Trypsiligase-Catalyzed Labeling of Proteins on Living Cells

Fluorescent fusion proteins are powerful tools for studying biological processes in living cells, but universal application is limited due to the voluminous size of those tags, which might have an impact on the folding, localization or even the biological function of the target protein. The designed biocatalyst trypsiligase enables site-directed linkage of small-sized fluorescence dyes on the N terminus of integral target proteins located in the outer membrane of living cells through a stable native peptide bond. The function of the approach was tested by using the examples of covalent derivatization of the transmembrane proteins CD147 as well as the EGF receptor, both presented on human HeLa cells. Specific trypsiligase recognition of the site of linkage was mediated by the dipeptide sequence Arg-His added to the proteins’ native N termini, pointing outside the cell membrane. The labeling procedure takes only about 5 minutes, as demonstrated for couplings of the fluorescence dye tetramethyl rhodamine and the affinity label biotin as well.     

Deliberately Losing Control of C−H Activation Processes in the Design of Small-Molecule-Fragment Arrays Targeting Peroxisomal Metabolism

Combined photochemical arylation, “nuisance effect” (S_NAr) reaction sequences have been employed in the design of small arrays for immediate deployment in medium-throughput X-ray protein–ligand structure determination. Reactions were deliberately allowed to run “out of control” in terms of selectivity; for example the ortho-arylation of 2-phenylpyridine gave five products resulting from mono- and bisarylations combined with S_NAr processes. As a result, a number of crystallographic hits against NUDT7, a key peroxisomal CoA ester hydrolase, have been identified.     

Contribution of the Internal and External Oxygen to the Oxidation of Microencapsulated Fish Oil

Microencapsulation aims to protect polyunsaturated fatty acids against oxidation by embedding oil droplets in a solid matrix. In such a system the internal (dissolved and entrapped) and external (in the environment) oxygen can be differentiated. The study aims to quantify the impact of both oxygen sources on the oxidation of microencapsulated fish oil. The impact of the solubilized oxygen in bulk fish oil is investigated by saturating the oil with nitrogen, synthetic air, and pure oxygen. Even though more dissolved oxygen results in more oxidation products, the difference between the oxidation of the nitrogen and air saturated oil is significant but low. For encapsulated fish oil powders, the internal oxygen is modified by preparing oil‐in‐water emulsions under atmospheric and inert conditions. The feed is atomized and spray dried with either nitrogen or air. Powders are stored under vacuum and in vials and the hydroperoxides and anisidine value are determined in the total‐ and encapsulated oil. The internal oxygen has a minor impact, whereas the external oxygen is the main determinant for autoxidation. Apart from oxidizing the non‐encapsulated oil, the external O₂ penetrates into the particle and reacts with the encapsulated oil. Practical Applications: Comparing the contribution of the internal and external oxygen to the oxidative stability shows that the internal O₂ plays a minor role and can be neglected. This means that the emulsion preparation as well as the spray drying process can be conducted under ambient conditions. An inert production is not extending the shelf life significantly as long as the external O₂ determines oxidation. The focus should be on optimizing the diffusion barrier properties of the wall matrix to reduce the penetration of the external oxygen into the particle system. Alternatively, packaging solution reducing the external O₂ will extend the shelf life of the microencapsulated oil.     

Deactivation of V2O5-WO3-TiO2 SCR catalyst at a biomass-fired combined heat and power plant

The deactivation of a commercial type V₂O₅-WO₃-TiO₂ monolith catalyst under biomass combustion was studied at a full-scale grate-fired power plant burning straw/wood using a slip stream pilot scale reactor. The aerosols in the flue gas consisted of a mixture of potassium chloride and sulphate. Three catalyst elements were exposed at 350 °C, and one element was exposed at 250 °C for comparison. The catalyst activity was measured in the reactor at the exposure temperature by addition of NH₃ and extra NO. The activity, in terms of a first-order rate constant, dropped by 52% after about 1140 h indicating a very fast deactivation compared to coal firing. It was also found that the reactor temperature was not of importance for the deactivation rate. SEM-EDX analysis showed that particle deposition and pore blocking contributed to the deactivation by decreasing the diffusion rate of NO and NH₃ into the catalyst. However, potassium also penetrated into the catalyst wall and the resulting average K/V ratio in the catalyst structure was high enough (about 0.3–0.5) for a significant chemical deactivation. Chemisorption studies carried out in situ showed that the amount of chemisorbed NH₃ on the catalyst decreased as a function of exposure time, which reveals that Brøndsted acid sites had reacted with potassium compounds and thereby rendered inactive. When washed by 0.5 M H₂SO₄ the regenerated catalyst regains a higher activity than that of the fresh catalyst at temperatures higher than 300 °C, but even though reactivation is possible, the deactivation rate appears too high for practical use of the SCR process in straw combustion.     

