentially dangerous IFV phenotypes. In this study, we developed a novel R library called “SNPer” to analyze quantitative variants in SNPs among IFV subpopulations. The computational SNPer program was applied to three different subpopulations of published IFV genomic information. SNPer queried SNPs data and grouped the SNPs into (1) universal SNPs, (2) likely common SNPs, and (3) unique SNPs. SNPer outperformed manual visualization in terms of time and labor. SNPer took only three seconds with no errors in SNP comparison events compared with 40 hours with errors using manual visualization. The SNPer tool can accelerate the capacity to capture new and potentially dangerous IFV strains to mitigate future influenza outbreaks.     

Structural Brain Changes after Traditional and Robot-Assisted Multi-Domain Cognitive Training in Community-Dwelling Healthy Elderly

The purpose of this study was to investigate if multi-domain cognitive training, especially robot-assisted training, alters cortical thickness in the brains of elderly participants. A controlled trial was conducted with 85 volunteers without cognitive impairment who were 60 years old or older. Participants were first randomized into two groups. One group consisted of 48 participants who would receive cognitive training and 37 who would not receive training. The cognitive training group was randomly divided into two groups, 24 who received traditional cognitive training and 24 who received robot-assisted cognitive training. The training for both groups consisted of daily 90-min-session, five days a week for a total of 12 weeks. The primary outcome was the changes in cortical thickness. When compared to the control group, both groups who underwent cognitive training demonstrated attenuation of age related cortical thinning in the frontotemporal association cortices. When the robot and the traditional interventions were directly compared, the robot group showed less cortical thinning in the anterior cingulate cortices. Our results suggest that cognitive training can mitigate age-associated structural brain changes in the elderly.

Trial Registration

ClnicalTrials.gov NCT01596205     

Development of a serum-free medium for the production of erythropoietin by suspension culture of recombinant Chinese hamster ovary cells using a statistical design.

In order to develop a serum-free (SF) medium for the production of erythropoietin (EPO) by suspension culture of recombinant Chinese hamster ovary (rCHO) cells, a statistical optimization approach based on a Plackett-Burman design was adopted. A basal medium was prepared by supplementing Iscove's modified Dulbecco's medium (IMDM) with Fe(NO3)3.9H2O, CuCl2 and ZnSO4.7H2O which are generally contained in SF medium formulations. Insulin, transferrin and ethanolamine were also supplemented to the basal medium to determine their optimal concentrations. From this statistical analysis, glutamate, serine, methionine, phosphatidylcholine, hydrocortisone and pluronic F68 were identified as positive determinants for cell growth. The SF medium was formulated by supplementing the basal medium with components showing positive effects on cell growth in suspension culture. An EPO titer in this optimized SF medium was 79% of that in IMDM supplemented with 5% dialyzed fetal bovine serum (dFBS). Furthermore, the in vitro and in vivo biological activities of EPO produced in the SF medium were comparable to those produced in the serum-supplemented medium. Taken together, the results obtained here show that a Plackett-Burman design facilitates the development of SF media for the production of EPO by suspension culture of rCHO cells.     

Both E6 and E7 oncoproteins of human papillomavirus 16 inhibit IL-18-induced IFN-gamma production in human peripheral blood mononuclear and NK cells.

