Publikationen

Technologies for regulated expression of multiple transgenes in mammalian cells have gathered momentum for bioengineering, gene therapy, drug discovery, and gene-function analyses. Capitalizing on recently developed mammalian transgene modalities (QuoRex) derived from Streptomyces coelicolor, we have designed a flexible and highly compatible expression vector set that enables desired transgene/siRNA control in response to the nontoxic butyrolactone SCB1. The construction-kit-like expression portfolio includes (i) multicistronic (pTRIDENT), (ii) autoregulated, (iii) bidirectional (pBiRex), (iv) oncoretro- and lentiviral transduction, and (v) RNA polymerase II-based siRNA transcription-fine-tuning vectors for straightforward implementation of QuoRex-controlled (trans)gene modulation in mammalian cells.

Inducible transgene expression technologies are of unmatched potential for biopharmaceutical manufacturing of unstable, growth-impairing and cytotoxic proteins as well as conditional metabolic engineering to improve desired cell phenotypes. Currently available transgene dosing modalities which rely on physical parameters or small-molecule drugs for transgene fine-tuning compromise downstream processing and/or are difficult to implement technologically. The recently designed gas-inducible acetaldehyde-inducible regulation (AIR) technology takes advantage of gaseous acetaldehyde to modulate product gene expression levels. At regulation effective concentrations gaseous acetaldehyde is physiologically inert and approved as food additive by the Federal Drug Administration (FDA). During standard bioreactor operation, gaseous acetaldehyde could simply be administered using standard/existing gas supply tubing and eventually eliminated by stripping with inducer-free air. We have determined key parameters controlling acetaldehyde transfer in three types of bioreactors and designed a mass balance-based model for optimal product gene expression fine-tuning using gaseous acetaldehyde. Operating a standard stirred-tank bioreactor set-up at 10 L scale we have validated AIR technology using CHO-K1-derived serum-free suspension cultures transgenic for gas-inducible production of human interferon-β (IFN-β). Gaseous acetaldehyde- inducible IFN-β production management was fully reversible while maintaining cell viability at over 95% during the entire process. Compatible with standard bioreactor design and downstream processing procedures AIR-based technology will foster novel opportunities for pilot and large-scale manufacturing of difficult-to-produce protein pharmaceuticals. © 2005 Elsevier Inc. All rights reserved.

Capitalizing on components evolved to metabolize ethanol in Aspergillus nidulans, we previously designed the first molecular gas-gene expression interface using gaseous acetaldehyde as the major inducer. This fungus-derived acetaldehyde-inducible gene regulation (AIR) system operated perfectly and enabled precise and reversible transgene expression dosing in a variety of mammalian cells. We now validate the use of mainstream cigarette smoke typically containing acetaldehyde at regulation-effective nontoxic concentrations as a non-invasive modality to adjust transgene transcription in mammalian cells and mice. Indeed, tobacco smoke-induced expression fine-tuning of AIR-driven transgenes was successful in mammalian cells. Even mice implanted with cells transgenic for AIR-controlled SEAP (human secreted alkaline phosphatase) production showed serum SEAP levels correlating with inhaled tobacco smoke doses. Tobacco smoke-controlled gene expression may foster clinical opportunities as well as advances in understanding smoke-related pathologies. © 2005 Wiley Periodicals, Inc.

This presentation will introduce laser interference lithography to prepare a periodic line and point micropatterns for study of cell-surface interactions. This process provides a straightforward micropatterning technique based on selective laser ablation of polymers utilizing the periodic energy distribution of two or more beam interference patterns. The micropatterns were characterized by atomic force microscopy, while the surface chemical modification was analyzed using X-ray photoelectron spectroscopy. Human pulmonary fibroblasts cultured on the surface of polycarbonate bearing line micropatterns were elongated, spindlelike, and oriented themselves along the line patterns with all different groove widths. In contrast, cells cultured on point patterns were also bipolar but showed no orientation. Further investigations demonstrated that human pulmonary fibroblast cells cultured on line and point micropatterns showed inflammatory response.

