Novel Old Yellow Enzyme Subclasses

Many drug candidate molecules contain at least one chiral centre, and consequently, the development of biocatalytic strategies to complement existing metal- and organocatalytic approaches is of high interest. However, time is a critical factor in chemical process development, and thus, the introduction of biocatalytic steps, even if more suitable, is often prevented by the limited availability of off-the-shelf enzyme libraries. To expand the biocatalytic toolbox with additional ene reductases, we screened 19 bacterial strains for double bond reduction activity by using the model substrates cyclohexanone and carvone. Overall, we identified 47 genes coding for putative ene reductases. Remarkably, bioinformatic analysis of all genes and the biochemical characterization of four representative novel ene reductases led us to propose the existence of two new Old Yellow Enzyme subclasses, which we named OYE class III and class IV. Our results demonstrate that although, on a DNA level, each new OYE subclass features a distinct combination of sequence motifs previously known from the classical and the thermophilic-like group, their substrate scope more closely resembles the latter subclass.     

Two Subtle Amino Acid Changes in a Transaminase Substantially Enhance or Invert Enantiopreference in Cascade Syntheses

Amine transaminases (ATAs) are powerful enzymes for the stereospecific production of chiral amines. However, the synthesis of amines incorporating more than one stereocenter is still a challenge. We developed a cascade synthesis to access optically active 3‐alkyl‐substituted chiral amines by combining two asymmetric synthesis steps catalyzed by an enoate reductase and ATAs. The ATA wild type from Vibrio fluvialis showed only modest enantioselectivity (14 % de) in the amination of (S)‐3‐methylcyclohexanone, the product of the enoate‐reductase‐catalyzed reaction step. However, by protein engineering we created two variants with substantially improved diastereoselectivities: variant Leu56Val exhibited a higher R selectivity (66 % de) whereas the Leu56Ile substitution caused a switch in enantiopreference to furnish the S‐configured diastereomer (70 % de). Addition of 30 % DMSO further improved the selectivity and facilitated the synthesis of (1R,3S)‐1‐amino‐3‐methylcyclohexane with 89 % de at 87 % conversion.     

uSafe: A privacy-aware and participative mobile application for citizen safety in urban environments

Recent mobile applications empower citizens to monitor noise pollution or report on features of their urban environment. One important aspect of urban life has, however, not been sufficiently addressed, namely the safety of citizens. We present a privacy-aware application called uSafe, in which users indicate how safe they feel in geographical locations. These feelings are then consolidated into summary maps accessible by other users and urban planners. We evaluate our concept with a questionnaire-based study involving 183 participants. The results confirm the utility of uSafe and show that privacy protection is a decisive factor in their decision to contribute to it.     

The use of computing technology in highway construction as a total jobsite management tool

The integration of Global Positioning Systems (GPS) and Information and Communications Technology (ICT) through the creation of a Total Jobsite Management Tool (TJMT) can revolutionize the way contractors do business. The key to this integration is the collection and processing of real-time GPS data that is produced on the jobsite for use in project management applications. This paper establishes the state-of-practice of GPS data collection and utilization by heavy construction contractor companies in the United States and presents an implementation framework to assist construction contractor companies in navigating the terrain of GPS and ICT use. The proposed framework, at a macro level, outlines the required ICT infrastructure, addresses the organizational restructuring requirements, presents the related benefits and costs, and provides guidelines for successful implementation.     

Capillary electrophoresis and fluorescence anisotropy for quantitative analysis of peptide-protein interactions using JAK2 and SH2-Bbeta as a model system

Fluorescence anisotropy capillary electrophoresis (FACE) and affinity probe capillary electrophoresis (APCE) with laser-induced fluorescence detection were evaluated for analysis of peptide-protein interactions with rapid binding kinetics. The Src homology 2 domain of protein SH2-Bbeta (SH2-Bbeta (525-670)) and a tyrosine-phosphorylated peptide corresponding to the binding sequence of JAK2 were used as a model system. For peptide labeled with fluorescein, the K(d) = 82 +/- 7 nM as measured by fluorescence anisotropy (FA). APCE assays had a limit of detection (LOD) of 100 nM or 12 amol injected for SH2-Bbeta (525-670). The separation time of 4 s, achieved using an electric field of 2860 V/cm on 7-cm-long capillaries, was on the same time scale as complex dissociation allowing K(d) (101 +/- 12 nM in good agreement with FA measurements) and dissociation rate (k(off) = 0.95 +/- 0.02 s(-)(1) corresponding to a half-life of 0.73 s) to be determined. This measurement represents a 30-fold higher rate of complex dissociation than what had previously been measurable by nonequilibrium CE analysis of equilibrium mixtures. Using FACE, the protein was detected with an LOD of 300 nM or 7.5 fmol injected. FACE was not used for determining K(d) or k(off); however, this method provided better separation resolution for multiple forms of the protein than APCE. Both methods were found suitable for analysis of cell lysate. These results demonstrate that FACE and APCE may be useful complements to existing techniques for exploring binding interactions with rapid kinetics.     

