Direct Cloning, Genetic Engineering, and Heterologous Expression of the Syringolin Biosynthetic Gene Cluster in E. coli through Red/ET Recombineering

The reconstruction of a natural product biosynthetic pathway from bacteria in a vector and subsequent heterologous expression in a technically amenable microbial system represents an efficient alternative to empirical traditional methods for functional discovery, yield improvement, and genetic engineering to produce "unnatural" derivatives. However, the traditional cloning procedure based on genomic library construction and screening are complicated due to the large size (>10 kb) of most biosynthetic pathways. Here, we describe the direct cloning of a partial syringolin biosynthetic gene cluster (sylCDE, 19 kb) from a digested genomic DNA mixture of Pseudomonas syringae into a plasmid in which sylCDE is under the control of an inducible promoter by one step linear-plus-linear homologous recombination (LLHR) in Escherichia coli. After expression in E. coli GB05-MtaA, two new syringolin derivatives were discovered. The complete syringolin gene cluster was assembled by addition of sylAB and exchange of a synthetic bidirectional promoter against the native promoter to drive sylB and sylC expression by using Red/ET recombineering. The varying production distribution of syringolin derivatives showed the different efficiencies of native and synthetic promoters in E. coli. The successful reconstitution and expression of the syringolin biosynthetic pathway shows that Red/ET recombineering is an efficient tool to clone and engineer secondary metabolite biosynthetic pathways.     

Erythropoietin Modulates Autophagy Signaling in the Developing Rat Brain in an In Vivo Model of Oxygen-Toxicity

Autophagy is a self-degradative process that involves turnover and recycling of cytoplasmic components in healthy and diseased tissue. Autophagy has been shown to be protective at the early stages of programmed cell death but it can also promote apoptosis under certain conditions. Earlier we demonstrated that oxygen contributes to the pathogenesis of neonatal brain damage, which can be ameliorated by intervention with recombinant human erythropoietin (rhEpo). Extrinsic- and intrinsic apoptotic pathways are involved in oxygen induced neurotoxicity but the role of autophagy in this model is unclear. We analyzed the expression of autophagy activity markers in the immature rodent brain after exposure to elevated oxygen concentrations. We observed a hyperoxia-exposure dependent regulation of autophagy-related gene (Atg) proteins Atg3, 5, 12, Beclin-1, microtubule-associated protein 1 light chain 3 (LC3), LC3A-II, and LC3B-II which are all key autophagy activity proteins. Interestingly, a single injection with rhEpo at the onset of hyperoxia counteracted these oxygen-mediated effects. Our results indicate that rhEpo generates its protective effect by modifying the key autophagy activity proteins.     

Desktop manufacturing of complex objects,prototypes and biomedical scaffolds by means of computer‐assisted design combined with computer‐guided 3D plotting of polymers and reactive oligomers

Computer‐assisted design and image processing were combined with computer‐guided one‐ and two‐component air‐driven 3D dispensing of hotmelts, solutions, pastes, dispersions of polymers as well as monomers and reactive oligomers to produce solid objects with complex shapes and tailor‐made internal structures. During the 3D plotting process either individual microdots or microstrands were positioned in order to construct complex objects, fibers, tubes and scaffolds similar to non‐wovens. The resolution was in the range of 200 μm and depended upon inner nozzle diameter, air pressure, plotting speed, rheology, and plotting medium. Plotting in liquid media with densities similar to that of the dispensing liquid eliminated the need for construction of temporary support structures. The design capabilities of this computer‐guided 3D plotting process was demonstrated using conventional moisture‐curable silicone resin.     