Laser interference lithography (LIL) is a straightforward technique to prepare linear micropatterns for regulating cellular adhesion behaviors on polymer substratum. This process is based on selective laser ablation directly duplicating the interference patterns of two or more coherent laser beams onto the polymer surface. Micropatterns prepared by LIL on poly(ethylene terephthalate) and Thermanox((R)) were characterized using atomic force microscopy (AFM) and white light interferometer while the chemical surface modification induced by laser was analyzed by X-ray photoelectron spectroscopy (XPS). The AFM photographs show that the micropattems are well-defined and of great consistency. Polymer properties and laser parameters related to LIL as well as laser ablation mechanisms are discussed in this technical note.

Iron oxide films have been deposited on Si(100) substrates by chemical vapour deposition (CVD) of iron(Ill) tert-butoxide ([Fe(OtBu)3]2) in the temperature range 350-450 °C. The precursor flux and substrate temperature were varied to control the phase composition, average grain size and film thickness. The nature of substrate and deposition temperature markedly influence the morphology and iron-oxygen stoichiometry in the CVD deposits. Phase transformations in iron oxide films were achieved through precise local and periodic heating of the films by interfering laser beams. The interaction of iron oxide films with short laser pulses (Nd:YAG, 355 nm) induced partial transformation of hematite (α-Fe2O3) to magnetite (Fe3O4) or magnetite to wustite (Fe1-xO), respectively. The phase characterization and morphology of the hematite and magnetite films were investigated before and after laser irradiation by X-ray diffractometry, high resolution scanning electron microscopy and white light interferometry.

A total of 81 doped and undoped anatase nano-particles were synthesised by a precipitation/co-precipitation process followed by a hydrothermal treatment to obtain increased visible light photocatalytic activity. The screening process was performed utilising a high throughput analysis system based on the photometric monitoring of the photocatalytic degradation of organic dyes (Rhodamine B, Malachite Green, Acid Blue 29). Photocatalytically active coatings were prepared with selected catalysts with high and low rankings from the screening. Degradation experiments with stearic acid could confirm the varying grades of visible light activity as seen in the screening process.

Complexation reactions of titanium tetraethoxide [Ti(OEt)4] and titanium tetra-n-butoxide [Ti(OBun)4] with 3-pentenoic acid (PA) and allylacetoacetate (AAA), in a 1 : 1M ratio, were studied in ethanol solution at room temperature. 13C-NMR and FTIR spectra showed that all PA and AAA completely reacted with both titanium alkoxides. Hydridosilane compounds such as triethoxysilane and triethylsilane were added to titanium chelate complexes in a 1 : 1M ratio. The investigation of products by 13C- and 29Si-NMR and FTIR showed additions of SiH to the CC double bond. The hydrolysis of titanium-PA and AAA complexes, by water in 1 : 4 ratios, resulted in released PA in an amount of 10% and AAA of 20%. The stability of hydrolyzed products was investigated by 13C-NMR, 29Si-NMR, and FTIR.

Titanium carbide/amorphous-carbon (TiC/a-C:H) nanocomposite coatings deposited by pulsed unbalanced reactive magnetron sputtering have been investigated in terms of structure, chemical and phase composition by AFM, TEM, XPS and XRD analyses. Subject to total carbon content, metallic titanium, titanium carbide and amorphous-carbon phases were found in the deposited coatings, which contributed to the observed microstructures and morphologies. The specific resistivity of nanocomposite coatings scales up with increasing amount of matrix-forming carbon. Hardness profiles of the different compositions revealed that nearly stoichiometric TiC films with average crystallite size of 70 nm exhibit the maximum hardness, whereas the lowest friction coefficient (µ < 0.1) was found in films rich in amorphous-carbon and containing smaller TiC nanocrystallites (<d> similar to 10 nm).