Cervical carcinoma is the predominant cancer among malignancies in women throughout the world, and human papillomavirus (HPV) 16 is the most common agent linked to human cervical carcinoma. The present study was performed to investigate the mechanisms of immune escape in HPV-induced cervical cancer cells. The presence of HPV oncoproteins E6 and E7 in the extracellular fluids of HPV-containing cervical cancer cell lines SiHa and CaSki was demonstrated by ELISA. The effect of HPV 16 oncoproteins E6 and E7 on the production of IFN-gamma by IL-18 was assessed. E6 and E7 proteins reduced IL-18-induced IFN-gamma production in both primary PBMCs and the NK0 cell line. FACS analysis revealed that the viral oncoproteins reduced the binding of IL-18 to its cellular surface receptors on NK0 cells, whereas there was no effect of oncoproteins on IL-1 binding to its surface IL-1 receptors on D10S, a subclone of the murine Th cell D10.G4.1. In vitro pull-down assays also revealed that the viral oncoproteins and IL-18 bound to IL-18R alpha-chain competitively. These results suggest that the extracellular HPV 16 E6 and E7 proteins may inhibit IL-18-induced IFN-gamma production locally in HPV lesions through inhibition of IL-18 binding to its alpha-chain receptor. Down-modulation of IL-18-induced immune responses by HPV oncoproteins may contribute to viral pathogenesis or carcinogenesis.     

Crystal structure of class I acetohydroxy acid isomeroreductase from Pseudomonas aeruginosa

Acetohydroxy acid isomeroreductase (AHIR) is a key enzyme in the biosynthesis of branched-chain amino acids. We have determined the first crystal structure of a class I AHIR from Pseudomonas aeruginosa at 2.0 A resolution. Its dodecameric architecture of 23 point group symmetry is assembled of six dimeric units and dimerization is essential for the formation of the active site. The dimeric unit of P.aeruginosa AHIR partially superimposes with a three-domain monomer of spinach AHIR, a class II enzyme. This demonstrates that the so-called plant-specific insert in the middle of spinach AHIR is structurally and functionally equivalent to the C-terminal alpha-helical domain of P.aeruginosa AHIR, and the C-terminal alpha-helical domain was duplicated during evolution from the shorter, class I AHIRs to the longer, class II AHIRs. The dimeric unit of P.aeruginosa AHIR possesses a deep figure-of-eight knot, essentially identical with that in the spinach AHIR monomer. Thus, our work lowers the likelihood of the previous proposal that "domain duplication followed by exchange of a secondary structure element can be a source of such a knot in the protein structure" being correct.     

Industrial attributes of β-glucanase produced by Bacillus sp. CSB55 and its potential application as bio-industrial catalyst

Bioprocess and Biosystems Engineering - The β-glucanase produced from Bacillus sp. CSB55 not only depicts the potent industrial characteristics but also relates as bio-industrial catalyst...     

Resolution and reconstitution of soluble components of rat liver microsomal vitamin D3-25-hydroxylase

Solubilized components of the vitamin D₃-25-hydroxylase, isolated from intact rat liver microsomes known to catalyze the C-25 oxidation of vitamin D₃in vitro, have been separated into two submicrosomal fractions enriched in detergent-solubilized NADPH-cytochrome c reductase and cytochrome P-450 or P-448. The P-450 hemoprotein-containing fraction was obtained by solubilization with cholic acid followed by treatment with the nonionic detergent, Emulgen 911, yielding a final preparation with a specific content of 7.25 nmol/mg microsomal protein. The reduced triphosphopyridine nucleotide-dependent cytochrome P-450 reductase activity, as detected by its ability to reduce the artificial electron acceptor, cytochrome c, was isolated free of cytochromes b₅ or P-450 by solubilization with deoxycholate and chromatography on DEAE-cellulose. The reductase component was found to exhibit kinetic properties with Michaelis constants: K_m(NADPH) = 3.14 μM, K_m(NADH) = 31.25 μM, and K_{m(cyt c)} = 12.34 μM. The NADPH-cytochrome c reductase activity was sensitive to NADPH-reversible inhibition by NADP, but not rotenone or cyanide. When the isolated components were incubated in the presence of an NADPH-generating system and carbon monoxide under anaerobic conditions, enzymatic reduction of the P-450 hemoprotein was measured by the appearance of characteristic absorbances at 420 and 450 nm of the reduced carbon monoxide vs. reduced difference spectrum. Furthermore, when the soluble submicrosomal components were reconstituted with excess reduced triphosphopyridine nucleotide, ³H-labeled vitamin D₃, and soluble cytosolic supernatant, full vitamin D₃-25-hydroxylase activity was restored at rates of up to 7.68 pmol/h/mg protein, with an apparent turnover number of cytochrome P-450 of 1.16 to 1.20 under conditions where the concentrations of the hemoprotein were rate limiting for net product formation. These results strongly support the hypothesis that the rat liver microsomal mixed-function oxidase, vitamin D₃-25-hydroxylase, consists of at least two membrane-bound protein components, NADPH-cytochrome c reductase and a cytochrome P-450 terminal oxidase, for the catalytic conversion of vitamin D₃ to 25-hydroxyvitamin D₃.     

