Etablierung eines Modellsystems zum Nachweis eines allergenen Potenzials in transgenen Pflanzen

Ziel dieser Arbeit war es, ein Modellsystem zur Untersuchung des allergenen Potenzials gentechnisch modifizierter Pflanzen (GMP) im Vergleich mit ihren nahen isogenen Varianten (NIV) zu entwickeln. Für die Etablierung eines auf In-silico-, In-vitro- und In-vivo-Methoden basierenden Entscheidungsbaumes wurden zwei nahe isogene und acht transgene Varianten der Kulturpflanze Kartoffel herangezogen. Auf Grundlage verschiedener Richtlinien der IFBC/ILSI, FAO/WHO und der EFSA zur allergologischen Risikoanalyse von GMP konnte eine Standardverfahrensweise zur Prüfung und experimentellen Testung der potenziellen Allergenität von GMP vor ihrer Verwendung durch den Verbraucher entwickelt werden

An endoplasmic reticulum (ER)-directed fusion protein comprising a bacterial subtilisin domain and the human cytokine interleukin 6 is efficiently cleaved in planta

A major limitation of plant bioreactors is the lack of suitable and cost-effective purification methods for the extraction of pharmaceutical-grade proteins. In contrast to that, there are numerous established purification systems for heterologous proteins, expressed in Escherichia coli, which are used for the commercial production of therapeutic proteins. Therefore, we wanted to adapt the BioRad Profinity eXact<sup>TM</sup> one-step protein purification system (originally designed for microbial expression platforms) to purify recombinant proteins in crude plant extracts. This system based on the prodomain of microbial subtilase as fusion partner and a column-bound subtilisin protease. The engineered protease captures and cleaves the fusion protein, retaining the tag and releasing the native protein into the eluate. The subtilase tag was fused to human interleukin 6 (IL6) and transiently expressed in Nicotiana benthamiana leaves using the MagnICON system. The fusion protein was expressed at lower levels than native IL6, suggesting it is expressed less efficiently and/or has a lower stability. However, free IL6 was also detected in the extract and was unaffected by the addition of protease inhibitors during extraction, suggesting that the fusion protein is cleaved in planta by endogenous proteases. Purification of the recombinant protein using the Profinity eXact<sup>TM</sup> system reduced the yield still further. The inefficient production of tagged IL6, coupled with the extensive losses during purification, indicate that the Profinity eXact<sup>TM</sup> system is not suitable for the extraction of IL6 from crude plant extracts.Keywords: Tobacco, transient expression, endoplasmic reticulum, Profinity protein purification, partial cleavageAfrican Journal of Biotechnology Vol. 12(3), pp. 311-31

BioOK – a Comprehensive System for Analysis and Risk Assessment of Genetically Modified Plants

