Engineering of Saccharomyces cerevisiae for the production of (+)-ambrein

The triterpenoid (+)-ambrein is the major component of ambergris, a coprolite of the sperm whale that can only be rarely found on shores. Upon oxidative degradation of (+)-ambrein, several fragrance molecules are formed, amongst them (−)-ambrox, one of the highest valued compounds in the perfume industry. In order to generate a Saccharomyces cerevisiae whole-cell biocatalyst for the production of (+)-ambrein, intracellular supply of the squalene was enhanced by overexpression of two central enzymes in the mevalonate and sterol biosynthesis pathway, namely the N-terminally truncated 3-hydroxy-3-methylglutaryl-CoA reductase 1 (tHMG) and the squalene synthase (ERG9). In addition, another key enzyme in sterol biosynthesis, squalene epoxidase (ERG1) was inhibited by an experimentally defined amount of the inhibitor terbinafine in order to reduce flux of squalene towards ergosterol biosynthesis while retaining sufficient activity to maintain cell viability and growth. Heterologous expression of a promiscuous variant of Bacillus megaterium tetraprenyl-β-curcumene cyclase (BmeTC-D373C), which has been shown to be able to catalyse the conversion of squalene to 3-deoxyachillol and then further to (+)-ambrein resulted in production of these triterpenoids in S. cerevisiae for the first time. Triterpenoid yields are comparable with the best microbial production chassis described in literature so far, the methylotrophic yeast Pichia pastoris. Consequently, we discuss similarities and differences of these two yeast species when applied for whole-cell (+)-ambrein production.     

Solution Structure and Dynamics of the Small Protein HVO_2922 from Haloferax volcanii

Past sequencing campaigns overlooked small proteins as they seemed to be irrelevant due to their small size. However, their occurrence is widespread, and there is growing evidence that these small proteins are in fact functionally very important in organisms found in all kingdoms of life. Within a global proteome analysis for small proteins of the archaeal model organism Haloferax volcanii, the HVO_2922 protein has been identified. It is differentially expressed in response to changes in iron and salt concentrations, thus suggesting that its expression is stress-regulated. The protein is conserved among Haloarchaea and contains an uncharacterized domain of unknown function (DUF1508, UPF0339 family protein). We elucidated the NMR solution structure, which shows that the isolated protein forms a symmetrical dimer. The dimerization is found to be concentration-dependent and essential for protein stability and most likely for its functionality, as mutagenesis at the dimer interface leads to a decrease in stability and protein aggregation.     

Trend Analysis and Spatial Prediction of Groundwater Levels Using Time Series Forecasting and a Novel Spatio-Temporal Method

Water Resources Management - Overexploitation of groundwater in the Malayer Plain has resulted in a continuous decline of groundwater levels over recent years with associated risks to water...     

Freshness Assessment of Thawed and Chilled Cod Fillets Packed in Modified Atmosphere Using Near-infrared Spectroscopy

Near-infrared reflectance (NIR) spectra was recorded of 105 samples of cod mince prepared from chill stored thawed cod fillets of varying quality in modified atmosphere packaging (MAP). Traditional chemical, physical, microbiological and sensory quality methods developed for assessing fresh fish products were determined on the same cod fillets. The purpose was to evaluate the potential of NIR spectroscopy for estimating (i) frozen storage temperature, (ii) frozen storage period and (iii) chill storage period of thawed-chilled MAP Barents Sea cod fillets. Furthermore, the potential for measuring of selected quality attributes as drip loss, water holding capacity and content of dimethylamine by NIR was evaluated. The results of the investigation were presented using multivariate modelling methods such as partial least-squares regression (PLSR) and discriminant partial least-squares regression (DPLSR). Systematic differences in the NIR measurements on minced cod fillets were primarily due to the chill storage duration (days at 2°C) on thawed-chilled MAP fillets. PLSR models based on wavelengths selected by a new Jack-knife method resulted in a correlation coefficient of 0.90 between measured and predicted duration of chill storage period (days at 2°C). The root-mean-square error of cross-validation (RMSECV) was 3.4 d at 2°C. NIR measurements provided promising results for evaluation of freshness for thawed-chilled MAP cod fillets completing the traditionally quality methods. However, it is necessary to study the effect of e.g. sample preparation, season, fishing ground and cod size together with more sophisticated pre-treatments of NIR spectra before the NIR method can be integrated as a method for evaluation of thawed-chilled MAP cod fillets.     

P3HT/PCBM Bulk Heterojunction Solar Cells: Impact of Blend Composition and 3D Morphology on Device Performance

The performance of polymer solar cells (PSC) strongly depends on the 3D morphological organization of the donor and acceptor compounds within the bulk heterojunction active layer. The technique of electron tomography is a powerful tool for studying 3D morphology of the layers composed of poly(3‐hexylthiophene) (P3HT) and a fullerene derivative ([6,6]‐phenyl‐C61‐butyric acid methyl ester; PCBM), especially to quantify the amount and distribution of fibrillar P3HT nanocrystals throughout the volume of the active layer. In this study, electron tomography is used to characterize P3HT/PCBM layers with different blend compositions, both before and after thermal annealing. The power conversion efficiency of the corresponding PSCs is strongly dependent on the overall crystallinity of P3HT and the way P3HT crystals are distributed throughout the thickness of the active layer.     

Capillary electrophoresis-mass spectrometry as a powerful tool in clinical diagnosis and biomarker discovery

Proteome analysis is now emerging as key technology for deciphering biological processes and the discovery of biomarkers for diseases from tissues and body fluids. The complexity and wide dynamic range of protein expression poses a formidable challenge to both peptide separation technologies and mass spectrometry (MS). Here we review the efforts that have been undertaken to date, focussing on capillary electrophoresis coupled to mass spectrometry (CE-MS). We discuss CE-MS from an application point of view evaluating its merits and vices in regard to biomarker discovery and clinical applications. As examples, we present the use of CE-MS for the determination of protein patterns in urine, serum, and other body fluids. Finally, the benefits and limitations of CE-MS for the analysis of proteins in clinical samples are discussed against the background of alternative technologies.     

Modulation of Aβ42 Aggregation Kinetics and Pathway by Low-Molecular-Weight Inhibitors

The aggregation of amyloid-β 42 (Aβ42) is directly related to the pathogenesis of Alzheimer's disease. Here, we have investigated the early stages of the aggregation process, during which most of the cytotoxic species are formed. Aβ42 aggregation kinetics, characterized by the quantification of Aβ42 monomer consumption, were tracked by real-time solution NMR spectroscopy (RT-NMR) allowing the impact that low-molecular-weight (LMW) inhibitors and modulators exert on the aggregation process to be analysed. Distinct differences in the Aβ42 kinetic profiles were apparent and were further investigated kinetically and structurally by using thioflavin T (ThT) and transmission electron microscopy (TEM), respectively. LMW inhibitors were shown to have a differential impact on early-state aggregation. Insight provided here could direct future therapeutic design based on kinetic profiling of the process of fibril formation.