Annual Report 2010 - Operation and Utilisation of the High Flux Reactor

The High Flux Reactor (HFR) at Petten is managed by the Institute for Energy (IE) of the EC - DG JRC and operated by NRG who are also licence holder and responsible for commercial activities. The HFR operates at 45 MW and is of the tank-in-pool type, light water cooled and moderated. It is one of the most powerful multi-purpose materials testing reactors in the world and one of the world leaders in target irradiation for the production of medical radioisotopes.JRC.F.4-Nuclear Reactor Integrity Assessment and Knowledge Managemen

Annual Report 2011 Operation and Utilisation of the High Flux Reactor

The High Flux Reactor (HFR) at Petten is managed by the Institute for Energy and Transport (IET) of the EC - DG JRC and operated by NRG who are also licence holder and responsible for commercial activities. The HFR operates at 45 MW and is of the tank-in-pool type, light water cooled and moderated. It is one of the most powerful multi-purpose materials testing reactors in the world and one of the world leaders in target irradiation for the production of medical radioisotopes.JRC.F-Institute for Energy and Transport (Petten

Operation and Utilisation of the High Flux Reactor - Annual Report 2009

The High Flux Reactor (HFR) at Petten is managed by the Institute for Energy (IE) of the EC - DG JRC and operated by NRG who are also licence holder and responsible for commercial activities. The HFR operates at 45 MW and is of the tank-in-pool type, light water cooled and moderated. It is one of the most powerful multi-purpose materials testing reactors in the world and one of the world leaders in target irradiation for the production of medical radioisotopes.JRC.F-Institute for Energy and Transport (Petten

Expression and analysis of the glycosylation properties of recombinant human erythropoietin expressed in Pichia pastoris

The Pichia pastoris expression system was used to produce recombinant human erythropoietin, a protein synthesized by the adult kidney and responsible for the regulation of red blood cell production. The entire recombinant human erythropoietin (rhEPO) gene was constructed using the Splicing by Overlap Extension by PCR (SOE-PCR) technique, cloned and expressed through the secretory pathway of the Pichia expression system. Recombinant erythropoietin was successfully expressed in P. pastoris. The estimated molecular mass of the expressed protein ranged from 32 kDa to 75 kDa, with the variation in size being attributed to the presence of rhEPO glycosylation analogs. A crude functional analysis of the soluble proteins showed that all of the forms were active in vivo

Real-Time Cytotoxicity Assay for Rapid and Sensitive Detection of Ricin from Complex Matrices

BACKGROUND: In the context of a potential bioterrorist attack sensitive and fast detection of functionally active toxins such as ricin from complex matrices is necessary to be able to start timely countermeasures. One of the functional detection methods currently available for ricin is the endpoint cytotoxicity assay, which suffers from a number of technical deficits. METHODOLOGY/FINDINGS: This work describes a novel online cytotoxicity assay for the detection of active ricin and Ricinus communis agglutinin, that is based on a real-time cell electronic sensing system and impedance measurement. Characteristic growth parameters of Vero cells were monitored online and used as standardized viability control. Upon incubation with toxin the cell status and the cytotoxic effect were visualized using a characteristic cell index-time profile. For ricin, tested in concentrations of 0.06 ng/mL or above, a concentration-dependent decrease of cell index correlating with cytotoxicity was recorded between 3.5 h and 60 h. For ricin, sensitive detection was determined after 24 h, with an IC50 of 0.4 ng/mL (for agglutinin, an IC50 of 30 ng/mL was observed). Using functionally blocking antibodies, the specificity for ricin and agglutinin was shown. For detection from complex matrices, ricin was spiked into several food matrices, and an IC50 ranging from 5.6 to 200 ng/mL was observed. Additionally, the assay proved to be useful in detecting active ricin in environmental sample materials, as shown for organic fertilizer containing R. communis material. CONCLUSIONS/SIGNIFICANCE: The cell-electrode impedance measurement provides a sensitive online detection method for biologically active cytotoxins such as ricin. As the cell status is monitored online, the assay can be standardized more efficiently than previous approaches based on endpoint measurement. More importantly, the real-time cytotoxicity assay provides a fast and easy tool to detect active ricin in complex sample matrices

