Shyang-Chwen Sheu

Composition analysis and immuno-modulatory effect of okra (Abelmoschus esculentus L.) extract.

The aim of this study was to analyse the composition of okra (Abelmoschus esculentus L.) extract and investigate the effect of A. esculentus L. polysaccharides (AE-PS) on the maturation and function of dendritic cells (DCs) derived from rat bone marrow hematopoietic cells (BMHCs) in vitro. BMHC-derived immature DCs (BMHC-imDCs) were extracted from rats and treated with AE-PS. The hydrolysed okra extract contained 0.6% β-1, 3-D-glucan. AE-PS induced the presence of polymorphic nuclei and elongated protrusion in the BHMC-imDCs, indicating DC activation. Treatment with 100 μg/mL of AE-PS increased the MHC class II and CD80/86 expression levels by 41% and 42%, respectively. Treated cells had reduced endocytosis activity. The secretion of IL-12 and IFN-γ increased significantly by 120% and 75%, respectively, when treated with 100 μg/mL of AE-PS. Moreover, IL-10 production was reduced by 66%. In conclusion, AE-PS exhibits stimulatory effects on rat dendritic cells and promotes the secretion of T(H)1 cytokines.

Estrogen degradation and sorption onto colloids in a constructed wetland with different hydraulic retention times

Endocrine disrupting compounds are a global concern, owing to their interference with the endocrine system of wildlife. In particular, natural estrogens at concentrations as low as ng/L level can interrupt the endocrine system of many organisms. A constructed wetland is an effective means of removing the residual levels of estrogen. This study investigates the estrogen degradation and sorption on colloids in a constructed wetland at hydraulic retention times (HRTs) of 27.5, 45.9, and 137.5h. Three natural estrogens (i.e. estrone (E1), 17β-estradiol (E2), and estriol (E3)) are analyzed with liquid chromatography/tandem mass spectrometry. At HRT=27.5h, no degradation occurs; at HRT=45.9h, the degradation rates are 0-46.2%; and at HRT=137.5h, the degradation rates are 40-84.3%. Additionally, estrogen sorption coefficients (logKCOC values) range from 3.37 to 4.89. Average logKCOC values are 4.08±0.33, 4.04±0.34, and 4.11±0.28 for E1, E2, and E3, respectively. At different HRTs, values of logKCOC increase with an increasing HRT. Analytical results indicate that constructed wetlands can remove residual natural estrogens. With an increasing HRT, the estrogen degradation rate increases as well as the estrogen sorption on colloids.

Production and partial characterization of monoclonal antibodies specific to cooked poultry meat

Monoclonal antibodies (MAbs) specific to cooked poultry muscle proteins have been developed for the detection of poultry adulterants in cooked mammalian meat. Saline (0.85% NaCl) extract of heat-treated (100 °C, 15min) chicken muscle proteins was used to immunize mice for MAb development. The specificity of MAbs was tested against chicken antigen and protein extracts from seven other meat species (pork, beef, lamb, deer, horse, turkey and duck) by indirect enzyme-linked immunosorbent immunoassay (ELISA). The immunogenic components in the poultry protein extracts were determined by SDS-PAGE followed by immunoblotting. A total of six hybridoma cell lines that secrete IgG class MAbs have been developed: MAbs 3E12 and 1A5 were able to distinguish between cooked poultry and mammalian meats, MAbs 9C6 and 6F7 reacted strongly with cooked chicken only, and MAbs, 5D2 and 6G8, reacted with both cooked turkey and chicken but not other species. All six MAbs demonstrated a proportional increased ELISA response to respective adulterated poultry samples in pork over a 0-100% range of aduleration.

