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Liu J.,Vicam | Yang Y.,Tongji Medical College | Li X.,Second Affiliated Hospital of Chongqin Medical University | Zhang P.,Syracuse University | And 2 more authors.
Methods in Enzymology | Year: 2010

Protein O-mannose N-acetylglucosaminyltransferase 1 (POMGnT1) is an enzyme that catalyzes the transfer of N-acetylglucosamine to O-mannose of glycoproteins. It is involved in posttranslational modification of α-dystroglycan (α-DG). POMGnT1-null mice were generated by gene trapping with a retroviral vector inserted into exon 2 of the POMGnT1 gene. Expression of POMGnT1 was completely disrupted as evidenced by absence of its mRNA expression. POMGnT1 knockout mice were viable but with reduced fertility and variable lifespan. The functional glycosylated form of α-DG was markedly reduced in POMGnT1 knockout mice along with impaired α-DG-laminin binding activity. Multiple developmental defects in muscle, brain, and eye were observed. In addition, the knockout mice exhibited extensive abnormalities in the neocortex, including changed neuron distribution, presence of ectopic fibroblasts, and GFAP-positive reactive astrocytes. Analysis of POMGnT1 knockout neocortex at several developmental stages revealed that these defects were secondary to disruptions of the pial basement membrane. © 2010 Elsevier Inc.

Lattanzio V.M.T.,CNR Institute of Sciences of Food Production | Ciasca B.,CNR Institute of Sciences of Food Production | Powers S.,Vicam | von Holst C.,European Commission
TrAC - Trends in Analytical Chemistry | Year: 2016

Recent advances in establishing harmonized guidelines for validation of screening methods are reviewed. Emphasis is given to guidelines set in the Regulation 519/2014/EU that specifies validation criteria for mycotoxin screening methods to be used for official control purposes. The application of this validation scheme is demonstrated through a case study. A commercial lateral flow immunoassay for deoxynivalenol has been and evaluated for its applicability to verify wheat compliance with EC maximum permitted level. The validation design provided information on the precision profile of the method, cut-off, false suspect and false negative rates of samples containing deoxynivalenol above the legal limit.The influence of the co-occurrence of major deoxynivalenol modified forms, i.e. 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, and deoxynivalenol-3-glucoside on test results, was evaluated by a factorial design. Finally, the applicability of the validated immunoassay was demonstrated by analysis of naturally contaminated wheat samples, and comparison with results obtained by a LC-MS/MS confirmatory method. © 2015 Elsevier B.V.

Lattanzio V.M.T.,CNR Institute of Sciences of Food Production | Ciasca B.,CNR Institute of Sciences of Food Production | Powers S.,Vicam | Visconti A.,CNR Institute of Sciences of Food Production
Journal of Chromatography A | Year: 2014

An improved method for the quantitative determination of aflatoxins (B1, B2, G1, G2), ochratoxin A, fumonisins (B1, B2), zearalenone, deoxynivalenol, nivalenol, T-2 and HT-2 toxins in cereals and derived products, at levels comparable with EU maximum permitted levels, was developed. The effective co-extraction of the mycotoxins under investigation was achieved in 4min by a double extraction approach, using water followed by methanol. Clean up of the extract was performed by a new multi-toxin immunoaffinity column. Analytical performance characteristics were evaluated through single laboratory validation. Raw wheat and maize, corn flakes and maize snacks were chosen as representative matrices for method validation. The validation assay was carried out at 50, 100 and 150% of EU maximum permitted levels for each mycotoxin. Statistical analysis of the results (ANOVA) provided the within laboratory reproducibility and the error contributions from repeatability, between day effects, and influences from different matrix composition. Recoveries generally higher than 70% were obtained for all tested mycotoxins with relative standard deviation (within laboratory reproducibility) lesser than 37%. Limits of quantification (calculated as the lowest amount of each analyte which could be determined with a precision of 10%) ranged from 1μg/kg to 30μg/kg. The trueness of generated data was assessed by analysis of reference materials. The proposed method was proven to be suitable to assess, with a single analysis, compliance of the selected cereal based foods with the EU maximum permitted or recommended levels for all regulated mycotoxins. © 2014 Elsevier B.V.