Engineering Yarrowia lipolytica to Produce Glycoproteins Homogeneously Modified with the Universal Man3GlcNAc2 N-Glycan Core

Yarrowia lipolytica is a dimorphic yeast that efficiently secretes various heterologous proteins and is classified as “generally recognized as safe.” Therefore, it is an attractive protein production host. However, yeasts modify glycoproteins with non-human high mannose-type N-glycans. These structures reduce the protein half-life in vivo and can be immunogenic in man. Here, we describe how we genetically engineered N-glycan biosynthesis in Yarrowia lipolytica so that it produces Man3GlcNAc2 structures on its glycoproteins. We obtained unprecedented levels of homogeneity of this glycanstructure. This is the ideal starting point for building human-like sugars. Disruption of the ALG3 gene resulted in modification of proteins mainly with Man5GlcNAc2 and GlcMan5GlcNAc2 glycans, and to a lesser extent with Glc2Man5GlcNAc2 glycans. To avoid underoccupancy of glycosylation sites, we concomitantly overexpressed ALG6. We also explored several approaches to remove the terminal glucose residues, which hamper further humanization of N-glycosylation; overexpression of the heterodimeric Apergillus niger glucosidase II proved to be the most effective approach. Finally, we overexpressed an α-1,2-mannosidase to obtain Man3GlcNAc2 structures, which are substrates for the synthesis of complex-type glycans. The final Yarrowia lipolytica strain produces proteins glycosylated with the trimannosyl core N-glycan (Man3GlcNAc2), which is the common core of all complex-type N-glycans. All these glycans can be constructed on the obtained trimannosyl N-glycan using either in vivo or in vitro modification with the appropriate glycosyltransferases. The results demonstrate the high potential of Yarrowia lipolytica to be developed as an efficient expression system for the production of glycoproteins with humanized glycans

Genome sequencing and analysis of the versatile cell factory Aspergillus niger CBS 513.88

The filamentous fungus Aspergillus niger is widely exploited by the fermentation industry for the production of enzymes and organic acids, particularly citric acid. We sequenced the 33.9-megabase genome of A. niger CBS 513.88, the ancestor of currently used enzyme production strains. A high level of synteny was observed with other aspergilli sequenced. Strong function predictions were made for 6,506 of the 14,165 open reading frames identified. A detailed description of the components of the protein secretion pathway was made and striking differences in the hydrolytic enzyme spectra of aspergilli were observed. A reconstructed metabolic network comprising 1,069 unique reactions illustrates the versatile metabolism of A. niger. Noteworthy is the large number of major facilitator superfamily transporters and fungal zinc binuclear cluster transcription factors, and the presence of putative gene clusters for fumonisin and ochratoxin A synthesis

Human gut Bacteroidetes can utilize yeast mannan through a selfish mechanism

Yeasts, which have been a component of the human diet for at least 7,000 years, possess an elaborate cell wall α-mannan. The influence of yeast mannan on the ecology of the human microbiota is unknown. Here we show that yeast α-mannan is a viable food source for the Gram-negative bacterium Bacteroides thetaiotaomicron, a dominant member of the microbiota. Detailed biochemical analysis and targeted gene disruption studies support a model whereby limited cleavage of α-mannan on the surface generates large oligosaccharides that are subsequently depolymerized to mannose by the action of periplasmic enzymes. Co-culturing studies showed that metabolism of yeast mannan by B. thetaiotaomicron presents a ‘selfish’ model for the catabolism of this difficult to breakdown polysaccharide. Genomic comparison with B. thetaiotaomicron in conjunction with cell culture studies show that a cohort of highly successful members of the microbiota has evolved to consume sterically-restricted yeast glycans, an adaptation that may reflect the incorporation of eukaryotic microorganisms into the human diet

