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We experienced a rare case of Munchausen syndrome with peculiar symmetric ulcers on the whole body. When encountering similar cases with factitious ulceration of the skin, a thorough understanding of the patient's psychiatric condition and a cordial consultation to present illness rather than physical medication might lead to disease stability.

Resistance-associated substitutions (RASs) of hepatitis C virus (HCV) in the NS5A region impair the efficacy of NS5A inhibitors. In this study, we evaluated the characteristics of the novel RASs observed in treatment-failure patients, A92K and a deletion at P32 (P32del), and the susceptibility of viruses with these RASs to various anti-HCV reagents by using JFH-1 based recombinant HCV with NS5A from a genotype 1b Con1 strain (JFH1/5ACon1). We introduced A92K or P32del solely or in combination with Q24K, L28M, R30Q or L31F into the NS5A of JFH1/5ACon1. Viruses harboring R30Q/A92K showed high extracellular core antigens and infectivity titers, whereas the other viruses with RASs showed low replication levels and infectivity titers. All the viruses with A92K or P32del were markedly resistant to ledipasvir, velpatasvir and elbasvir. Interestingly, viruses with R30Q/A92K were more susceptible to grazoprevir than viruses without RAS. All the viruses had a similar susceptibility to ribavirin and sofosbuvir. In conclusion, combination RASs R30Q/A92K enhanced virus production whereas other RASs impaired virus replication. Both A92K and P32del conferred severe resistance even to second generation NS5A inhibitors. However, these viruses were susceptible to grazoprevir, ribavirin and sofosbuvir. Thus, combination regimens with these reagents may eradicate viruses harboring A92K or P32del.

Background: The risk factors associated with the development of hepatocellular carcinoma (HCC) in nonalcoholic fatty liver disease (NAFLD) are still unclear. The aim of the present study was to identify such risk factors in NAFLD patients who developed HCC. Methods: Between April 2000 and ­December 2016, a total of 182 patients with NAFLD were enrolled in this study; of these, only 22 patients had HCC. To identify risk factors, univariate and multivariate analyses were performed. To identify risk factors other than the degree of fibrosis, propensity matched analysis adjusted by the NAFLD fibrosis score (NFS) was carried out on 44 patients. Multivariate and survival analyses were also performed in HCC patients. Results: In 182 patients, multivariate analysis highlighted the NFS (OR 2.275; p < 0.001) and hypertension (OR 5.868; p = 0.037) as independent factors that were significantly associated with the development of HCC. After adjustment for the NFS, multivariate analysis identified diabetic retinopathy (OR 8.654; p = 0.017) as an independent factor that was significantly associated with the development of HCC. For predicting the development of HCC, the area under the receiver operating characteristic curve of diabetic retinopathy was significantly higher than that of diabetes (0.731 vs. 0.615; p < 0.001). In patients with HCC, multivariate analysis indicated that the NFS were significantly associated with diabetic retinopathy. Conclusions: Diabetic retinopathy as well as liver fibrosis is a risk factor that associates with the development of HCC in NAFLD patients. Therefore, NAFLD patients with diabetic retinopathy should undergo careful screening for HCC.

Pouchitis is the most common long-term complication of restorative proctocolectomy with ileal pouch-anal anastomosis. We investigated alterations in the expression of microRNAs, noncoding RNAs that act as potent negative regulators of gene expression, in pouchitis. The subjects of this study were 16 patients with diagnosed pouchitis and 48 patients without pouchitis after restorative proctocolectomy, performed for ulcerative colitis. Total RNA was extracted from biopsies and microRNAs were quantified using a real-time polymerase chain reaction. The expression of microRNA 21 and 223 was higher, whereas that of microRNA 192 and 196a was lower, in the inflamed mucosa from the pouchitis patients than in the mucosa from the non-pouchitis patients. The levels of 14 microRNAs were significantly lower in the mucosa from the pouchitis patients, than in the non-inflamed proximal ileal mucosal samples. The expression of microRNA 192 was remarkably reduced in pouchitis. A significant negative correlation was found between microRNA 192 and interleukin 17 receptor A mRNA levels. Significant alteration in miRNA expression in line with inflammatory bowel disease was evident in the mucosa from the pouchitis patients. Interleukin 17 receptor A may be involved in the pathogenesis of pouchitis through the downregulation of microRNA 192.

