EXPOSE, an Astrobiological Exposure Facility on the International Space Station - from Proposal to Flight

Following an European Space Agency announcement of opportunity in 1996 for ”Externally mounted payloads for 1st utilization phase” on the International Space Station (ISS), scientists working in the fields of astrobiology proposed experiments aiming at long-term exposure of a variety of chemical compounds and extremely resistant microorganisms to the hostile space environment. The ESA exposure facility EXPOSE was built and an operations´ concept was prepared. The EXPOSE experiments were developed through an intensive pre-flight experiment verification test program. 12 years later, two sets of astrobiological experiments in two EXPOSE facilities have been successfully launched to the ISS for external exposure for up to 1.5 years. EXPOSE-E, now installed at the balcony of the European Columbus module, was launched in February 2008, while EXPOSE-R took off to the ISS in November 2008 and was installed on the external URM-D platform of the Russian Zvezda module in March 2009.     

Polychlorinated biphenyl (PCB)-degrading bacteria associated with trees in a PCB-contaminated site

The abundance, identities, and degradation abilities of indigenous polychlorinated biphenyl (PCB)-degrading bacteria associated with five species of mature trees growing naturally in a contaminated site were investigated to identify plants that enhance the microbial PCB degradation potential in soil. Culturable PCB degraders were associated with every plant species examined in both the rhizosphere and root zone, which was defined as the bulk soil in which the plant was rooted. Significantly higher numbers of PCB degraders (2.7- to 56.7-fold-higher means) were detected in the root zones of Austrian pine (Pinus nigra) and goat willow (Salix caprea) than in the root zones of other plants or non-root-containing soil in certain seasons and at certain soil depths. The majority of culturable PCB degraders throughout the site and the majority of culturable PCB degraders associated with plants were identified as members of the genus Rhodococcus by 16S rRNA gene sequence analysis. Other taxa of PCB-degrading bacteria included members of the genera Luteibacter and Williamsia, which have not previously been shown to include PCB degraders. PCB degradation assays revealed that some isolates from the site have broad congener specificities; these isolates included one Rhodococcus strain that exhibited degradation abilities similar to those of Burkholderia xenovorans LB400. Isolates with broad congener specificity were widespread at the site, including in the biostimulated root zone of willow. The apparent association of certain plant species with increased abundance of indigenous PCB degraders, including organisms with outstanding degradation abilities, throughout the root zone supports the notion that biostimulation through rhizoremediation is a promising strategy for enhancing PCB degradation in situ.     

Biocatalytic conversion of avermectin to 4'-oxo-avermectin: improvement of cytochrome p450 monooxygenase specificity by directed evolution

Discovery of the CYP107Z subfamily of cytochrome P450 oxidases (CYPs) led to an alternative biocatalytic synthesis of 4'-oxo-avermectin, a key intermediate for the commercial production of the semisynthetic insecticide emamectin. However, under industrial process conditions, these wild-type CYPs showed lower yields due to side product formation. Molecular evolution employing GeneReassembly was used to improve the regiospecificity of these enzymes by a combination of random mutagenesis, protein structure-guided site-directed mutagenesis, and recombination of multiple natural and synthetic CYP107Z gene fragments. To assess the specificity of CYP mutants, a miniaturized, whole-cell biocatalytic reaction system that allowed high-throughput screening of large numbers of variants was developed. In an iterative process consisting of four successive rounds of GeneReassembly evolution, enzyme variants with significantly improved specificity for the production of 4'-oxo-avermectin were identified; these variants could be employed for a more economical industrial biocatalytic process to manufacture emamectin.     