Gentechnisch veränderte (GV) Pflanzen müssen im Rahmen des Zulassungsverfahrens in der EU auf ihre potentiellen Auswirkungen auf die Umwelt und die mensch­liche oder tierische Gesundheit analysiert werden. Der gegenwärtige Zulassungsprozess ist ein Konglo­merat verschiedenster Analysemethoden und extrem zeit- und kostenaufwendig. Das Anliegen von BioOK als ein multidisziplinäres wissenschaftliches Netzwerk ist die Entwicklung von maßgeschneiderten Ansätzen zur Risikoanalyse von GV Pflanzen auf der Grundlage von Ursache-Wirkungs­hypothesen mit dem Ziel des Aufbaus eines effektiven und qualifizierten Risikobewertungssystems. Die Forschungsaktivitäten von BioOK zielen auf einen Paradigmenwechsel im aktuellen Zulassungsprozess. Sie basieren auf einem modularen System, das alle Aspekte des Risikomanagements umfasst: molekulare Charakterisierung, Inhaltsstoffanalyse, agronomische Eigenschaften, Ziel- und Nichtzielorganismen, Boden und Mikroorganismen, Toxikologie, Allergenität und Überwachung nach Markt­einführung, wobei jeder Modul unterschiedliche Analysemethoden beinhaltet. Die durch BioOK angestrebte Reform des Risikobewertungsprozesses von GV Pflanzen umfasst zwei Phasen: zunächst die Optimierung der Analysemethoden selbst und dann die Etablierung eines Entscheidungsunterstützungssystems (Test Decision System – DSS), basierend auf biologischen Schwankungsbreiten (baselines), Zeigermerkmalen (indicators) und Grenzwerten (thresholds) für jede Analysemethode. BioOK hat in einer ersten Entwicklungsphase bereits optimierte Testmethoden entwickelt: Für die Inhaltsstoffanalyse wurde die Untersuchung auf substantielle Äquivalenz durch GC-MS, LC-MS und HPLC/RI Methoden vereinfacht. Ein neu eingeführtes Analyseschema zur Ermittlung potentieller Effekte von GV Pflanzen auf den Boden kombiniert ein in vitro System zur Beprobung von Rhizodepositaten von Pflanzen, die unter kontrollierten Umweltbedingen gewachsen sind, sowie die entsprechenden Bodentypen und deren Charakterisierung mit offenen und hochsensitiven molekular-chemischen Screening und Fingerprinting-Methoden. Ein neues in vitro System zur Simulation des Transports von Substanzen aus dem Darm ins Blut, das das Risiko der Aufnahme durch Mensch oder Tier zu einem frühen Zeitpunkt misst, wurde entwickelt. Um die Effektivität und Reproduzierbarkeit von Probenahmen an der Pflanze zu erhöhen, wird ein genau definiertes Probenahmeschema entwickelt. Schließlich, in Ergänzung der aktuellen Methodik zur Allgemeinen Überwachung (General Surveillance) von GV Pflanzen im Anbau, wurde eine Herangehensweise zur Abschätzung der Notwendigkeit für ein europaweites fallspezifisches (Case Specific) Monitoring beruhend auf Ursache-Wirkungsszenarien, erarbeitet. Die zweite Phase der BioOK F&E-Arbeiten konzentriert sich auf die Entwicklung eines Entscheidungsunterstützungssystems (Decision Support System, DSS). Dazu wird ein computergestütztes System implementiert, in dem alle standardisierten und validierten Methoden zu einem Entscheidungsbaum mit Knotenpunkten, definiert über biologische Schwankungsbreiten und potentielle Risiken definierenden Grenzwerten für Zeigermerkmale, zusammengeführt sind.    Genetically modified (GM) plants have to be analyzed for their potential impacts on the environment and on human or animal health before authorisation by the EU. The approval process currently refers to a conglomeration of diverse analytical methods and is intensive in time and costs. The intention of BioOK as a multidisciplinary scientific network is the development of tailor-made approaches for GM plants based on a cause-effect hypothesis to obtain an effective and qualified risk assessment system. The research activity of BioOK aims to renew the current approval process. It is based on a modular system covering all aspects of risk assessment: molecular characterisation, compound analysis, agronomic traits, target and non-target organisms, soil and micro organisms, toxicology, allergenicity and post-market monitoring, each module containing several test methods. The renewal of the risk assessment procedure intended by BioOK consists of two phases: first the optimization of test methods and second the establishment of a decision support system (DSS) based on baselines, indicators and thresholds developed for each of the methods. Optimized test methods have been developed mainly during the first phase: For compound analysis methods have been developed to ease the analysis of substantial equivalence of the events by GC-MS, LC-MS and HPLC/RI. A newly introduced testing scheme for the detection of potential effects of GM plants on soil combines an in-vitro system to collect rhizodeposits from plants grown under controlled environmental conditions and the correspon­ding bulk soil, and their characterisation by untargeted and highly sensitive molecular-chemical screening and fingerprinting technique. A novel in vitro system simula­ting the transport of substances from the gut into the blood that detects the risk of incorporation in human or animal at an early time point was developed. In order to increase the effectiveness and reproducibility of the sampling procedure we developed a valid defined sampling scheme. Finally, complementing the actual General Surveillance methodology, an approach for a Europe-wide case specific monitoring referring to cause-effect sce­narios was developed. The second phase concentrates on the development of a Decision Support System (DSS). A computer-based system will implement and merge all standardized methods in a decision tree system following decision rules defined by baseline and thresholds for indicators.   &nbsp