Intercalation of metals at a graphene/substrate interface can modify the electronic and vibration properties of the graphene. We investigated the intercalation of Au at the graphene/polycrystalline Ni interface by scanning electron microscopy, transmission electron microscopy, and Raman scattering spectroscopy. Graphene coverage induces step bunching on the poly-crystalline Ni surface. Au intercalation selectively occurs at the graphene/Ni(111) facets and changes the secondary electron contrast as well as recovering intrinsic graphene Raman signals. Au nanoparticles are observed only on the Ni(111) facets and are absent on the narrow step-bunched regions. High-angle annular dark field images indicates the presence of monolayer Au only at the graphene/Ni(111) interface.

AIM Although the efficacy of hepatitis C virus (HCV) treatment is improved dramatically by direct-acting antiviral agents (DAAs), the assessment of hepatocellular carcinoma (HCC) remains important. Interferon lambda 3 (IFN-λ3) is associated with liver fibrosis and inflammation in chronic hepatitis C (CHC) patients, but its impact on carcinogenesis remains controversial and little is known about its effects after viral clearance. To determine the contribution of IFN-λ3 to hepatocarcinogenesis after HCV clearance, we analyzed IFNL3 genotypes and serial serum IFN-λ3 levels in CHC patients who achieved sustained virologic responses (SVR). METHODS This study comprised 201 CHC patients treated with DAAs. Serum samples were collected sequentially and IFN-λ3 levels were quantified by chemiluminescence enzyme immunoassay. The IFNL3 polymorphism (rs8099917) was genotyped in 195 patients. RESULTS One hundred and twenty-five patients were rs8099917 T/T and 70 were non-T/T. Serum IFN-λ3 levels did not differ significantly with IFNL3 genotype, dropped markedly by 1 week and remained low up to 24 weeks after the end of treatment. Interferon-λ3 levels were significantly higher after viral clearance in patients who developed HCC and were associated with a higher potential for hepatocarcinogenesis, such as a higher frequency of non-hypervascular hypointensive nodules (P = 0.046), higher stages of liver fibrosis (P

Lichenoid drug eruptions have been previously reported. Here, we report two cases of bilateral oral lichenoid lesions on the buccal mucosa that appeared during low-dose methotrexate treatment for rheumatoid arthritis. The first case was a 71-year-old woman on methotrexate for rheumatoid arthritis, who presented with bilateral oral ulceration on the buccal mucosa. On examination, erythematous ulcers surrounded by white plaques and striae were seen. Oral lichen planus was suspected clinically but topical steroids were ineffective. However, an improvement was observed after reducing the dose of methotrexate, and epithelialization occurred after drug cessation. Reintroduction of methotrexate resulted in recurrence. Histopathological examination of a biopsy taken from the oral mucosa confirmed the lesion as an oral lichenoid lesion. The second case was an 87-year-old woman on methotrexate for rheumatoid arthritis, who presented with pain of the oral mucosa. On examination, ulceration, erythema, and white plaques were observed bilaterally on the buccal mucosa. The disease was diagnosed clinically as oral lichen planus but no improvement was seen after topical steroid treatment. The lesions disappeared after discontinuation of methotrexate. This is the first report of oral lichenoid lesions due to methotrexate.

