Structural elucidation and analysis of thermal degradation products of the Fusarium mycotoxin nivalenol

The major class of mycotoxins produced by Fusarium moulds are trichothecenes, a large group of sesquiterpenes sharing the same basic chemical structure, a 12,13-epoxytrichothec-9-ene ring system. Their toxic effects range from causing diarrhoea, vomiting and gastro-intestinal inflammation to noncompetitive inhibition of the biosynthesis of proteins in eukaryotic cells. Trichothecenes in general are relatively stable compounds, their degradation is observed only at high temperatures and prolonged heating time. In order to investigate the stability of the trichothecene nivalenol (NIV) under food processing conditions such as cooking or baking, we performed model heating experiments and screened the residue for degradation products using gas chromatography-mass spectrometry (GC-MS). Heating of nivalenol, especially under mild alkaline conditions, gave a mixture of four compounds (norNIV A, norNIV B, norNIV C, and NIV lactone), which where isolated and identified by nuclear magnetic resonance (NMR) and MS experiments. Although their formation was also demonstrated in heating experiments with spiked flour samples, only norNIV B was detectable in a screening of several commercially available samples, possibly due to the very low contamination with nivalenol. Furthermore, cell culture experiments using immortalized human kidney epithelial (IHKE) cells showed that the four compounds are less cytotoxic (formazan dye cytotoxicity assay) compared to nivalenol. Whereas nivalenol revealed an EC50 at 0.9 micromol, all other compounds did not show any significant effect up to 100 micromol.     

Enhanced Osteoblast Adhesion to Epoxide‐Functionalized Surfaces

As an alternative to expensive extracellular matrix (ECM) proteins generally applied as coatings in Petri dishes used for cell binding, an innovative system based on epoxide‐functionalized monolayers capable of protein binding is proposed. Since cells bind to material surfaces through proteins, protein‐binding surfaces should also promote cell binding. Here we investigate how the cell‐binding properties of an epoxide‐functionalized surface compares with ECM protein gel coated surfaces and tissue culture polystyrene control surfaces. Glass surfaces are functionalized with glycidoxypropyltriethoxysilane (GOPS), which results in an epoxide‐functionalized surface capable of binding proteins through an epoxide–amine reaction. Advancing contact angle measurements and atomic force microscopy measurements confirm the formation of a homogeneous GOPS monolayer. This monolayer is micropatterned with fluorescein‐labeled ECM protein gel by microcontact printing (µCP). Confocal laser scanning microscopy (CLSM) shows accurately transferred ECM protein gel micropatterns. Osteoblasts that are seeded on these micropatterned substrates show a clear preference for adhering to the epoxide‐functionalized areas. The morphology of these cultured osteoblasts is needle‐like with high aspect ratios. As controls, osteoblasts are cultured on GOPS‐functionalized surfaces, unstructured ECM protein gel surfaces, and tissue culture polystyrene (TCPS). The GOPS surfaces demonstrate a drastic increase in cell adhesion after 2 h, whilst the other tests show no adverse effects of this surface on the osteoblasts as compared to ECM and TCPS. CLSM shows healthy cell morphologies on each surface. It is demonstrated for the first time that epoxide groups outperform ECM protein gel in cell adhesion, thereby providing new routes for cost‐effective coatings that improve biocompatibility as well as exciting, new methodologies to control and direct cell adhesion.     

Effects of the mycotoxin deoxynivalenol on human primary hepatocytes

Toxic effects of the mycotoxin deoxynivalenol (DON) observed in animals range from diarrhea, vomiting, gastro-intestinal inflammation to necrosis of several tissues. In the last years, DON has been tested in hepatocytes of several animal species for its cytotoxicity. However, these tests are limited to the use of animal cells. No studies using human hepatocytes are available. Further investigations with the human hepatocellular liver carcinoma cell line HepG2 might be limited due to the disadvantages of cell lines (e. g. immortalization, tumor derivation, longtime cultivation) and do not necessarily reflect the response of normal human cells. In order to overcome this problem and to be closer to the human situation, we studied the effect of DON in human primary hepatocytes and compared these data to the effects in the HepG2 cell line. Cell viability, apoptotic and necrotic cell death, albumin secretion and metabolic activity were determined. It could be demonstrated that DON has a distinct cytotoxic effect on human primary hepatocytes. Viability, protein content and albumin secretion were reduced in a dose-dependent manner. The apoptotic key enzyme caspase-3 was activated, while LDH release occurred only after long incubation time due to a secondary necrosis. Furthermore, we studied the metabolism of DON using LC-MS/MS. DON was neither metabolized by primary hepatocytes cells nor by the HepG2 cell line.     

