On-line reaction monitoring in the liquid phase using two mid-infrared quantum cascade lasers simultaneously

On-line monitoring of a model reaction was performed by employing two pulsed mid-infrared Fabry-Pérot quantum cascade lasers (QCL). The emission maxima of the QCLs were located at 1393 and 1080 cm(-1). An optical system of parabolic mirrors and a ZnSe beam splitter combined the two laser beams and allowed a transmission cell to be probed with both QCLs simultaneously. The reaction mixture was pumped continuously through a cell that had an optical path of 48 microm. This dual QCL system allowed fast absorption measurements of the reaction mixture at two distinct wavenumbers. The reaction under study was the oxidation of sulfite to sulfate with hydrogen peroxide acting as oxidant. On-line measurements of the chemical reaction allowed direct, real-time monitoring of sulfate formation and hydrogen peroxide depletion.     

Parallel manipulation of bifunctional DNA molecules on structured surfaces using kinesin-driven microtubules

We have developed a technique to manipulate bifunctional DNA molecules: One end is thiolated to bind to a patterned gold surface and the other end is biotinylated to bind to a microtubule gliding over a kinesin-coated surface. We found that DNA molecules can be stretched and overstretched between the gold pads and the motile microtubules, and that they can form dynamic networks. This serves as a proof-of-principle that biological machineries can be used in vitro to accomplish the parallel formation of structured DNA templates that will have applications in biophysics and nanoelectronics.     

Natural products from marine organisms and their associated microbes

The marine environment is distinguished by unique groups of organisms being the source of a wide array of fascinating structures. The enormous biodiversity of marine habitats is mirrored by the molecular diversity of secondary metabolites found in marine animals, plants and microbes. The recognition that many marine invertebrates contain endo- and epibiotic microorganisms and that some invertebrate-derived natural products are structurally related to bacterial metabolites suggests a microbial origin for some of these compounds. Other marine natural products, however, are clearly located in invertebrate tissue and microbial involvement in the biosynthetic process seems unlikely. The complexity of associations in marine organisms, especially in sponges, bryozoans and tunicates, makes it extremely difficult to definitively state the biosynthetic source of many marine natural products or to deduce their ecological significance. Whereas many symbiotic marine microorganisms cannot be isolated and cultured, numerous epi- and endobiotic marine fungi produce novel secondary metabolites in laboratory cultures. The potent biological activity of many marine natural products is of relevance for their ecological function but is also the basis of their biomedical importance.     

C-terminal modifications of a protein by UAG-encoded incorporation of puromycin during in vitro protein synthesis in the absence of release factor 1

Deactivation of release factor 1 by polyclonal antibodies in an in vitro translation system, which was used to express the esterase gene, led to the reversible elimination of naturally occurring termination. This technique allowed the antibiotic puromycin to be used as an acceptor substrate for the peptidyl residue in the peptidyl-transferase reaction. This resulted in more than 80 % yield of protein with C-terminally incorporated puromycin. pCpPuromycin that was either conjugated with the Cy3 fluorophor or biotin by N4 alkylation of cytosine, also acted as an acceptor substrate for the peptidyl-transferase reaction and was incorporated into the protein C terminus. The resulting conjugates possessed Cy3-specific fluorescence and affinity to streptavidin-coated surfaces, respectively. This left the enzymatic activity of the reporter protein unaffected. It was also shown that extension of puromycin on its 5'-hydroxyl end by up to ten deoxyoligonucleotides also allowed conjugation with the C terminus of in vitro translated protein when RF1-dependent termination was suppressed. However, the conjugation yield decreased upon addition of more than six nucleotides.     