Successful and failed international mediation

Diese Arbeit möchte durch politische Analysen und eine Synthese rechtswissenschaftlicher Bestimmungen eine Einsicht in Gelingensfaktoren internationaler Mediation geben. Die Evaluation der Richtlinie ‘UN Guidance for Effective Mediation’ bezogen auf zwei verschiedene Kosovo-Missionen unter der Leitung von Martti Ahtisaari einerseits und Catherine Ashton andererseits ergibt, dass die Zustimmung und der lokale Besitz entscheidend sind für den Erfolg , sofern sich Gelegenheiten bieten und genutzt werden. Unter Berücksichtigung der dargestellten Einsichten in den kosovarischen Einheitsprozess verdeutlicht diese Arbeit, dass viele – vornehmlich mediatorische – Neuentwicklungen einen dritten Weg aus dem Dilemma zwischen einerseits der ‘staatlichen Souveränität und territorialen Integrität’ und andererseits dem ‘Selbstbestimmungsrecht der Völker’ zeigen, einer “paradigmatischen Verschiebung der rechtswissenschaftlichen Grundlagen des Sicherheitsrates zur Autorisierung von Macht” (Haines 2009, 486, übersetzt). Da aber das Vetorecht diese Verschiebung inkonsequent erscheinen lässt und eine Änderung der UN-Charta sehr unwahrscheinlich ist, empfiehlt diese Arbeit, dass die UN die Leitung übernimmt, die internationale Mediation als eigenständige Profession zu entwickeln, weil “es sein kann, dass die Mediation den einzigen zur Verfügung stehenden Ansatz bietet, mit Konflikten des 21. Jahrhunderts umzugehen.” (Bercovitch 2011, Intro, übersetzt).Through policy analysis and legal synthesis this thesis pursues insight into what makes the difference between success and failure in international mediation. In addition to applying the ‘UN Guidance for Effective Mediation’ to two Kosovo missions, a first one led by Martti Ahtisaari and a second one by Catherine Ashton, comparing and contrasting the findings thereof reveals that ‘consent’ and ‘local ownership’ are decisive for success given opportunities present themselves and are seized. Bringing together prominent insights into Kosovo’s legalistic road to independence, it appears that many, mainly mediatory, innovations to finding a third way out of the legal dilemma between on the one hand ‘Sovereignty and Territorial Integrity of States’, and on the other hand ‘Self-Determination of Peoples’, led to “a paradigm shift in the jurisprudential underpinning in the Security Council’s power to authorize force” (Haines 2009, 486). While veto power has left that shift largely inconsequential and a change to the UN Charter is highly unlikely, this thesis recommends the United Nations to take the lead in fully developing international mediation as a profession in its own right, because “it may well be that mediation is the closest thing we have to an effective technique for dealing with conflicts in the twenty-first century” (Bercovitch 2011, Intro)

“A Giant Leap for Mankind”, but What About Women? The Role of System-Justifying Ideologies in Predicting Attitudes Toward Sexist Language

Sexist language excludes, trivializes or diminishes either gender. Despite efforts by many professional bodies to encourage the use of nonsexist alternatives, sexist language use persists across many languages. Further, research has shown that men are less supportive of nonsexist language alternatives than women, and that this effect is mediated by attitudes toward women. We propose that broader ideologies related to the perceived legitimacy of dominance hierarchies and existing social systems also explain this gender gap. British undergraduate participants completed measures of attitudes toward women, gender-specific system justification, and social dominance orientation. They also completed an inventory of attitudes toward sexist language. There was a strong gender difference in attitudes toward sexist language that was significantly mediated by gender-specific system justification and social dominance orientation. The relationship between gender and attitudes toward sexist language therefore appears to be driven by broader ideologies that serve to keep women “in their place”