The bovine leukemia virus (BLV) Tax protein is believed to play a crucial role in leukemogenesis by the virus. BLV usually causes asymptomatic infections in cattle, but only one-third develop persistent lymphocytosis that rarely progress after a long incubation period to lymphoid tumors, namely enzootic bovine leucosis (EBL). In the present study, we demonstrated that the BLV tax genes could be divided into two alleles and developed multiplex PCR detecting an L233P mutation of the Tax protein. Then, in order to define the relationship between the Tax protein and leukemogenicity, we examined 360 tumor samples randomly collected from dairy or breeding cattle in Japan, of which Tax proteins were categorized, for age at the time of diagnosis of EBL. The ages of 288 animals (80.0%) associated with L233-Tax and those of 70 animals (19.4%) with P233-Tax individually followed log-normal distributions. Only the two earliest cases (0.6%) with L233-Tax disobeyed the log-normal distribution. These findings suggest that the animals affected by EBL were infected with the virus at a particular point in life, probably less than a few months after birth. Median age of those with P233-Tax was 22 months older than that with L233-Tax and geometric means exhibited a significant difference (P

Pandemic influenza H1N1 virus (A[H1N1]pdm09) emerged in 2009. To determine the phylogeography of A(H1N1)pdm09 in a single population, 70 strains of the virus were isolated from university students or trainee doctors at Tobetsu, Hokkaido, Japan, between September and December 2009. The nucleotide sequences of the HA1 region of the HA genes and described phylogenetic relationships of the strains circulating among them were analyzed. It was found that the 70 isolates could be phylogenetically separated into three groups and that two epidemics were caused by different groups of the virus. The three groups were also distinguishable from each other by three amino acid changes: A197T, S203T and Q293H. The substitution of S203T, which is located in the antigenic site, suggests antigenic drift of the virus.

Bovine leukemia virus (BLV) infection in cattle causes persistent lymphocytosis, and a few percent of infected animals develop lymphoid tumors, namely enzootic bovine leukosis (EBL). In this study, a 440-bp fragment of the env gene was amplified from 204 tumor samples collected from different regions of Japan and analyzed by restriction fragment length polymorphism (RFLP) to determine the association of BLV with EBL. Of the seven RFLP types defined, types I, II, and III were dominant and found in 12.7, 75.0, and 8.3% of tumor samples, respectively. Cattle harboring type III virus were significantly older than other animals at the time of diagnosis of EBL. Type III viruses were found in approximately 33% and 5.5% of Japanese Black and Holstein cattle, respectively, with EBL. These findings indicate that genetically distinct BLV was associated with EBL in Japan and that the genetic profile may influence the leukemogenicity of the virus.

Bovine leukemia virus (BLV) infection of cattle has been increasing yearly in Japan although several European countries have successfully eradicated the infection. In the present study, phylogenetic analysis on the env gene obtained from 64 tumor samples found in different regions in Japan was carried out in order to define the genetic background of BLV strains prevailing in the country. Most of the Japanese isolates were found to reside in the consensus cluster or genotype 1 of BLV strains (Rodriguez et al., 2009). Out of them, 21 isolates and 10 isolates exhibited the identical sequences, respectively. Only one isolate was classified into the different genotype related to the US isolates. Analysis on the deduced amino acids of gp51 demonstrated the sequence diversity in the neutralizing domain. These data may indicate that two major populations of BLV prevailed throughout Japan, whereas antigenic variants also exist. It was further proved that multiple invasion of the genetically different BLV strains have occurred in Japan.

Influenza A virus has eight-segmented RNA molecules as a genome and, among all strains of the virus, both ends of each segment have 13 and 12 nucleotide sequences conserved. In the present study, a simple RT-PCR method to amplify all eight segments of the virus and determine the HA and NA subtype using a single primer set based on the conserved terminal sequences has been established. This method is also capable of detecting subgenomic defective interfering RNA of the influenza A virus. Since the primers used here cope with each and every RNA segment of influenza A virus, this simple RT-PCR method is valuable not only for cloning each gene of the virus, but also for identifying subtypes, including subtypes other than 16 HA and 9 NA subtypes.

In this study, 520 serum samples from Yezo-deer in the Hidaka district, Hokkaido, Japan were examined by enzyme-linked immunosorbent assay to investigate whether the animals were infected with hepatitis E virus (HEV). The distribution of optical density values showed a bimodal pattern and 181 samples (34.8%) were deemed to be antibody-positive against HEV. At least five (2.8%) of the positive sera gave specific bands by Western blot analysis. An age-dependent increase in prevalence of the antibodies was found among the animals. These findings indicate that Yezo-deer are a possible host for HEV infection. To avoid the risk of becoming HEV infected, the consumption of raw Yezo-deer meat must be prohibited.