A unique mechanism for methyl ester formation via an amide intermediate found in myxobacteria

Secondary metabolism involves a broad diversity of biochemical reactions that result in a wide variety of biologically active compounds. Terminal amide formation during the biosynthesis of the myxobacterial electron-transport inhibitor, myxothiazol, was analyzed by heterologous expression of the unique nonribosomal-peptide synthetase, MtaG, and incubation with a synthesized substrate mimic. These experiments provide evidence that the terminal amide is formed from a carrier protein-bound myxothiazol acid that is thioesterified to MtaF. This intermediate is transformed to an amide by extension with glycine and subsequent oxidative cleavage by MtaG. The final steps of melithiazol assembly involve a highly similar protein-bound intermediate (attached to MelF, a homologue of MtaF), which is transformed to an amide by MelG (homologue of MtaG). In this study, we also show that the amide moiety of myxothiazol A can be hydrolyzed in vivo to the formerly unknown free myxothiazol acid by heterologous expression of melJ in the myxothiazol producer Stigmatella aurantiaca DW4/3-1. The methyltransferase MelK can finally methylate the acid to give rise to the methyl ester, which is produced as the final product in the melithiazol A biosynthetic pathway. These experiments clarify the role of MelJ and MelK during melithiazol assembly.     

Polyaza crown ethers as non-nucleosidic building blocks in DNA conjugates: synthesis and remarkable stabilization of dsDNA

The synthesis of amphiphilic polyaza crown ether monomers X (benzyl-substituted), Y (palmityl-substituted) and Z (cholesteryl-substituted) and their incorporation into oligonucleotides are described. Their effects on thermal duplex stability were investigated by UV melting curve analysis in different alkaline metal buffer solutions. Thermal-denaturation experiments showed remarkable stabilization of dsDNA by polyaza crown ether monomers when incorporated in opposite positions. The series of polyaza crown ether monomers (X, Y and Z) with different overall lipophilicities showed a trend of increased stability of the corresponding dsDNA with increasing lipophilicity of the polyaza crown ether monomer. Multiple incorporations of benzyl-substituted polyaza crown ether monomer X as dangling ends on both sides of dsDNA resulted in strongly increased stability of the corresponding duplex.     

Hydrogen energy from coupled waste gasification and cement production—a thermochemical concept study

A plant concept for hydrogen production from waste gasification coupled with cement manufacturing is presented. Hot precalcined cement meal, from the operating cement process, is used as heat carrier to provide energy required by the parallel arranged gasifier. The amount of CaO present in the cement meal operates simultaneously as an effective in situ _CO2${\mathrm{CO}}_2$-sorbent. First, a practical case study was devised to be able to perform simulations for estimation of expected hydrogen yield. The influence of different operation parameters of the gasifier and the hydrogen separation unit (steam-to-fuel ratio, pyrolysis temperature, PSA efficiency) was studied based on chemical equilibrium calculations. The simulation results indicate, that the coupling provides advantages for both processes. The production of a hydrogen-rich gas via thermal gasification benefits from the continuously available fresh CaO, which improves fuel conversion reactions and captures _CO2${\mathrm{CO}}_2$ in situ. High-calorific streams from gasification process remaining after hydrogen separation may substitute fossil fuels needed for cement process. For a steam/fuel ratio of 0.3 and a PSA efficiency of 0.7, the calculated hydrogen energy yield is 46% of fuel energy input.     

High-resolution ab initio three-dimensional x-ray diffraction microscopy

Coherent x-ray diffraction microscopy is a method of imaging nonperiodic isolated objects at resolutions limited, in principle, by only the wavelength and largest scattering angles recorded. We demonstrate x-ray diffraction imaging with high resolution in all three dimensions, as determined by a quantitative analysis of the reconstructed volume images. These images are retrieved from the three-dimensional diffraction data using no a priori knowledge about the shape or composition of the object, which has never before been demonstrated on a nonperiodic object. We also construct two-dimensional images of thick objects with greatly increased depth of focus (without loss of transverse spatial resolution). These methods can be used to image biological and materials science samples at high resolution with x-ray undulator radiation and establishes the techniques to be used in atomic-resolution ultrafast imaging at x-ray free-electron laser sources.     

Physical, electrical, and optical properties of SF-PECVD-grown hydrogenated microcrystalline silicon with growth surface electrical bias

Characterization results on hydrogenated microcrystalline silicon (μc-Si:H) thin films grown in a Saddle Field (SF) PECVD system are presented. The microcrystalline content of the films is controlled by the application of a positive electrical bias to the film growth surface. The results of photoluminescence, atomic force microscopy, infrared-absorption, and electrical conductivity studies are presented. The results correlate to the changing microcrystalline content of the films in the same way as when microcrystalline content is influenced through growth parameters such as hydrogen dilution in other CVD techniques.

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