Utility of Ethylene-Diamine-Tetraacetic Acid Buffer Solution With Boric Acid for Immunostaining of Specimens Stored for an Extended Period

Antigen modification and denaturation are recognized causes of false negatives in immunostaining. Specimens that have been stored for an extended period at room temperature show decreased immunoreactivity and may mislead the diagnosis. Studies of the molecular targeting of drugs often involve immunostaining of previous samples and, in some situations, only unstained specimens can be used. The present study aimed to develop an effective staining method to recover antigen activation in unstained specimens stored for an extended period by using ethylene-diamine-tetraacetic acid (EDTA) buffer solution with boric acid. We compared several commonly used antigen retrieval solutions and found that Tris-borate-EDTA (TBE) buffer solution with a pH ≥8.3 provided sufficient antigen retrieval. However, pH values higher than 8.3 (9.0, 10.0, and 11.0) frequently caused severe tissue damage. Thus, TBE with pH 8.3 was the most suitable antigen retrieval solution for recovering the antigenicity of specimens stored for an extended period. This procedure may allow useful immunohistochemical information, even from sections that have been stored for an extended period

Facilitatory effect of insulin treatment on hepatocellular carcinoma development in diabetes

Background: To evaluate the effect of insulin treatment on the incidence and/or severity of hepatocellular carcinoma (HCC) in a mouse model of HCC based on diabetes. Methods: We recently reported that neonatal streptozotocin (STZ) treatment causes type 1 diabetes and subsequent HCC in ddY, Institute for Animal Reproduction (DIAR) mice. Newborn male DIAR mice were divided into three groups based on STZ and insulin (INS) treatment. STZ was subcutaneously injected (60 mg/g) into the STZ-treated group (DIAR-nSTZ mice, N = 13) and the STZ/insulin-treated group (DIAR-nSTZ/INS mice, N = 20). A physiologic solution was injected into the control group (DIAR-control mice, N = 8) 1.5 days after birth. Insulin was subcutaneously injected into the DIAR-nSTZ/INS mice according to the following protocol: 2 IU/day at 4–5 weeks of age, 3 IU/day at 5–7 weeks of age, and 4 IU/day at 7–12 weeks of age. All mice were fed a normal diet and were subjected to physiological and histopathological assessments at 12 weeks of age. Results: DIAR-nSTZ mice had significantly lower body weight and higher blood glucose levels than DIAR-control mice, whereas no significant differences were observed between DIAR-nSTZ/INS mice and control mice. At 12 weeks of age, lower weight of paratesticular fat and higher levels of total cholesterol, triglyceride, and free fatty acids were observed in DIAR-nSTZ mice compared to DIAR-control mice, whereas there were no significant differences between DIAR-nSTZ/INS mice and DIAR-control mice. In the livers of DIAR-nSTZ mice, HCC was observed in 15% of cases, and dysplastic nodules were observed in 77% of cases. In the livers of DIAR-nSTZ/INS mice, HCC was observed in 39% of cases and dysplastic nodules were observed in 61% of cases (p = 0.011). Moreover, the average tumor size was significantly larger in STZ/INS-treated mice than in STZ-treated mice. Immunohistochemical analysis demonstrated that the expression of ERK1/2, downstream substrates of insulin signaling that activate cell proliferation, was significantly higher in STZ/INS-treated mice compared to STZ-treated mice. Conclusions: Insulin treatment promoted, rather than inhibited, the progression of liver carcinogenesis in DIAR-nSTZ mice. Hyperinsulinemia rather than hyperglycemia can accelerate the progression of HCC via insulin signaling

Melatonin sensitises shikonin-induced cancer cell death mediated by oxidative stress via inhibition of the SIRT3/SOD2-AKT pathway

Recent research suggests that melatonin (Mel), an endogenous hormone and natural supplement, possesses anti-proliferative effects and can sensitise cells to anti-cancer therapies. Although shikonin (SHK) also possesses potential anti-cancer properties, the poor solubility and severe systemic toxicity of this compound hinders its clinical usage. In this study, we combined Mel and SHK, a potentially promising chemotherapeutic drug combination, with the aim of reducing the toxicity of SHK and enhancing the overall anti-cancer effects. We demonstrate for the first time that Mel potentiates the cytotoxic effects of SHK on cancer cells by inducing oxidative stress via inhibition of the SIRT3/SOD2-AKT pathway. Particularly, Mel-SHK treatment induced oxidative stress, increased mitochondrial calcium accumulation and reduced the mitochondrial membrane potential in various cancer cells, leading to apoptosis. This drug combination also promoted endoplasmic reticulum (ER) stress, leading to AKT dephosphorylation. In HeLa cells, Mel-SHK treatment reduced SIRT3/SOD2 expression and SOD2 activity, while SIRT3 overexpression dramatically reduced Mel-SHK-induced oxidative stress, ER stress, mitochondrial dysfunction and apoptosis. Hence, we propose the combination of Mel and SHK as a novel candidate chemotherapeutic regimen that targets the SIRT3/SOD2-AKT pathway in cancer