The hypothesis of this research was that elderly people with many remaining teeth and good occlusion (8020 achievers) would be able to maintain proper head and body posture, despite aging. The purpose of this study, as a first stage, was to clarify the aging phenomenon of cervical curvature in 8020 achievers in comparison with that in young adults. Subjects consisted of twenty-eight 8020 achievers, with a mean age of 82.96+/-3.3 years and 26.5+/-4.0 teeth. For comparison, forty adults in their 20's with a mean age of 22.9+/-0.7 years and 28.2+/-0.6 teeth were also enrolled. The cervical vertebra was assessed based on the distance from the CV line (tangential line of the 2nd and 6th cervical vertebra) to each cervical vertebra and the angles formed by the cervical and reference lines in the cranial bone. Every distance from the CV line to each cervical vertebra in the 8020 group was bigger than that in the 20's group (p0.01-0.001). The distance from the CV line to CV-3 and CV-5 in 8020 women was larger than that in 8020 men (p0.05). Every distance from the CV line to each cervical vertebra in 8020 women was larger than that in 20's women (p0.01-0.001). There was no significant difference between 8020 men and 20's men. The difference between the women's group was more marked than that between age groups for men. The cervical curvature in 8020 achievers showed a greater tendency toward cervical lordosis than that in young adults. In the 8020 achievers, the curvature in women was greater than that in men. The curvature in 8020 women seemed was marked, showing strong cervical lordosis, despite the presence of many remaining teeth and good occlusion. It remains to be determined by comparing 8020 achievers with ordinary elderly whether the condition of the teeth influences spinal curvature with aging.

Bovine leukemia virus (BLV) is the causative agent of enzootic bovine leucosis (EBL), to which animals are most susceptible at 4–8 years of age. In this study, we examined tumor cells associated with EBL in an 18-year-old cow to reveal that the cells carried at least two different copies of the virus, one of which was predicted to encode a reverse transcriptase (RT) lacking ribonuclease H activity and no integrase. Such a deficient enzyme may exhibit a dominant negative effect on the wild-type RT and cause insufficient viral replication, resulting in delayed tumor development in this cow.

Objective— The acquisition of arterial or venous identity is highlighted in vascular development. Previously, we have reported an embryonic stem (ES) cell differentiation system that exhibits early vascular development using vascular endothelial growth factor (VEGF) receptor-2 (VEGFR2)-positive cells as common vascular progenitors. In this study, we constructively induced differentiation of arterial and venous endothelial cells (ECs) in vitro to elucidate molecular mechanisms of arterial-venous specification. Methods and Results— ECs were induced from VEGFR2 + progenitor cells with various conditions. VEGF was essential to induce ECs. Addition of 8bromo-cAMP or adrenomedullin (AM), an endogenous ligand-elevating cAMP, enhanced VEGF-induced EC differentiation. Whereas VEGF alone mainly induced venous ECs, 8bromo-cAMP (or AM) with VEGF supported substantial induction of arterial ECs. Stimulation of cAMP pathway induced Notch signal activation in ECs. The arterializing effect of VEGF and cAMP was abolished in recombination recognition sequence binding protein at the Jκ site deficient ES cells lacking Notch signal activation or in ES cells treated with γ-secretase inhibitor. Nevertheless, forced Notch activation by the constitutively active Notch1 alone did not induce arterial ECs. Conclusions— Adrenomedullin/cAMP is a novel signaling pathway to activate Notch signaling in differentiating ECs. Coordinated signaling of VEGF, Notch, and cAMP is required to induce arterial ECs from vascular progenitors.