Antibody microarray-based profiling of complex specimens: systematic evaluation of labeling strategies

Antibody microarrays have often had limited success in detection of low abundant proteins in complex specimens. Signal amplification systems improve this situation, but still are quite laborious and expensive. However, the issue of sensitivity is more likely a matter of kinetically appropriate microarray design as demonstrated previously. Hence, we re-examined in this study the suitability of simple and inexpensive detection approaches for highly sensitive antibody microarray analysis. N-hydroxysuccinimidyl ester (NHS)- and Universal Linkage System (ULS)-based fluorescein and biotin labels used as tags for subsequent detection with anti-fluorescein and extravidin, respectively, as well as fluorescent dyes were applied for analysis of blood plasma. Parameters modifying strongly the performance of microarray detection such as labeling conditions, incubation time, concentrations of anti-fluorescein and extravidin and extent of protein labeling were analyzed and optimized in this study. Indirect detection strategies whether based on NHS- or ULS-chemistries strongly outperformed direct fluorescent labeling and enabled detection of low abundant cytokines with many dozen-fold signal-to-noise ratios. Finally, particularly sensitive detection chemistry was applied to monitoring cytokine production of stimulated peripheral T cells. Microarray data were in accord with quantitative cytokine levels measured by ELISA and Luminex, demonstrating comparable reliability and femtomolar range sensitivity of the established microarray approach.     

Thermodynamic characterization of allosteric glycogen phosphorylase inhibitors

Glycogen phosphorylase (GP) is a validated target for the treatment of type 2 diabetes. Here we describe highly potent GP inhibitors, AVE5688, AVE2865, and AVE9423. The first two compounds are optimized members of the acyl urea series. The latter represents a novel quinolone class of GP inhibitors, which is introduced in this study. In the enzyme assay, both inhibitor types compete with the physiological activator AMP and act synergistically with glucose. Isothermal titration calorimetry (ITC) shows that the compounds strongly bind to nonphosphorylated, inactive GP (GPb). Binding to phosphorylated, active GP (GPa) is substantially weaker, and the thermodynamic profile reflects a coupled transition to the inactive (tense) conformation. Crystal structures confirm that the three inhibitors bind to the AMP site of tense state GP. These data provide the first direct evidence that acyl urea and quinolone compounds are allosteric inhibitors that selectively bind to and stabilize the inactive conformation of the enzyme. Furthermore, ITC reveals markedly different thermodynamic contributions to inhibitor potency that can be related to the binding modes observed in the cocrystal structures. For AVE5688, which occupies only the lower part of the bifurcated AMP site, binding to GPb (Kd = 170 nM) is exclusively enthalpic (Delta H = -9.0 kcal/mol, TDelta S = 0.3 kcal/mol). The inhibitors AVE2865 (Kd = 9 nM, Delta H = -6.8 kcal/mol, TDelta S = 4.2 kcal/mol) and AVE9423 (Kd = 24 nM, Delta H = -5.9 kcal/mol, TDelta S = 4.6 kcal/mol) fully exploit the volume of the binding pocket. Their pronounced binding entropy can be attributed to the extensive displacement of solvent molecules as well as to ionic interactions with the phosphate recognition site.     

Impact of the FcgammaII-receptor on quartz uptake and inflammatory response by alveolar macrophages

The inflammatory response following particle inhalation is described as a key event in the development of lung diseases, e.g., fibrosis and cancer. The essential role of alveolar macrophages (AM) in the pathogenicity of particles through their functions in lung clearance and mediation of inflammation is well known. However, the molecular mechanisms and direct consequences of particle uptake are still unclear. Inhibition of different classic phagocytosis receptors by flow cytometry shows a reduction of the dose-dependent quartz particle (DQ12) uptake in the rat AM cell line NR8383. Thereby the strongest inhibitory effect was observed by blocking the FcgammaII-receptor (FcgammaII-R). Fluorescence immunocytochemistry, demonstrating FcgammaII-R clustering at particle binding sites as well as transmission electron microscopy, visualizing zippering mechanism-like morphological changes, confirmed the role of the FcgammaII-R in DQ12 phagocytosis. FcgammaII-R participation in DQ12 uptake was further strengthened by the quartz-induced activation of the Src-kinase Lyn, the phospho-tyrosine kinases Syk (spleen tyrosine kinase) and PI3K (phosphatidylinositol 3-kinase), as shown by Western blotting. Activation of the small GTPases Rac1 and Cdc42, shown by immunoprecipitation, as well as inhibition of tyrosine kinases, GTPases, or Rac1 provided further support for the role of the FcgammaII-R. Consistent with the uptake results, FcgammaII-R activation with its specific ligand caused a similar generation of reactive oxygen species and TNF-alpha release as observed after treatment with DQ12. In conclusion, our results indicate a major role of FcgammaII-R and its downstream signaling cascade in the phagocytosis of quartz particles in AM as well as in the associated generation and release of inflammatory mediators.     