Effect of Continuous Feeding of Ayu-Narezushi on Lipid Metabolism in a Mouse Model of Metabolic Syndrome

Ayu-narezushi, a traditional Japanese fermented food, comprises abundant levels of lactic acid bacteria (LAB) and free amino acids. This study aimed to examine the potential beneficial effects of ayu-narezushi and investigated whether ayu-narezushi led to improvements in the Tsumura Suzuki obese diabetes (TSOD) mice model of spontaneous metabolic syndrome because useful LAB are known as probiotics that regulate intestinal function. In the present study, the increased body weight of the TSOD mice was attenuated in those fed the ayu-narezushi-comprised chow (ayu-narezushi group) compared with those fed the normal rodent chow (control group). Serum triglyceride and cholesterol levels were significantly lower in the Ayu-narezushi group than in the control group at 24 weeks of age. Furthermore, hepatic mRNA levels of carnitine-palmitoyl transferase 1 and acyl-CoA oxidase, which related to fatty acid oxidation, were significantly increased in the ayu-narezushi group than in the control group at 24 weeks of age. In conclusion, these results suggested that continuous feeding with ayu-narezushi improved obesity and dyslipidemia in the TSOD mice and that the activation of fatty acid oxidation in the liver might contribute to these improvements

An LSI implementation of an adaptive genetic algorithm with on-the-fly crossover operator selection

ABSTRACT This paper describes an LSI implementation of a genetic algorithm (GA), called the Genetic Algorithm Accelerator (GAA) chip. The GAA chip is an LSI implementation of a GA, in which two types of crossover operators are supported, and the operator to be actually used in the algorithm is not fixed in advance, but dynamically selected for each pair of chromosomes in the algorithm execution. The GAA chip has been designed with the Verilog HDL and simulated with some benchmark functions. According to the simulation, the GAA chip will run with 50MHz clock in maximum. The chip has been fabricated with the CMOS 0.5 m standard cell technology

Global genomic analyses of wheat powdery mildew reveal association of pathogen spread with historical human migration and trade

The fungus Blumeria graminis f. sp. tritici causes wheat powdery mildew disease. Here, we study its spread and evolution by analyzing a global sample of 172 mildew genomes. Our analyses show that B.g. tritici emerged in the Fertile Crescent during wheat domestication. After it spread throughout Eurasia, colonization brought it to America, where it hybridized with unknown grass mildew species. Recent trade brought USA strains to Japan, and European strains to China. In both places, they hybridized with local ancestral strains. Thus, although mildew spreads by wind regionally, our results indicate that humans drove its global spread throughout history and that mildew rapidly evolved through hybridization.Centro de Investigaciones AgropecuariasFil: Sotiropoulos, Alexandros G. University of Zurich. Department of Plant and Microbial Biology; SuizaFil: Arango-Isaza, Epifanía. University of Zurich. Department of Evolutionary Biology and Environmental Studies; SuizaFil: Ban, Tomohiro. Yokohama City University. Kihara Institute for Biological Research; JapónFil: Barbieri, Chiara. University of Zurich. Department of Evolutionary Biology and Environmental Studies; SuizaFil: Barbieri, Chiara. Max Planck Institute for Evolutionary Anthropology. Department of Linguistic and Cultural Evolution; AlemaniaFil: Bourras, Salim. University of Zurich. Department of Plant and Microbial Biology; SuizaFil: Bourras, Salim. University of Agricultural Sciences. Department of Forest Mycology and Plant Pathology; SueciaFil: Cowger, Christina. North Carolina State University; Estados Unidos. USDA-ARS Department of Plant Pathology; Estados UnidosFil: Czembor, Paweł C. National Research Institute. Plant Breeding and Acclimatization Institute; PoloniaFil: Ben-David, Roi. ARO-Volcani Center. Institute of Plant Sciences. Department of Vegetables and Field Crops; IsraelFil: Dinoor, Amos. University of Jerusalem. The Robert H. Smith Faculty of Agriculture, Food & Environment. Department of Plant Pathology and Microbiology; IsraelFil: Ellwood, Simon R. Curtin University. School of Molecular and Life Sciences. Centre for Crop and Disease Management; AustraliaFil: Graf, Johannes. University of Zurich. Department of Plant and Microbial Biology; SuizaFil: Hatta, Koichi. National Agricultural Research Organization. Hokkaido Agricultural Research Center Field Crop Research and Development; JapónFil: Helguera, Marcelo. Instituto Nacional de Tecnología Agropecuaria (INTA). Centro de Investigaciones Agropecuarias; ArgentinaFil: Wicker, Thomas. University of Zurich. Department of Plant and Microbial Biology; Suiz