Immunomodulating Activity of Nymphaea rubra Roxb. Extracts: Activation of Rat Dendritic Cells and Improvement of the TH1 Immune Response

Polysaccharides play a key role in enhancing immune function and facilitating cellular communication. Here, we purified Nymphaea rubra Roxb. polysaccharides (NR-PS) by treating them with pullulanase. They were then cultured with immature dendritic cells (DCs) derived from rat bone marrow hematopoietic cells (BMHCs). After treatment with bioactive NR-PS with a degree of polymerization (DP) value of 359.8, we found that the DCs underwent morphological changes indicative of activation. CD80/86 (87.16% ± 8.49%) and MHC class II (52.01% ± 10.11%) expression levels were significantly up-regulated by this treatment compared to the controls (65.45% ± 0.97% and 34.87% ± 1.96%). In parallel, endocytosis was also reduced (167.94% ± 60.59%) after treatment with 25 μg/mL of NR-PS as measured by the medium fluorescence intensity compared to the control (261.67% ± 47.26%). Furthermore, the DCs after treatment with 25 μg/mL NR-PS showed increased IL-12 (102.09 ± 10.16 to 258.78 ± 25.26 pg/mL) and IFN-γ (11.76 ± 0.11 to 15.51 ± 1.66 pg/mL) secretion together with reduced IL-10 secretion (30.75 ± 3.35 to 15.37 ± 2.35 pg/mL), which indicates a TH1 immune response. In conclusion, NR-PS exhibits stimulatory effects on rat DCs and promotes the secretion of TH1 cytokines. Taken together, our studies are the first to show that NR-PS is an immunomodulator affecting the maturation and functioning of DCs.

Identification of the NLS and NES motifs of VP2 from chicken anemia virus and the interaction of VP2 with mini-chromosome maintenance protein 3

BackgroundVP2 of chicken anemia virus (CAV) is a dual-specificity phosphatase required for virus infection, assembly and replication. The functions of the nuclear localization signal (NLS) and nuclear export signal (NES) of VP2 in the cell, however, are poorly understood. Our study identified the presence of a NLS in VP2 and showed that the protein interacted significantly with mini-chromosome maintenance protein 3 (MCM3) in the cell.ResultsAn arginine-lysine rich NLS could be predicted by software and spanned from amino acids 133 to 138 of VP2. The critical amino acids residues between positions 136 and 138, and either residue 133 or 134 are important for nuclear import in mammalian cells based on systematic mutagenesis. A NES is also predicted in VP2; however the results suggest that no functional NES is present and that this protein is CRM1 independent. It was also shown that VP2 is a chromatin binding protein and, notably, using a co-immunoprecipitation assay, it was found that VP2 association with MCM3 and that this interaction does not require DSP activity.ConclusionsVP2 contains a NLS that span from amino acids 133 to 138. VP2 is a CRM1 independent protein during nuclear export and associates with MCM3 in cells.

Production of Enzyme and Growth of Aspergillus oryzae S. on Soybean Koji

 Abstract—Soybean koji is an important ingredient for traditional fermented food in South-East Asia and East Asia. It provides large amount of enzyme from koji mold, Aspergillus oryzae S., to digest nutrients in substrates. This study aimed at production of certain enzymes in soybean koji and potential to be applied for accelerating fish sauce fermentation. Koji containing 60% soybean was used as substrate to investigate the enzyme production by A. oryzae S. The growth of this mold was enumerated by potato dextrose agar. The mycelium development of A. oryzae S. was observed by scanning electron microscope. During koji production, pH of soybean koji increased from 6.32 to 6.07. It was caused by extracellular proteins production. The highest neutral protease, alkaline protease and amylase activities were 84.38, 41.35 and 200 unit/g of dry weight, respectively. Moreover, growing of enzyme activities on soybean koji correlated with the growth of this mold. Electron micrograph showed that spores of A. oryzae S. were formed after 48 h of cultivation period. Additionally, the highest enzymes activities were also shown in this stage.

