Mass production of rubusoside using a novel stevioside-specific β-glucosidase from Aspergillus aculeatus

Rubusoside (R) is a natural sweetener and a solubilizing agent with antiangiogenic and antiallergic properties. However, currently, its production is quite expensive, and therefore, we have investigated nine commercially available glycosidases to optimize an economically viable R-production method. A stevioside (ST)-specific β-glucosidase (SSGase) was selected and purified 7-fold from Aspergillus aculeatus Viscozyme L by a two-step column chromatography procedure. The 79 kDa protein was stable from pH 3.0 to pH 7.0 at 50-60 °C. Hydrolysis of ST by SSGase produced R and steviol monoglucosyl ester as determined by (1)H and (13)C nuclear magnetic resonance (NMR). Importantly, SSGase showed higher activity toward ST than other β-linked glucobioses. The optimal conditions for R production were 280 mM ST and 16.6 μL of SSGase at pH 5.1 and 63 °C. This is the first discussion detailing the production of R by enzymatic hydrolysis of ST and is useful for the food additive and pharmaceutical industries.     

Processing of microcellular nanocomposite foams by using a supercritical fluid

Polystyrene/layered silicate nanocomposites were prepared by melt intercalation. To examine the distribution of the clay in polymer matrix, small angle X-ray scattering (SAXS) and transmission electron microscopy (TEM) were used. Intercalated nanocomposites were obtained and their rheological properties were investigated. Microcellular nanocomposite foams were produced by using a supercritical fluid. As clay contents increased, the cell size decreased and the cell density increased. It was found that layered silicates could operate as heterogeneous nucleation sites. As the saturation pressure increased and the saturation temperature decreased, the cell size decreased and the cell density increased. Microcellular foams have different morphology depending upon the dispersion state of nanoclays.     

Molecular characterization of low pathogenic avian influenza viruses, isolated from food products imported into Singapore

We have completed the genetic characterization of all eight gene segments for four low pathogenic avian influenza (LPAI) viruses. The objective of this study was to detect the presence of novel signatures that may serve as early warning indicators of the conversion of LPAI viruses to high pathogenic avian influenza (HPAI) viruses. This study included three H5N2 and one H5N3 viruses that were isolated from live poultry imported into Singapore as part of the national avian influenza virus (AIV) surveillance program. Based on the molecular criterion of the World Organisation for Animal Health (OIE), sequence analysis with the translated amino acid (aa) sequence of the hemagglutinin (HA) gene revealed the absence of multibasic aa at the HA cleavage site, identifying all four virus isolates as LPAI. Detailed phylogenetic tree analyses using the HA and neuraminidase (NA) genes clustered these isolates in the Eurasian H5 lineage, but away from the HPAI H5 subtypes. This analysis further revealed that the internal genes clustered to different avian and swine subtypes, suggesting that the four isolates may possibly share their ancestry with these different influenza subtypes. Our results suggest that the four LPAI isolates in this study contained mainly avian signatures, and the phylogenetic tree for the internal genes further suggests the potential for reassortment with other different circulating avian subtypes. This is the first comprehensive report on the genetic characterization of LPAI H5N2/3 viruses isolated in South-East Asia.     

An outbreak of highly pathogenic avian influenza subtype H5N1 in broiler breeders, Korea

Highly pathogenic avian influenza (HPAI) was diagnosed in broiler breeders, submitted to the National Veterinary Research and Quarantine Service in South Korea. Grossly, the dead breeders had lesions consistent with HPAI, including pancreatic mottling, splenomegaly, pulmonary edema and congestion, and hemorrhages in the mucosa of the proventriculus, gizzard and small intestine, and on the serosal surface. Microscopically, there were necrotized hepatitis and pancreatitis, lymphocytic meningoencephalitis, myocarditis, and interstitial pneumonia. Influenza viral antigen was demonstrated in areas closely associated with histopathologic lesions. The AI virus was isolated from cecal tonsils, feces, trachea, and kidney of the chickens. The isolated virus was identified as the highly pathogenic H5N1, with a hemagglutinin proteolytic cleavage site deduced amino acid sequences of QREKRKKR/GLFGAGLFGAIAG. In order to determine the pathogenicity of the isolate, eight 6-week-old specific pathogen free chickens were inoculated intravenously with the virus, and all the birds died within 24 hr after inoculation. This is the first report of an outbreak of HPAI in the chickens in South Korea.     

Inflammatory cytokines IL-6 and TNF-α regulate lymphocyte trafficking through the local lymph node

Lymphocyte trafficking from blood to lymph and back is a tightly regulated process. Given appropriate stimuli, trafficking of cells through the lymph node changes from a 'steady-state' to a bimodal flow. Initially, a 'shutdown' phase occurs, leading to a dramatic reduction in efferent cell output. This is followed by a 'recruitment' phase whereby the efferent cell output becomes greatly elevated before returning to baseline levels. The shutdown/recruitment process is hypothesised to promote encounters between Ag-specific lymphocytes and APCs in an environment conducive to immune response induction. Cytokines, such as TNF-α have been shown to play an important role in regulating lymphocyte trafficking. Here, we unravel the role of cytokines in the regulation of cell trafficking using an in vivo sheep lymphatic cannulation model whereby the prefemoral lymph nodes were cannulated and recombinant cytokines were injected subcutaneously into the draining area of the cannulated node. We demonstrate that local injection of purified IL-6 or TNF-α stimulates shutdown/recruitment in the draining lymph node. While the effect of IL-6 appears to be direct, TNF-α may mediate shutdown/recruitment through IL-6.     

