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A simple and precise genotyping system based on PCR using type-specific primers was developed for the determination of genotypes A through F of hepatitis B virus (HBV). This assay system is considered to be a useful tool for the molecular diagnosis of HBV infection and for large-scale surveys.Hepatitis B virus (HBV) is a well-known agent of acute and chronic hepatitis, with an estimated 350 million chronic carriers around the world. HBV has a circular and partially doublestranded DNA genome of 3.2 kb containing four overlapping open reading frames. HBV strains isolated worldwide have been classified into six genomic groups deduced from genome comparisons and designated genotypes A to F (3, 6, 7). The HBV genotypes have a characteristic geographic distribution. HBV genotyping by phylogenetic analysis based on nucleotide sequences produces the most reliable and certain genotyping results. However, this is not an appropriate method for largescale genotyping. On the other hand, several groups have reported the genotyping of HBV by the restriction fragment length polymorphism method (1, 2, 4, 8). However, their methods were not so sensitive and specific compared with our PCR genotyping method. In this paper, we report a simpler, more rapid, and more specific genotyping system for HBV involving PCR using type-specific primers.A total of 55 HBV DNA-positive serum samples obtained from individuals in six different countries, including Japan, Vietnam, the United States, Egypt, Ghana, and Bolivia, were used for the evaluation of our genotyping system. We selected the HBV DNA-positive samples by nested PCR. The sequences of PCR primers used in this study are shown in Table 1. The first-round PCR primers (outer primer pairs) and second-round PCR primers (inner primer pairs) were designed on the basis of the conserved nature of nucleotide sequences in regions of the pre-S1 through S genes, irrespective of the six HBV genotypes. P1 (sense) and S1-2 (antisense) were universal outer primers (1,063 bases). B2 was used as the inner primer (sense) with a combination called mix A for genotypes A, B, and C. Mix A consisted of antisense primers BA1R (type A specific), BB1R (type B specific), and BC1R (type C specific). B2R was used as the inner primer (antisense) with a combination called mix B for genotypes D, E, and F. Mix B consisted of sense primers BD1 (type D specific), BE1 (type E specific), and BF1 (type F specific). These primer combinations for second-round PCR were designed on the basis of the differences in the sizes of the genotype-specific bands. The type-specific primers were designed on the basis of the conserved nature of those sequences within a genotype and on the basis of their poor homology with the sequences derived from other HBV genotypes. The strategy for HBV genotyping is illustrated in Fig.

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La 0.6 Sr 0.4 MnO 3 thin films were fabricated on SrTiO3 (001) substrates using pulsed laser deposition with observing persistent intensity oscillation of reflection high-energy electron diffraction. By atomic force microscopy, the surface of resulting films was confirmed to be extremely flat, showing atomically smooth terraces and 0.4 nm high steps corresponding to a unit cell height of perovskite. The surface terminating atomic layer was unambiguously assigned to the MnO2 layer by coaxial impact collision ion scattering spectroscopy. Crystal symmetry of the films is distorted into a tetragonal one due to the strain to fulfill perfect in-plane matching with the substrate even for films as thick as 100 nm. Even for films as thin as 4 nm (10 unit cells), ferromagnetic transition takes place to induce a metallic state and large negative magnetoresistance is observed as well.

Interaction of advanced glycation end products (AGE) with AGE receptors induces several cellular phenomena potentially relating to diabetic complications. We here show that AGE-modi¢ed bovine serum albumin (BSA) is endocytosed by adipocytes via CD36. Upon di¡erentiation, 3T3-L1 and human subcutaneous adipose cells showed marked increases in endocytic uptake and subsequent degradation of [125 I]AGE-BSA, which were inhibited e¡ectively by the anti-CD36 antibody. Ligand speci¢city of CD36 for modi¢ed BSAs was compared with that of LOX-1 and scavenger receptor class A. Effect of fucoidan on [125 I]AGE-BSA binding showed a sharp contrast to that on [125 I]-oxidized low density lipoprotein. These results implicate that CD36-mediated interaction of AGE-modi¢ed proteins with adipocytes might play a pathological role in obesity or insulin-resistance. ß

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Objective To examine recent trends of acute infection with hepatitis B virus (HBV) in Japan by nationwide surveillance and phylogenetic analyses. Methods During 1991 through 2009, a sentinel surveillance was conducted in 28 national hospitals in a prospective cohort study. Genotypes of HBV were determined in 547 patients with acute hepatitis B. Nucleotide sequences in the preS1/S2/S gene of genotype A and B isolates were determined for phylogenetic analyses. Results HBV genotype A was detected in 137 (25% (accompanied by genotype G in one)) patients, B in 48 (9%), C in 359 (66%), and other genotypes in the remaining three (0.5%). HBV persisted in five with genotype A including the one accompanied by genotype G; another was co-infected with HIV type 1. The genotype was A in 4.8% of patients during 1991e1996, 29.3% during 1997e2002, and 50.0% during 2003e2008 in the capital region, as against 6.5%, 8.5% and 33.1%, respectively, in other regions. Of the 114 genotype A isolates, 13 (11.4%) were subgenotype A1, and 101 (88.6%) were A2, whereas of the 43 genotype B isolates, 10 (23.3%) were subgenotype B1, 28 (65.1%) were B2, two (4.7%) were B3, and three (7.0%) were B4. Sequences of 65 (64%) isolates of A2 were identical, as were three (23%) of A1, and five (18%) of B2, but none of the B1, B3 and B4 isolates shared a sequence. Conclusions Acute infection with HBV of genotype A, subgenotype A2 in particular, appear to be increasing, mainly through sexual contact, and spreading from the capital region to other regions in Japan nationwide. Infection persisted in 4% of the patients with genotype A, and HBV strains with an identical sequence prevailed in subgenotype A2 infections. This study indicates the need for universal vaccination of young people to prevent increases in HBV infection in Japan.Hepatitis B virus (HBV) has been classified into 10 genotypes, designated AeJ, based on a >8% divergence in the full-genome sequence. Significance of this studyWhat is already known about this subject?< In Japan, a national prevention programme was started in 1986 with selective vaccination of babies born to mothers who carry hepatitis B virus (HBV). Since then, the prevalence of hepatitis B surface antigen among younger generations has decreased sharply. < However, retrospective studies indicate that the frequency of HBV genotype A is increasing among patients with acute hepatitis B (AHB) within the capital region of Japan. < Infection with genotype A more often persists than infection with other genotypes. < Because there is no reliable and comprehensive surveillance system for AHB in Japan, the incidence of AHB and factors responsible for changes over many years are not known. What are the new findings?< This is a prospective cohort study for surveillance of AHB throughout Japan in a national research programme. < The incidence of AHB in Japan has not decreased, because genotype A infections have increased over time. < Genotype A infections started to increase in the capital region of Japan, and then spread to other r...