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G protein-coupled receptors (GPCRs) comprise the largest family of transmembrane receptors and represent major drug targets. Upon ligand stimulation, GPCRs activate G proteins and undergo a complex regulation by interaction with GPCR kinases (GRKs) and formation of receptor–arrestin complexes. For many GPCRs, this mechanism triggers receptor desensitisation, internalisation, and possibly a second intracellular signalling wave. Here we created eleven different HEK293 knockout cell clones for GRK2, 3, 5, and 6 individually and in combination. These include four single, two double, four triple, and the quadruple GRK knockout. The statistical evaluation of β-arrestin1/2 interactions for twelve different receptors grouped the tested GPCRs into two main subsets: those for which β-arrestin interaction was mediated by either GRK2, 3, 5, or 6 and those that are mediated by GRK2 or 3 only. Interestingly, the overexpression of specific GRKs was found to induce a robust, ligand-independent β-arrestin interaction with the V2R and AT1R. Finally, using GRK knockout cells, PKC inhibitors, and β-arrestin mutants, we present evidence for differential AT1R–β-arrestin2 complex configurations mediated by selective engagement of PKC, GRK2, or GRK6. We anticipate our novel GRK-knockout platform to facilitate the elucidation of previously unappreciated details of GRK-specific GPCR regulation and β-arrestin complex formation.

Different batches of biomass/feed quality maize contaminated by aflatoxins were processed at the industrial scale (a continuous process and separate discontinuous steps) to evaluate the effect of different cleaning solutions on toxin reduction. The investigated cleaning solutions included: (i) mechanical size separation of coarse, small and broken kernels, (ii) removal of dust/fine particles through an aspiration channel, (iii) separation of kernels based on gravity and (iv) optical sorting of spatial and spectral kernel defects. Depending on the sampled fraction, dynamic or static sampling was performed according to the Commission Regulation No. 401/2006 along the entire cleaning process lines. Aflatoxin analyses of the water–slurry aggregate samples were performed according to the AOAC Official Method No. 2005.008 based on high-performance liquid chromatography and immunoaffinity column cleanup of the extracts. A significant reduction in aflatoxin content in the cleaned products, ranging from 65% to 84% with respect to the uncleaned products, was observed when continuous cleaning lines were used. Additionally, an overall aflatoxin reduction from 55% to 94% was obtained by combining results from separate cleaning steps. High levels of aflatoxins (up to 490 µg/kg) were found in the rejected fractions, with the highest levels in dust and in the rejected fractions from the aspirator and optical sorting. This study shows that a cleaning line combining both mechanical and optical sorting technologies provides an efficient solution for reducing aflatoxin contamination in maize.

Mycotoxins in the feed and food chain are a major threat both for human and animal health. Just a few highly contaminated grains can make a whole lot unsafe for use as human or animal nutrition. For efficient mycotoxin reduction, a full value chain approach is needed, starting from measures to prevent contamination in the field, to control measures to avoid mycotoxin production during storage and over the process line, until final consumption. Grain cleaning is the most effective post-harvest mitigation strategy to reduce high levels of mycotoxins due to the efficient removal of mold-infected grains and grain fractions with high mycotoxin content. Several studies have been performed during the last years to investigate the reduction of deoxynivalenol in wheat and barley, ergot in rye, and total aflatoxins in peanuts and maize. In this study, the reduction of Fusarium mycotoxins content in maize, i.e. deoxynivalenol (DON), zearalenone (ZEA) and fumonisins (FBs), was tested. Three online cleaning processes were used: (i) mechanical size separation and dust removal by aspiration, (ii) separation based on density differences, and finally (iii) optical sorting. Samples were taken dynamically according to the Commission Regulation No. 401/2006 along the entire process line, including cleaned and rejected project streams. Mycotoxin analyses were performed of water-slurry aggregate samples by validated HPLC methods based on immunoaffinity column clean-up of extracts. The overall reduction rate were up to 55% for DON, up to 100% for ZEA, and up to 65% for FBs. High levels of mycotoxins were found in all rejected fractions.