The Effect of Olive Leaf Extract on Hepatic Fat Accumulation in Sprague-Dawley Rats Fed a High-fat Diet

Oleuropein, the active constituent of olive leaf extract, possesses anti-oxidant, hypoglycemic, and hypolipidemic activities. We aimed to assess whether the effect of olive leaf extract on hepatic fat accumulation is preventive or therapeutic. Sprague-Dawley (SD) rats were fed a high-fat diet with (ODOD group) or without (HDHD group) olive leaf extract (1,000 mg/kg diet) for 38 weeks. Another group of rats were fed a high-fat diet for 23 weeks, followed by a high-fat diet with olive leaf extract (1,000 mg/kg diet) for 15 weeks (HDOD group). Serology, histopathology, anti-oxidative activity, and liver fatty acid synthesis were compared to those fed a standard diet (LDLD group) at 26 and 41 weeks of age. The serum levels of total cholesterol, triglyceride and aspartate aminotransferase tended to be lower in the ODOD group as compared to the HDHD and HDOD groups, although there were no significant differences. Histopathologically, hepatic steatosis tended to be less evident in the HDOD and ODOD groups as compared to the HDHD group, and lobular inflammation was not observed in the ODOD group at 26 weeks of age. Hepatic thioredoxin-1 staining tended to be less evident in the ODOD group than in the HDHD and HDOD groups at 41 weeks of age. There were no significant differences in hepatic lipogenic enzyme activities between the ODOD group and HDHD/HDOD groups. Our data suggest that olive leaf extract had a preventive, rather than therapeutic, effect on hepatic steatohepatitis in SD rats fed a high-fat diet

Global genomic analyses of wheat powdery mildew reveal association of pathogen spread with historical human migration and trade

The fungus Blumeria graminis f. sp. tritici causes wheat powdery mildew disease. Here, Sotiropoulos et al. analyze a global sample of 172 mildew genomes, providing evidence that humans drove global spread of the pathogen throughout history and that mildew rapidly evolved through hybridization with local fungal strains.The fungus Blumeria graminis f. sp. tritici causes wheat powdery mildew disease. Here, we study its spread and evolution by analyzing a global sample of 172 mildew genomes. Our analyses show that B.g. tritici emerged in the Fertile Crescent during wheat domestication. After it spread throughout Eurasia, colonization brought it to America, where it hybridized with unknown grass mildew species. Recent trade brought USA strains to Japan, and European strains to China. In both places, they hybridized with local ancestral strains. Thus, although mildew spreads by wind regionally, our results indicate that humans drove its global spread throughout history and that mildew rapidly evolved through hybridization

Molecular Characteristics of Extended-Spectrum Beta-Lactamases and qnr Determinants in Enterobacter Species from Japan

The incidence of extended-spectrum β-lactamases (ESBLs) has been increasing worldwide, but screening criteria for detection of ESBLs are not standardized for AmpC-producing Enterobacteriaceae such as Enterobacter species. In this study, we investigated the prevalence of ESBLs and/or AmpC β-lactamases in Japanese clinical isolates of Enterobacter spp. and the association of plasmid-mediated quinolone resistance (PMQR) determinants with ESBL producers. A total of 364 clinical isolates of Enterobacter spp. collected throughout Japan between November 2009 and January 2010 were studied. ESBL-producing strains were assessed by the CLSI confirmatory test and the boronic acid disk test. PCR and sequencing were performed to detect CTX-M, TEM, and SHV type ESBLs and PMQR determinants. For ESBL-producing Enterobacter spp., pulsed-field gel electrophoresis (PFGE) was performed using XbaI restriction enzyme. Of the 364 isolates, 22 (6.0%) were ESBL producers. Seven isolates of Enterobacter cloacae produced CTX-M-3, followed by two isolates producing SHV-12. Two isolates of Enterobacter aerogenes produced CTX-M-2. Of the 22 ESBL producers, 21 had the AmpC enzyme, and six met the criteria for ESBL production in the boronic acid test. We found a significant association of qnrS with CTX-M-3-producing E. cloacae. The 11 ESBL-producing Enterobacter spp. possessing blaCTX-M, blaSHV, or blaTEM were divided into six unique PFGE types. This is the first report about the prevalence of qnr determinants among ESBL-producing Enterobacter spp. from Japan. Our results suggest that ESBL-producing Enterobacter spp. with qnr determinants are spreading in Japan