Evaluation of real-time PCR results at the limit of detection

Real-time polymerase chain reaction (real-time PCR) facilitates to detect DNA fragments at very low copy numbers. Positive results, e.g. for unauthorized genetically modified organism (GMO) contamination or for allergens, may raise safety concerns and have far-reaching consequences. However, in case of very low concentrations of DNA samples, results for the same product lot or even for identical samples from different laboratories may differ. Therefore, an approach for a standardized interpretation and reporting of results obtained by real-time PCR at the limit of detection (LOD) is proposed. A quality control DNA sample containing the target at the LOD (95?%) is analysed in parallel with the real DNA sample and the respective C _T values are compared. In addition, practical approaches for in house and precision-based estimation of the LOD are presented. The proposed approach may also contribute to the current discussion on implementing a technical solution to handle DNA traces in specimen, e.g. for the detection of unauthorized GMO.     

Optimal Newton–Secant like methods without memory for solving nonlinear equations with its dynamics

We construct two optimal Newton–Secant like iterative methods for solving nonlinear equations. The proposed classes have convergence order four and eight and cost only three and four function evaluations per iteration, respectively. These methods support the Kung and Traub conjecture and possess a high computational efficiency. The new methods are illustrated by numerical experiments and a comparison with some existing optimal methods. We conclude with an investigation of the basins of attraction of the solutions in the complex plane.     

Growth kinetics and stable carbon isotope fractionation during aerobic degradation of cis-1,2-dichloroethene and vinyl chloride

Assessing changes in the isotopic signature of contaminants is a promising new tool to monitor microbial degradation processes. In this study, chloroethene degradation was proven by depletion of chloroethenes, formation of chloride, increase in protein content and stable carbon isotope fractionation. Aerobic degradation of vinyl chloride (VC) was found to proceed metabolically, with degradation rates of 0.48 and 0.29 d(-1); and growth yields of 9.7 and 6.4 g of protein/mol of VC at room and groundwater temperature, respectively. Cis-1,2-dichloroethene (cDCE) was degraded cometabolically under aerobic conditions when VC was provided as growth substrate. Aerobic degradation was associated with significant stable carbon isotope fractionation, with enrichment factors ranging from -5.4+/-0.4 per thousand for metabolic degradation of VC to -9.8+/-1.7 per thousand for cometabolic degradation of cDCE. Thus, it was demonstrated that stable carbon isotope fractionation is suitable for assessing aerobic chloroethene degradation, which can contribute significantly to site remediation.     

Immunostimulatory CpG oligonucleotides form defined three-dimensional structures: results from an NMR study

The DNA eicosamer 5'-TCCATGACGTTCCTGATGCT-3' is known to stimulate the innate immune system of vertebrae. The immunostimulatory activity is based on the activation of Toll-like receptor 9 (TLR9). While it is known that the CG dinucleotide of the eicosamer has to be unmethylated, the structural basis of the recognition of the DNA through the receptor remains unclear. Oligodeoxynucleotides containing the sequence of the eicosamer, or a portion thereof, ranging in length from hexamer to pentaeicosamer were studied by (1)H NMR spectroscopy. Based on two-dimensional NMR spectra, a number of resonances could be unambiguously assigned. For all oligonucleotides, structural transitions were detected upon heating, as monitored by the line width and chemical shift of low-field resonances. This includes the TC dinucleotide of the 5'-terminal portion, which does not have any clear base-pairing partners. The melting transitions, together with the NOESY cross-peaks, demonstrate that structure formation occurs well beyond the core hexamer 5'-GACGTT-3', a fact that may be important for understanding the molecular recognition by the Toll-like receptors of the innate immune system.     

Metabolism of quercetin and rutin by the pig caecal microflora prepared by freeze-preservation

Several studies confirm a protection of cardiovascular diseases and certain forms of cancer by dietary flavonoid intake. The bioavailability of flavonoids is influenced by the metabolism of the microflora in the intestine. Using a new in vitro model system the deglycosylation of the flavonol rutin and the degradation of its aglycone quercetin were investigated by using fresh pig caecal inocula in comparison to inocula prepared before by freeze-preservation between 6 wk and 5 months. The incubation experiments led to the same pattern of phenolic degradation products in comparable amounts in both preparations using HPLC-DAD and GC-flame ionization detection (GC-FID) or GC-MS detection within 24-48 h of incubation. With the preservation of the microbial vitality and the metabolic efficiency by freeze-preparation over several months the experimental design of microbial metabolism studies will be independent in time and locality.