Binding of an antitumor drug to DNA, Netropsin and C-G-C-G-A-A-T-T-BrC-G-C-G

The antitumor antibiotic netropsin has been co-crystallized with a double-helical B-DNA dodecanucleotide of sequence: C-G-C-G-A-A-T-T-BrC-G-C-G, and the structure of the complex has been solved by X-ray diffraction at a resolution of 2.2 A. The structure has been refined independently by Jack-Levitt and Hendrickson-Konnert least-squares methods, leading to a final residual error of 0.257 by the Jack-Levitt approach (0.211 for two-sigma data) or 0.248 by the Hendrickson-Konnert approach, with no significant difference between refined structures. The netropsin molecule displaces the spine of hydration and fits snugly within the minor groove in the A-A-T-T center. It widens the groove slightly and bends the helix axis back by 8 degrees, but neither unwinds nor elongates the double helix. The drug molecule is held in place by amide NH hydrogen bonds that bridge adenine N-3 and thymine O-2 atoms, exactly as with the spine of hydration. The requirement of A X T base-pairs in the binding site arises because the N-2 amino group of guanine would demand impermissibly close contacts with netropsin. It is proposed that substitution of imidazole for pyrrole in netropsin should create a family of "lexitropsins" capable of reading G X C-containing base sequences.     

Lafutidine versus Lansoprazole in Combination with Clarithromycin and Amoxicillin for One versus Two Weeks for Helicobacter pylori Eradication in Korea

Background and Aims: Lafutidine is a novel H₂‐receptor antagonist with gastroprotective activity that includes enhancement of gastric mucosal blood flow. The aim of the present study was to test the efficacy of 7‐ or 14‐day lafutidine–clarithromycin–amoxicillin therapy versus a lansoprazole‐based regimen for Helicobacter pylori eradication. Methods: Four hundred and sixty‐three patients with H. pylori‐infected peptic ulcer disease were randomized to one of four regimens: (1) lafutidine (20 mg b.i.d.), clarithromycin (500 mg b.i.d.) and amoxicillin (1000 mg b.i.d.) for 7 days (the 7LFT group) or (2) for 14 days (the 14LFT group); (3) lansoprazole (30 mg b.i.d.), clarithromycin (500 mg b.i.d.), and amoxicillin (1000 mg b.i.d.) for 7 days (the 7LPZ group); or (4) for 14 days (the 14LPZ group). The eradication rates, drug compliance, and adverse effects among the four regimens were compared. Results: The eradication rates by the intention‐to‐treat and per‐protocol analyses in the 7LFT and 7LPZ groups were 76.5% and 81.6%, and 76.9% and 82.0% (p = .94 and .95), respectively. The eradication rates by intention‐to‐treat and per‐protocol analyses in the 14LFT and 14LPZ groups were 78.2% and 82.2%, and 80.4% and 85.9% (p = .70 and .49), respectively. The treatment duration for 7 days or 14 days did not affect the eradication rates. In addition, the adverse effect rates and discontinuation rates were similar among the four groups. Furthermore, the ulcer cure rate and symptom response rate were similar in the lafutidine and lansoprazole groups. Conclusion: The results of this study showed that lafutidine–clarithromycin–amoxicillin therapy was a safe and effective as lansoprazole‐based triple therapy for the eradication rate of H. pylori, and could be considered as an additional treatment option.