Analysis of Human TAAR8 and Murine Taar8b Mediated Signaling Pathways and Expression Profile

The thyroid hormone derivative 3-iodothyronamine (3-T₁AM) exerts metabolic effects in vivo that contradict known effects of thyroid hormones. 3-T₁AM acts as a trace amine-associated receptor 1 (TAAR1) agonist and activates G_s signaling in vitro. Interestingly, 3-T₁AM-meditated in vivo effects persist in Taar1 knockout-mice indicating that further targets of 3-T₁AM might exist. Here, we investigated another member of the TAAR family, the only scarcely studied mouse and human trace-amine-associated receptor 8 (Taar8b, TAAR8). By RT-qPCR and locked-nucleic-acid (LNA) in situ hybridization, Taar8b expression in different mouse tissues was analyzed. Functionally, we characterized TAAR8 and Taar8b with regard to cell surface expression and signaling via different G-protein-mediated pathways. Cell surface expression was verified by ELISA, and cAMP accumulation was quantified by AlphaScreen for detection of G_s and/or G_i/o signaling. Activation of G-proteins G_q/11 and G_12/13 was analyzed by reporter gene assays. Expression analyses revealed at most marginal Taar8b expression and no gender differences for almost all analyzed tissues. In heart, LNA-in situ hybridization demonstrated the absence of Taar8b expression. We could not identify 3-T₁AM as a ligand for TAAR8 and Taar8b, but both receptors were characterized by a basal G_i/o signaling activity, a so far unknown signaling pathway for TAARs.     

A pH‐Based High‐Throughput Assay for Transketolase: Fingerprinting of Substrate Tolerance and Quantitative Kinetics

A pH‐based high‐throughput assay method has been developed for the rapid and reliable measurement of transketolase (TK) activity. The method is based on the decarboxylation of lithium hydroxypyruvate (HPA) as a hydroxyacetyl donor with an aldehyde acceptor, using phenol red as the pH indicator. Upon release of carbon dioxide from HPA, the pH increase in the reaction mixture can be determined photometrically by the color change of the pH indicator. At low buffer concentration (2 mM triethanolamine, pH 7.5), the method is highly sensitive and allows continuous monitoring, for quantitative determination of the kinetic parameters. By using this method, the substrate specificities of the TK enzymes from Escherichia coli and Saccharomyces cerevisiae, as well as two active‐site‐modified variants of the E. coli TK (D469E, H26Y) were evaluated against a panel of substrate analogues; specific activities and kinetic constants could be rapidly determined. Substrate quality indicated by assay determination was substantiated with novel TK applications by using achiral 3‐hydroxypropanal and 4‐hydroxybutanal for preparative synthesis of chiral deoxyketose‐type products. Determination of ee for the latter could be performed by chiral GC analysis, with an unambiguous correlation of the absolute configuration from rotation data. This pH‐based assay method is broadly applicable and allows rapid, sensitive, and reliable screening of the substrate tolerance of known TK enzymes and variants obtained from directed evolution.     

Occurrence and composition of extracellular lipids and polysaccharides in a full-scale membrane bioreactor

The aim of this study was to characterize the polysaccharides and lipid fractions of membrane foulants in a full-scale membrane bioreactor (MBR) treating municipal wastewater. Both of these polymeric compounds are major components of bacterial lipopolysaccharides and are impacting membrane fouling; however most of the data so far have been collected by determining sum parameters rather than the detailed composition of these polymers.Photometric analysis of sugars showed that uronic acids (glucuronic, mannuronic and galacturonic acid) as common units of bacterial polysaccharides accounted for 8% (w/w) of extracellular polymeric substances (EPS) in activated sludge flocs. Further the so-called polysaccharide peak of EPS, with a molecular weight >10 kDa according to size exclusion chromatography, was proven to contain bacterial sugar units as shown by high resolution LC-MS. Interestingly, only traces of uronic acids could be detected in EPS of the membrane fouling layer.A far more dramatic enrichment in the fouling layer was revealed for the lipid fraction of EPS, which was determined as fatty acid methyl esters by GC-MS. The weight percentage of fatty acids in EPS extracted from fouled ultrafiltration membranes was much higher (10%) than in the activated sludge itself (1-3%). The fatty acids accumulated on the membrane fouling layer were obviously not only of microbial origin (C16:0, C18:0) but also derived from the raw wastewater itself (C9:0). Hydrophobic interaction of lipids with the PVDF (polyvinylidene fluoride) membrane material therefore seems a plausible explanation for the observed fouling phenomenon. The results suggest that fatty acids from bacterial lipopolysaccharides as well as from synthetic sources are of much higher relevance to membrane fouling than previously assumed.     

Occurrence and removal of N-nitrosamines in wastewater treatment plants

The presence of nitrosamines in wastewater might pose a risk to water resources even in countries where chlorination or chloramination are hardly used for water disinfection. We studied the variation of concentrations and removal efficiencies of eight N-nitrosamines among 21 full-scale sewage treatment plants (STPs) in Switzerland and temporal variations at one of these plants. N-nitrosodimethylamine (NDMA) was the predominant compound in STP primary effluents with median concentrations in the range of 5-20 ng/L, but peak concentrations up to 1 μg/L. N-nitrosomorpholine (NMOR) was abundant in all plants at concentrations of 5-30 ng/L, other nitrosamines occurred at a lower number of plants at similar levels. From concentrations in urine samples and domestic wastewater we estimated that human excretion accounted for levels of <5 ng/L of NDMA and <1 ng/L of the other nitrosamines in municipal wastewater, additional domestic sources for <5 ng/L of NMOR. Levels above this domestic background are probably caused by industrial or commercial discharges, which results in highly variable concentrations in sewage. Aqueous removal efficiencies in activated sludge treatment were in general above 40% for NMOR and above 60% for the other nitrosamines, but could be lower if concentrations were below 8-15 ng/L in primary effluent. We hypothesize that substrate competition in the cometabolic degradation explains the occurrence of such threshold concentrations. An additional sand filtration step resulted in a further removal of nitrosamines from secondary effluents even at low concentrations. Concentrations released to surface waters were largely below 10 ng/L, suggesting a low impact on Swiss water resources and drinking water generation considering the generally high environmental dilution and possible degradation. However, local impacts in case a larger fraction of wastewater is present cannot be ruled out.     

杭州RAFFLES城

杭州Raffles城发展坐落于浙江省省会杭州市的钱塘江边，是嘉德置地继新加坡、上海、北京、成都和巴林之后的第六栋，将零售店、办公楼、住户和酒店设施融于一体，成为新城区的文化地标。