Carotenoids and chlorophylls in processed xanthophyll-rich food

Processing of food modifies nutrient contents and food matrices. The present analyses provide information about the effects of heat steam sterilization (HSS), high pressure processing (HPP) and common household cooking on the stability of carotenoids and chlorophylls in spinach, parsley, dill and kale. For HSS, samples were heated in an autoclave for 5, 10, 15 and 20 min at 121 °C. Additionally, one sample was treated via HPP at 200, 400 and 600 MPa for 5, 10 and 40 min at room temperature. Carotenoid and chlorophyll contents were determined by reversed-phase HPLC. Lutein was the most abundant carotenoid in all samples. Furthermore, violaxanthin, zeaxanthin, β-carotene, chlorophylls a and b were quantified. Thermal treatment via HSS led to a significant decline of contents of lutein, chlorophylls a and b. HPP did not reduce the amounts of xanthophylls and chlorophylls. Conclusively, HPP seems to be a good alternative to HSS for the preservation of xanthophyll-rich food.     

Pima Indian males have lower beta-adrenergic sensitivity than Caucasian males

The sympathetic nervous system controls cardiovascular homeostasis and regulates energy metabolism. Pima Indians, a population with a low prevalence of hypertension and a high prevalence of obesity, have low sympathetic nervous activity, compared with Caucasians. Preliminary findings suggest that they may also have a low beta-adrenergic sensitivity. We studied beta-adrenergic sensitivity in 87 nondiabetic normotensive individuals [52 Pima Indians (35 males/17 females) and 35 Caucasians (24 males/11 females)], matched for age and body weight. Chronotropic sensitivity to beta-adrenergic stimulation was assessed by the dose of isoproterenol necessary to increase heart rate by 25 beats per minute [chronotropic dose-25 (CD25)]. Despite a similar basal heart rate and arterial blood pressure, Pimas tended to have lower beta-adrenergic sensitivity than Caucasians (CD25 = 2.37 +/- 2.27 vs. 1.57 +/- 1.38 microg, P = 0.07; mean +/- SD). This difference was significant in males (CD25 = 3.03 +/- 2.39 vs. 1.85 +/- 1.56 microg, P = 0.02) but not in females (CD25 = 1.01 +/- 1.17 vs. 0.96 +/- 0.61 microg, P = 0.99). In males only, CD25 was positively correlated to percent body fat (r = 0.36, P < 0.01). After adjustment for percent body fat, beta-adrenergic sensitivity was still significantly lower in Pima than in Caucasian males (CD25 = 3.44 +/- 2.24 vs. 2.57 +/- 1.60 microg, P = 0.05). In conclusion, our data suggest that increased adiposity is accompanied by decreased beta-adrenergic sensitivity in males only. However, at each level of adiposity, Pima Indian males have lower beta-adrenergic sensitivity than Caucasian males. In combination with a low sympathetic nervous system activity, a reduced beta-adrenergic sensitivity may contribute to the low prevalence of hypertension and the high prevalence of obesity observed in Pima Indians.     

A rotating PET scanner using BGO block detectors: design, performance and applications

Recent advances in fully three-dimensional reconstruction for multi-ring PET scanners have led us to explore the potential of a prototype scanner based on the rotation of two opposing arrays of BGO block detectors. The prototype contains only one-third of the number of detectors in the equivalent full ring scanner, resulting in reduced cost. With a lower energy threshold at 250 keV, the absolute efficiency of the scanner is 0.5% and the scatter fraction is 35% for a 20-cm cylinder. Transaxial and axial spatial resolution is about 6 mm. The maximum noise equivalent count rate estimated for a 15-cm diameter cylinder is 36,000 cps at a concentration of 26 kBq/ml. The minimum scan time for a 18F-fluoro-2-deoxyglucose (FDG) brain study is 55 sec. The camera has been validated for clinical applications using both FDG and 82Rb.