Anesthetic considerations in the child with Gaucher disease

Alpidem, an imidazopyridine that acts at the gamma-aminobutyric acid/benzodiazepine receptor complex, has been reported to be an effective anxiolytic with a more favorable side effect profile than benzodiazepines. The effect of alpidem was investigated in an 8-week, open, clinical trial in 13 patients with panic disorder, with or without agoraphobia. Three patients were responders (much improved or very much improved), five patients were nonresponders, and five patients dropped out after less than 6 weeks of treatment. Significant improvement was seen in the sample as a whole for spontaneous panic attacks, phobic avoidance, and anticipatory anxiety. Most improvement occurred during the first 4 weeks of treatment, and responders had milder panic disorder at baseline. Adverse effects were generally mild. After 8 weeks of treatment, taper of medication over 2 weeks occurred without significant worsening of panic disorder symptoms. The efficacy of alpidem in the treatment of panic disorder remains uncertain and requires assessment in a controlled trial.     

Purification and characterization of an endopeptidase from Lactobacillus delbrueckii subsp. bulgaricus B14

An intracellular endopeptidase was purified from cell-free extracts of Lactobacillus delbrueckii subsp. bulgaricus B14 by anion exchange chromatography on DEAE-Sepharose, hydroxyapatite chromatography, second anion exchange chromatography on Mono-Q, and metal-chelating affinity chromatography. The endopeptidase was a monomer with a molecular mass of approximately 70 kDa determined by SDS-PAGE and gel filtration. Various oligopeptides (e.g. Met-enkephalin, bradykinin) were hydrolysed by the endopeptidase. Exopeptidase activity and cleavage of dipeptides or tripeptides was not observed. The K_M value for the cleavage of Metenkephalin was 1.2 mM. Temperature and pH optima were 47 °C and pH 7.7, respectively. The endopeptidase was inhibited by the classical agents for metal-dependent (EDTA) and serine (DFP) enzymes. Activity was increased by Co²⁺ and Mg²⁺, no effect was observed with Ca²⁺. After inhibition with EDTA, enzyme activity could be restored fully by Co²⁺. Activity was inhibited by Zn²⁺, Mn²⁺, Fe²⁺, Cu²⁺, Cd²⁺ and Hg²⁺. The N-terminal sequence of the endopeptidase was determined as: H₂N-Val-Arg-Gly-Gly-Ser-Gly-Asp-Thr-Thr-Val-0H.     

Performance-Analyse und -Optimierung in der Softwareentwicklung

Nicht-funktionale Anforderungen an ein Softwaresystem beschreiben Aspekte, die nicht direkt die Funktionalit?t, wie sie der Benutzer sieht, betreffen.     

The high-resolution time-of-flight spectrometer TOFTOF

The TOFTOF spectrometer is a multi-disc chopper time-of-flight spectrometer for cold neutrons at the research neutron source Heinz Maier-Leibnitz (FRM II). After five reactor cycles of routine operation the characteristics of the instrument are reported in this article. The spectrometer features an excellent signal to background ratio due to its remote position in the neutron guide hall, an elaborated shielding concept and an s-shaped curved primary neutron guide which acts i.a. as a neutron velocity filter. The spectrometer is fed with neutrons from the undermoderated cold neutron source of the FRM II leading to a total neutron flux of 10¹⁰n/cm²/s in the continuous white beam at the sample position distributed over a continuous and particularly broad wavelength spectrum. A high energy resolution is achieved by the use of high speed chopper discs made of carbon-fiber-reinforced plastic. In the combination of intensity, resolution and signal to background ratio the spectrometer offers new scientific prospects in the fields of inelastic and quasielastic neutron scattering.     

The State of the Art of Spatial Interfaces for 3D Visualization

We survey the state of the art of spatial interfaces for 3D visualization. Interaction techniques are crucial to data visualization processes and the visualization research community has been calling for more research on interaction for years. Yet, research papers focusing on interaction techniques, in particular for 3D visualization purposes, are not always published in visualization venues, sometimes making it challenging to synthesize the latest interaction and visualization results. We therefore introduce a taxonomy of interaction technique for 3D visualization. The taxonomy is organized along two axes: the primary source of input on the one hand and the visualization task they support on the other hand. Surveying the state of the art allows us to highlight specific challenges and missed opportunities for research in 3D visualization. In particular, we call for additional research in: (1) controlling 3D visualization widgets to help scientists better understand their data, (2) 3D interaction techniques for dissemination, which are under‐explored yet show great promise for helping museum and science centers in their mission to share recent knowledge, and (3) developing new measures that move beyond traditional time and errors metrics for evaluating visualizations that include spatial interaction.