Beneficial combination of wet oxidation,membrane separation and biodegradation processes for treatment of polymer processing wastewaters

The treatment of a model wastewater containing polyethylene glycol of molecular weight (MW) 10 000 by means of combined chemical oxidative pretreatment, membrane separation and biological post‐treatment was investigated. Wet oxidation was employed as a chemical pretreatment process to convert the original, high MW polymer to lower MW compounds in an attempt to improve the biotreatability of the waste‐water. The partially oxidized effluents formed during wet oxidation at temperatures up to 403 K were separated by nanofiltration where larger molecules were recycled into the wet oxidation reactor, while the permeate leaving the filtration step was treated biologically. At a biological residence time (τ_B) of 12 h and 3 h, the resulting total organic carbon (TOC) removal in the biological step was as high as 94% and 87%, respectively. Conversely, a continuous aerobic biological process was found inadequate to completely mineralize the original wastewater, since at τ_b of 96 h only about 60% to 70% TOC removal was achieved, while at τ_b of 12 h the original wastewater was practically non‐biockgradable.     

Evaluation of the effects of injection timing and rate-shape on diesel low temperature combustion using advanced CFD modeling

It is well known that injection strategies including the injection timing and the injection rate-shape play an important role in determining engine performance, especially pollutant emissions. But how the injection timing and the injection rate-shape quantitatively affect the performance of diesel low temperature combustion (LTC) is still not well understood. In this paper, the KIVA-CHEMKIN computational fluid dynamics (CFD) code with an improved spray model is used to simulate the spray and combustion processes of diesel LTC with early and late injection timings and seven different injection rate-shapes. The validation of the models is based on comparisons with laser diagnostic and in-cylinder pressure data under a low load operating condition. It is concluded that the use of early injection provides lower soot, HC and CO emissions but higher NOx emissions than the late injection. A rectangular-type (case1) and boot-type (case 4) injection rate-shape displays the potential to reduce the soot, HC and CO emissions compared the other generic rate-shapes considered.     

Biological properties of iron oxide nanoparticles for cellular and molecular magnetic resonance imaging

Superparamagnetic iron-oxide particles (SPIO) are used in different ways as contrast agents for magnetic resonance imaging (MRI): Particles with high nonspecific uptake are required for unspecific labeling of phagocytic cells whereas those that target specific molecules need to have very low unspecific cellular uptake. We compared iron-oxide particles with different core materials (magnetite, maghemite), different coatings (none, dextran, carboxydextran, polystyrene) and different hydrodynamic diameters (20-850 nm) for internalization kinetics, release of internalized particles, toxicity, localization of particles and ability to generate contrast in MRI. Particle uptake was investigated with U118 glioma cells und human umbilical vein endothelial cells (HUVEC), which exhibit different phagocytic properties. In both cell types, the contrast agents Resovist, B102, non-coated Fe(3)O(4) particles and microspheres were better internalized than dextran-coated Nanomag particles. SPIO uptake into the cells increased with particle/iron concentrations. Maximum intracellular accumulation of iron particles was observed between 24 h to 36 h of exposure. Most particles were retained in the cells for at least two weeks, were deeply internalized, and only few remained adsorbed at the cell surface. Internalized particles clustered in the cytosol of the cells. Furthermore, all particles showed a low toxicity. By MRI, monolayers consisting of 5000 Resovist-labeled cells could easily be visualized. Thus, for unspecific cell labeling, Resovist and microspheres show the highest potential, whereas Nanomag particles are promising contrast agents for target-specific labeling.     

Towards anti-corrosion coatings from surfactant-free latexes based on maleic anhydride containing polymers

We report on the film formation of surfactant-free, artificial latexes based on copolymers containing maleic anhydride. Different metallic substrates, such as aluminum, steel and magnesium alloys, were coated with three different latexes. A commercial polyester based coating was used as a comparative sample. Two of the latexes were based on polymer with a high T_g (resp. 100 and 130 °C), and one on a polymer with a T_g of −70 °C. The wetting of the substrates could be optimized by etching the metal substrate, acidic or alkaline, leading to homogenous film formation and improved adhesion. For aluminum substrates an alkaline pretreatment improved the adhesion with the polymer films, whereas for magnesium the acidic pretreatment improved the adhesion. Furthermore, acid pretreatment increased the corrosion resistance of the coated magnesium substrate, when compared to an alkaline pretreatment. The films formed from latex displayed comparable or better anti-corrosive properties when compared to the commercial polyester.