Development and characterization of a potential diagnostic monoclonal antibody against capsid protein VP1 of the chicken anemia virus

Chicken anemia virus (CAV) is an important viral pathogen that causes anemia and severe immunodeficiency syndrome in chickens worldwide. In this study, a potential diagnostic monoclonal antibody against the CAV VP1 protein was developed which can precisely recognize the CAV antigen for diagnostic and virus recovery purposes. The VP1 gene of CAV encoding the N-terminus-deleted VP1 protein, VP1Nd129, was cloned into an Escherichia (E.) coli expression vector. After isopropyl-β-D-thiogalactopyronoside induction, VP1Nd129 protein was shown to be successfully expressed in the E. coli. By performing an enzyme-linked immunoabsorbent assay using two coating antigens, purified VP1Nd129 and CAV-infected liver tissue lysate, E3 monoclonal antibody (mAb) was found to have higher reactivity against VP1 protein than the other positive clones according to the result of limiting dilution method from 64 clones. Using immunohistochemistry, the presence of the VP1-specific mAb, E3, was confirmed using CAV-infected liver and thymus tissues as positive-infected samples. Additionally, CAV particle purification was also performed using an immunoaffinity column containing E3 mAb. The monoclonal E3 mAb developed in this study will not only be very useful for detecting CAV infection and performing histopathology studies of infected chickens, but may also be used to purify CAV particles in the future.

Development of loop-mediated isothermal amplification (LAMP) assays for the rapid detection of allergic peanut in processed food

Peanut is a widely and common used in many cuisines around the world. However, peanut is also one of the most important food allergen for causing anaphylactic reaction. To prevent allergic reaction, the best way is to avoid the food allergen or food containing allergic ingredient such as peanut before food consuming. Thus, to efficient and precisely detect the allergic ingredient, peanut or related product, is essential and required for maintain consumers health or their interest. In this study, a loop-mediated isothermal amplification (LAMP) assay was developed for the detection of allergic peanut using specifically designed primer sets. Two sets of the specific LAMP primers respectively targeted the internal transcribed sequence 1 (ITS1) of nuclear ribosomal DNA sequence regions and the ara h1 gene sequence of Arachia hypogeae (peanut) were used to address the application of LAMP for detecting peanut in processed food or diet. The results demonstrated that the identification of peanut using the newly designed primers for ITS 1 sequence is more sensitive rather than primers for sequence of Ara h1 gene when performing LAMP assay. Besides, the sensitivity of LAMP for detecting peanut is also higher than the traditional PCR method. These LAMP primers sets showed high specificity for the identification of the peanut and had no cross-reaction to other species of nut including walnut, hazelnut, almonds, cashew and macadamia nut. Moreover, when minimal 0.1% peanuts were mixed with other nuts ingredients at different ratios, no any cross-reactivity was evident during performing LAMP. Finally, genomic DNAs extracted from boiled and steamed peanut were used as templates; the detection of peanut by LAMP was not affected and reproducible. As to this established LAMP herein, not only can peanut ingredients be detected but commercial foods containing peanut can also be identified. This assay will be useful and potential for the rapid detection of peanut in practical food markets.

Development of qualitative and quantitative PCR analysis for meat adulteration from RNA samples

Total RNA samples were used to establish qualitative and quantitative PCR-based methods for assessing meat adulteration. The primers were designed based on the mRNA sequences of troponin I (TnI), mitochondrial ribosomal protein (MRP) and tropomodulin genes to distinguish chicken, pork, goat, beef and ostrich. There was no cross reaction between the primers, and the detection limit of the cDNA template was 0.01 and 20 ng in simplex PCR and multiplex PCR, respectively. In the low temperature storage test, the detection limits of cDNA template with 10 and 1 ng were determined at 4 °C and -80 °C. In quantitative assay, the precision of real-time PCR analysis expressed as a coefficient of variation (CV) ranged from 0.25% to 5.24% and the trueness, expressed as an error, ranged from 0.28% to 6.98% for adulteration. Thus, herein, we provided alternative tools for the assessment of meat adulteration using mRNA-based PCR methods.