Lateralization of hippocampal nitric oxide mediator system in people with Alzheimer disease, multi-infarct dementia and schizophrenia

There is evidence that brain lateralization underlying hemispheric specialization can be observed also at biochemical level. However, hemispheric differences in nitric oxide mediator system have not yet been evaluated. The hippocampus and planum temporale are highly asymmetrical regions but the degree of their laterality is altered in demented or psychotic people. In the study, l-glutamate/l-arginine/l-citrulline concentrations, nitric oxide synthase activities/expressions and nitrites/nitrates levels were estimated in autoptic hippocampi. Right/left laterality in endothelial synthase activity and in nitrites/nitrates was observed in controls. Lateral changes were estimated in patients with Alzheimer disease (a marked increase in activities of constitutive synthases and in expression of inducible enzyme in the left side) and schizophrenia (an increase in activities of all enzymes especially in the right side). Significant shifts from positive to negative correlations were found between laterality of some components of nitric oxide pathway and of planum temporale volumetry under pathological conditions. The hippocampal nitric oxide system appears to be globally right/left lateralized, especially via actions of highly asymmetrical endothelial synthase. The results suggest a specific involvement of all synthases in the development of selected diseases and show that lateral analyses are of sufficient sensitivity to reveal subtle links. The volumetric asymmetry of the planum temporale as a marker of handedness is not probably simply linked to brain laterality at biochemical level but reflects alterations due to pathological processes.     

Type I IFN-induced, NKT cell-mediated negative control of CD8 T cell priming by dendritic cells

We investigated the negative effect of type I IFN (IFN-I) on the priming of specific CD8 T cell immunity. Priming of murine CD8 T cells is down-modulated if Ag is codelivered with IFN-I-inducing polyinosinic:polycytidylic acid (pI/C) that induces (NK cell- and T/B cell-independent) acute changes in the composition and surface phenotype of dendritic cells (DC). In wild-type but not IFN-I receptor-deficient mice, pI/C reduces the plasmacytoid DC but expands the CD8(+) conventional DC (cDC) population and up-regulates surface expression of activation-associated (CD69, BST2), MHC (class I/II), costimulator (CD40, CD80/CD86), and coinhibitor (PD-L1/L2) molecules by cDC. Naive T cells are efficiently primed in vitro by IFN-I-stimulated CD8 cDC (the key APC involved in CD8 T cell priming) although these DC produced less IL-12 p40 and IL-6. pI/C (IFN-I)-mediated down modulation of CD8 T cell priming in vivo was not observed in NKT cell-deficient CD1d(-/-) mice. CD8 cDC from pI/C-treated mice inefficiently stimulated IFN-gamma, IL-4, and IL-2 responses of NKT cells. In vitro, CD8 cDC that had activated NKT cells in the presence of IFN-I primed CD8 T cells that produced less IFN-gamma but more IL-10. The described immunosuppressive effect of IFN-I thus involves an NKT cell-mediated change in the phenotype of CD8 cDC that favors priming of IL-10-producing CD8 T cells. In the presence of IFN-I, NKT cells hence impair the competence of CD8 cDC to prime proinflammatory CD8 T cell responses.