Sato T.,Shinshu University | Higashihara K.,Shinshu University | Sasaki A.,Shinshu University | Toth D.,Vicam | Goto T.,Shinshu University
World Mycotoxin Journal | Year: 2010

Citrinin (CIT) is a nephrotoxic secondary metabolite produced by several fungal species. CIT contamination has been reported in many cereals. Recently CIT contamination of red mold rice, used as an artificial colour, received attention because a CIT producing Monascus species was used in its production. Therefore, we developed a relatively simple method, immunoaffinity column clean-up followed by high performance liquid chromatographic (IAC-HPLC) detection, to determine CIT in red mold rice and several other commodities, and performed a single laboratory validation of the method. CIT was extracted by shaking for 30 min with 70% methanol, after which the extract was purified using an immunoaffinity column. The isolated CIT was quantitated by HPLC with fluorescence detection. Recoveries of CIT from red mold rice and koji-rice at levels of 50-2,500 μg/kg were 81-88%. RSDs, estimated with naturally contaminated red mold rice at 50, 200 and 400 μg/kg were 5.1%, 2.8% and 4.1%, respectively. Recoveries of CIT from cereals and mushroom samples at 50, 200 and 400 μg/kg were 60-123%, 69-88% and 73-100% with RSDs of 2.1-7.8%, 1.7-9.2% and 0.6-5.3%, respectively. © 2010 Wageningen Academic Publishers.

Pascale M.,CNR Institute of Sciences of Food Production | Panzarini G.,CNR Institute of Sciences of Food Production | Powers S.,Vicam | Visconti A.,CNR Institute of Sciences of Food Production
Food Analytical Methods | Year: 2014

An ultra-performance liquid chromatography (UPLC®) method has been developed for the simultaneous determination of deoxynivalenol (DON) and nivalenol (NIV) in wheat. Ground sample was extracted with water and the filtered extract was cleaned up through an immunoaffinity column containing a monoclonal antibody specific for DON and NIV. Toxins were separated and quantified by UPLC® with photodiode-array detector (λ = 220 nm) in less than 3 min. Mean recoveries from blank wheat samples spiked with DON and NIV at levels of 100-2,000 μg/kg (each toxin) ranged from 85 to 95 % for DON and from 81 to 88 % for NIV, with relative standard deviations less than 7 %. Similar recoveries were observed from spiked samples when methanol/water (80:20, v/v) was used as extraction solvent. However, by using a wheat sample naturally contaminated with DON and NIV, the one-way analysis of variance (Student-Newman-Keuls test) between different extraction solvents and modes showed that water extraction provided a significant increase (P < 0.001) in toxin concentrations (mean values of six replicate analyses) with respect to methanol/water (80:20, v/v). No significant difference was observed between shaking (60 min) and blending (3 min). The limit of detection (LOD) of the method was 30 μg/kg for DON and 20 μg/kg for NIV (signal-to-noise ratio 3:1). The immunoaffinity columns showed saturation of DON/NIV binding sites at levels higher than 2,000 ng in blank wheat extracts spiked with the corresponding amount of mycotoxin, as single mycotoxin or sum of DON and NIV. The range of applicability of the method was from LOD to 4,000 μg/kg, as single mycotoxin or sum of DON and NIV in wheat. The analyses of 20 naturally contaminated wheat samples showed DON contamination in all analyzed samples at level ranging from 30 to 2,700 μg/kg. NIV was detected in two samples at negligible toxin levels (up to 46 μg/kg). This is the first UPLC® method using immunoaffinity column cleanup for the simultaneous and sensitive determination of DON and NIV in wheat. © 2013 Springer Science+Business Media New York.

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