Cell-Free Synthesis of Selenoproteins in High Yield and Purity for Selective Protein Tagging

The selenol group of selenocysteine is much more nucleophilic than the thiol group of cysteine. Selenocysteine residues in proteins thus offer reactive points for rapid post-translational modification. Herein, we show that selenoproteins can be expressed in high yield and purity by cell-free protein synthesis by global substitution of cysteine by selenocysteine. Complete alkylation of solvent-exposed selenocysteine residues was achieved in 10 minutes with 4-chloromethylene dipicolinic acid (4Cl-MDPA) under conditions that left cysteine residues unchanged even after overnight incubation. Gd^III−Gd^III distances measured by double electron–electron resonance (DEER) experiments of maltose binding protein (MBP) containing two selenocysteine residues tagged with 4Cl-MDPA-Gd^III were indistinguishable from Gd^III−Gd^III distances measured of MBP containing cysteine reacted with 4Br-MDPA tags.     

Numerical experience with diagonalization algorithms for asymmetric demand traffic assignment

This paper reviews the concept of a diagonalization algorithm for use in solving traffic network equilibrium problems for which the arc cost and/or the origin-destination travel demand functions are asymmetric. Such functions are known to occur in realistic settings involving multiple modes or users. The computational performance of this algorithm for different degrees of travel demand asymmetry is then explored by a detailed numerical experiment since no previous results of this type have been reported. It is found that, through the use of progressive stopping tolerances, the impact of high degrees of travel demand function asymmetry on the computational burden associated with finding a traffic network equilibrium may be mitigated; in effect equilibrium problems with high degrees of demand asymmetry are little more difficult to solve than perfectly symmetric problems.     

MALDI Q-TOF CID MS for Diagnostic Ion Screening of Human Milk Oligosaccharide Samples

Human milk oligosaccharides (HMO) represent the bioactive components of human milk, influencing the infant’s gastrointestinal microflora and immune system. Structurally, they represent a highly complex class of analyte, where the main core oligosaccharide structures are built from galactose and N-acetylglucosamine, linked by 1–3 or 1–4 glycosidic linkages and potentially modified with fucose and sialic acid residues. The core structures can be linear or branched. Additional structural complexity in samples can be induced by endogenous exoglycosidase activity or chemical procedures during the sample preparation. Here, we show that using matrix-assisted laser desorption/ionization (MALDI) quadrupole-time-of-flight (Q-TOF) collision-induced dissociation (CID) as a fast screening method, diagnostic structural information about single oligosaccharide components present in a complex mixture can be obtained. According to sequencing data on 14 out of 22 parent ions detected in a single high molecular weight oligosaccharide chromatographic fraction, 20 different oligosaccharide structure types, corresponding to over 30 isomeric oligosaccharide structures and over 100 possible HMO isomers when biosynthetic linkage variations were taken into account, were postulated. For MS/MS data analysis, we used the de novo sequencing approach using diagnostic ion analysis on reduced oligosaccharides by following known biosynthetic rules. Using this approach, de novo characterization has been achieved also for the structures, which could not have been predicted.     

The Process Mining Manifesto—An interview with Wil van der Aalst

The IEEE Task Force on Process Mining has recently published its Process Mining Manifesto (PMM) in an effort to promote the topic of process mining. As this topic touches a number of areas in computer science, the editors of Information Systems have decided to conduct an interview with the person in charge of the task force, Prof. Wil van der Aalst of Eindhoven University of Technology (TU/e) in the Netherlands     

Gallium gradients in Cu(In,Ga)Se2 thin‐film solar cells

The gallium gradient in Cu(In,Ga)Se₂ (CIGS) layers, which forms during the two industrially relevant deposition routes, the sequential and co‐evaporation processes, plays a key role in the device performance of CIGS thin‐film modules. In this contribution, we present a comprehensive study on the formation, nature, and consequences of gallium gradients in CIGS solar cells. The formation of gallium gradients is analyzed in real time during a rapid selenization process by in situ X‐ray measurements. In addition, the gallium grading of a CIGS layer grown with an in‐line co‐evaporation process is analyzed by means of depth profiling with mass spectrometry. This gallium gradient of a real solar cell served as input data for device simulations. Depth‐dependent occurrence of lateral inhomogeneities on the µm scale in CIGS deposited by the co‐evaporation process was investigated by highly spatially resolved luminescence measurements on etched CIGS samples, which revealed a dependence of the optical bandgap, the quasi‐Fermi level splitting, transition levels, and the vertical gallium gradient. Transmission electron microscopy analyses of CIGS cross‐sections point to a difference in gallium content in the near surface region of neighboring grains. Migration barriers for a copper‐vacancy‐mediated indium and gallium diffusion in CuInSe₂ and CuGaSe₂ were calculated using density functional theory. The migration barrier for the In_Cu antisite in CuGaSe₂ is significantly lower compared with the Ga_Cu antisite in CuInSe₂, which is in accordance with the experimentally observed Ga gradients in CIGS layers grown by co‐evaporation and selenization processes. Copyright © 2014 John Wiley & Sons, Ltd.     

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