Dissection of cardiomyocyte differentiation process at the cellular level is indispensable in the research for cardiac development and regeneration. Previously, we have established an embryonic stem cell differentiation system that reproduces early vascular development from progenitor cells that express Flk1, a vascular endothelial growth factor receptor, by the combinatory application of 2-dimensional culture and flowcytometry. Here we show that cardiomyocytes can be successfully induced from a single Flk1+ cell on 2-dimensional culture, enabling the direct observation of differentiating cardiomyocytes and the prospective identification of cardiac progenitor potentials. Flk1+ cells could give rise to cardiomyocytes, as well as endothelial cells, from a single cell by the co-culture on OP9 stroma cells in a fusion-independent manner. Among the cell populations in intermediate stages from Flk1+ cells to cardiomyocytes, Flk1+/CXCR4+/vascular endothelial cadherin- cells were cardiac-specific progenitors at the single cell level. Noggin, a bone morphogenetic protein inhibitor, abolished cardiomyocyte differentiation by inhibiting the cardiac progenitor induction. However, wnt inhibitors Dkk-1 or Frizzled-8/Fc chimeric protein augmented, but wnt3a inhibited, cardiomyocyte differentiation. In vitro reproduction of cardiomyocyte differentiation process should be a potent tool for the cellular and molecular elucidation of cardiac development, which would provide various targets for cardiac regeneration.

We identified a novel neuraminidase (NA)-deficient virus that was a 2009 pandemic influenza H1N1 virus mutant. The mutant virus had a deletion of 1,009 nt in the NA gene and lacked an enzymatic domain. Although the yield of the NA-deficient virus was limited, it formed large plaques when applied to MDCK cell cultures, indicating that the virus was able to spread to adjacent cells. Furthermore, the NA-deficient virus was eluted from chicken erythrocytes at 37 °C, even in the presence of the antiviral drug peramivir. Spread of this NA-deficient virus may pose a potential threat to anti-influenza therapies.

Summary: The nucleotide sequences of the proximal promoters of UDP-glucuronosyltransferase (UGT) 1A8 and 1A9 genes are very similar. However, UGT1A8 and 1A9 are mainly expressed in extra-hepatic and hepatic cells, respectively. Using mutants of UGT1A8 and 1A9 proximal promoters, we revealed their critical differences in terms of promoter activity and the role of the T-repeat region (T-region) conserved in both promoters. In extra-hepatic cells, Caco2, the activity of UGT1A9 proximal promoter increased to 73.4 ± 8.5% of that of the UGT1A8 proximal promoter with only 4 base changes: –160C, –152A, –62T, and –59G. The derivatives of the T-region showed that this region is not necessary for promoter activity, but the length of T repeats influences the activity somewhat. Therefore, the cause of the low activity of the UGT1A9 proximal promoter may be not only 4 base changes, but also the truncation of T repeats. From these results, the UGT1A9 proximal promoter was assumed to change into the non-active form from the original sequence, and this might be one of the reasons for the tissue-specific expression of UGT1A9.

Pandemic influenza H1N1 virus (A[H1N1]pdm09) emerged in 2009. To determine the phylogeography of A(H1N1)pdm09 in a single population, 70 strains of the virus were isolated from university students or trainee doctors at Tobetsu, Hokkaido, Japan, between September and December 2009. The nucleotide sequences of the HA1 region of the HA genes and described phylogenetic relationships of the strains circulating among them were analyzed. It was found that the 70 isolates could be phylogenetically separated into three groups and that two epidemics were caused by different groups of the virus. The three groups were also distinguishable from each other by three amino acid changes: A197T, S203T and Q293H. The substitution of S203T, which is located in the antigenic site, suggests antigenic drift of the virus.

Influenza A virus has eight-segmented RNA molecules as a genome and, among all strains of the virus, both ends of each segment have 13 and 12 nucleotide sequences conserved. In the present study, a simple RT-PCR method to amplify all eight segments of the virus and determine the HA and NA subtype using a single primer set based on the conserved terminal sequences has been established. This method is also capable of detecting subgenomic defective interfering RNA of the influenza A virus. Since the primers used here cope with each and every RNA segment of influenza A virus, this simple RT-PCR method is valuable not only for cloning each gene of the virus, but also for identifying subtypes, including subtypes other than 16 HA and 9 NA subtypes.