Temperature and food availability effects on spinosad and malathion against <Emphasis Type="Italic">Rhagoletis indifferens</Emphasis> (Diptera: Tephritidae) in the laboratory

Western cherry fruit fly, Rhagoletis indifferens Curran (Diptera: Tephritidae), is a quarantine pest of cherries (Prunus spp.) in western North America that is managed primarily using insecticides. Different insecticides could vary in efficacy and ability to control flies depending on environmental factors. Here, the objective was to determine if temperature and food availability affect the efficacies of spinosad and malathion against R. indifferens in the laboratory. Fourteen- to 18-day old flies were exposed to sweet cherries with dried residues of spinosad and malathion at 19 or 21 versus 27 °C with or without yeast extract + sucrose food (‘food’). Deaths and oviposition were recorded over four days. In spinosad treatments, fly kill was greater at 27 °C than at lower temperatures when there was no food, but in the malathion treatments, kill did not differ between temperatures and it was greatest when there was no food. In spinosad treatments, lower oviposition occurred at 19 or 21 °C than 27 °C, with differences larger when there was food. However, in malathion treatments, oviposition was not affected by temperature although it was lower when there was no food. Results imply temperature and food availability could be factors affecting R. indifferens control in cherries, but whether temperature is such a factor depends on the insecticide used.     

Antibiogram and heavy metal resistance of pathogenic bacteria isolated from moribund cage cultured silver catfish (<Emphasis Type="Italic">Pangasius sutchi</Emphasis>) and red hybrid tilapia (<Emphasis Type="Italic">Tilapia</Emphasis> sp.)

Antibiogram and heavy metal resistance patterns of pathogenic bacteria isolated from moribund cage cultured silver catfish (Pangasius sutchi) and red hybrid tilapia (Tilapia sp.) from Sungai Manir, Terengganu, Malaysia were studied and characterized. Sungai Manir is one of the famous rivers in Terengganu for its wide variety of cage cultured freshwater fish. However, to date, the baseline information of antibiogram and heavy metal resistance patterns of the pathogenic bacteria attacking the freshwater fish cultured in Sungai Manir is still lacking. Therefore, this study was carried out, which may be useful for fish farmers as a guideline for fish prophylactic and treatment purposes. Furthermore, present studies also provide information on the safety level of consuming freshwater fish produced from Sungai Manir. In the present study, bacteria were isolated from 100 fish of each moribund silver catfish and red hybrid tilapia using seven media including tryptic soy agar (TSA), Mac Conkey, thiosulphate citrate bile salt (TCBS), eosin methylene blue (EMB), glutamate starch pseudomonas (GSP), xylose lysine deoxycholate (XLD) and Baird Parker media. Identification of bacteria was carried out using conventional biochemical tests and confirmed by commercial bacterial identification kit. Antibiogram of the bacterial isolates against 18 antibiotics; oxolinic acid (2 μg), ampicillin (10 μg), erythromycin (15 μg), furazolidone (15 μg), lincomycin (15 μg), oleandomycin (15 μg), amoxicillin (25 μg), colistin sulphate (25 μg), sulphamethoxazole (25 μg), chloramphenicol (30 μg), doxycycline (30 μg), florfenicol (30 μg), flumequine (30 μg), kanamycin (30 μg), nalidixic acid (30 μg), tetracycline (30 μg), nitrofurantoin (50 μg) and spiramycin (100 μg) was carried out using disk diffusion method, whereas heavy metal resistance patterns (Hg²⁺, Cd²⁺, Cr^{6 +} and Cu²⁺) of the bacterial isolates was determined through twofold agar dilution method. The results showed that the percentage of sensitivity case of the 120 bacterial isolates to the tested antibiotics was 62.7%. This was followed by resistance (26.9%) and intermediary sensitive (10.4%) cases. In terms of the heavy metal resistance patterns, all bacterial isolates were resistant to Hg²⁺ and Cr^{6 +}. However, only 27.8% and 16.7% of the bacterial isolates were sensitive to Cu²⁺ and Cd²⁺, respectively. The multiple antibiotic resistance (MAR) indices indicated that the cage cultured silver catfish and red hybrid tilapia were under high exposure to the tested antibiotic. Overall, the results of the present studies showed that Sungai Manir may be polluted with heavy metal and antibiotic residues.     

TRB3 links the E3 ubiquitin ligase COP1 to lipid metabolism

During fasting, increased concentrations of circulating catecholamines promote the mobilization of lipid stores from adipose tissue in part by phosphorylating and inactivating acetyl-coenzyme A carboxylase (ACC), the rate-limiting enzyme in fatty acid synthesis. Here, we describe a parallel pathway, in which the pseudokinase Tribbles 3 (TRB3), whose abundance is increased during fasting, stimulates lipolysis by triggering the degradation of ACC in adipose tissue. TRB3 promoted ACC ubiquitination through an association with the E3 ubiquitin ligase constitutive photomorphogenic protein 1 (COP1). Indeed, adipocytes deficient in TRB3 accumulated larger amounts of ACC protein than did wild-type cells. Because transgenic mice expressing TRB3 in adipose tissue are protected from diet-induced obesity due to enhanced fatty acid oxidation, these results demonstrate how phosphorylation and ubiquitination pathways converge on a key regulator of lipid metabolism to maintain energy homeostasis.