Long-term follow-up of IPEX syndrome patients after different therapeutic strategies : an international multicenter retrospective study

Background: Immunodysregulation polyendocrinopathy enteropathy x-linked(IPEX) syndrome is a monogenic autoimmune disease caused by FOXP3 mutations. Because it is a rare disease, the natural history and response to treatments, including allogeneic hematopoietic stem cell transplantation (HSCT) and immunosuppression (IS), have not been thoroughly examined. Objective: This analysis sought to evaluate disease onset, progression, and long-term outcome of the 2 main treatments in long-term IPEX survivors. Methods: Clinical histories of 96 patients with a genetically proven IPEX syndrome were collected from 38 institutions worldwide and retrospectively analyzed. To investigate possible factors suitable to predict the outcome, an organ involvement (OI) scoring system was developed. Results: We confirm neonatal onset with enteropathy, type 1 diabetes, and eczema. In addition, we found less common manifestations in delayed onset patients or during disease evolution. There is no correlation between the site of mutation and the disease course or outcome, and the same genotype can present with variable phenotypes. HSCT patients (n = 58) had a median follow-up of 2.7 years (range, 1 week-15 years). Patients receiving chronic IS (n 5 34) had a median follow-up of 4 years (range, 2 months-25 years). The overall survival after HSCT was 73.2% (95% CI, 59.4-83.0) and after IS was 65.1% (95% CI, 62.8-95.8). The pretreatment OI score was the only significant predictor of overall survival after transplant (P = .035) but not under IS. Conclusions: Patients receiving chronic IS were hampered by disease recurrence or complications, impacting long-term.disease-free survival. When performed in patients with a low OI score, HSCT resulted in disease resolution with better quality of life, independent of age, donor source, or conditioning regimen

Nature of High- and Low-Affinity Metal Surface Sites on Birnessite Nanosheets

International audienceBirnessite nanosheets (δ-MnO2) are key reactive nanoparticles that regulate metal cycling in terrestrial and marine settings, yet there is no molecular explanation for the sorption selectivity of metals which controls their enrichment. This fundamental question was addressed by optimizing the structure of Ni, Cu, Zn, and Pb surface complexes on δ-MnO2 and by calculating the Gibbs free-energy change (ΔG) of the sorption reactions with density functional theory. The sorption selectivity follows the order Pb > Cu > Ni > Zn in good agreement with experimental data. Cu, Ni, and Zn bind preferentially to layer edges at low surface coverage forming double-edge-sharing (DES) complexes, whereas Pb binds extensively with high selectivity over the three transition metals to both layer edges (DES bonding) and to basal planes forming triple-corner-sharing (TCS) complexes. Pb has a similar affinity for the DES and TCS sites at pH 5 and a higher affinity for the TCS sites at circumneutral pH. The Pb DES and TCS complexes are both dehydrated at the δ-MnO2–water interface and feature a trigonal pyramidal geometry with three surface O atoms. The high stability of the two new Pb complexes arises from the hybridization between the Pb 6s/6p and O 2p states, forming a strong covalent Pb–O/OH bond at the δ-MnO2 surface. The quantum chemical results provide a mechanistic and energetics insight into the metal uptake on δ-MnO2 that extends what extended X-ray absorption fine structure (EXAFS) spectroscopy alone can provide

Density Functional Theory Modeling of the Oxidation Mechanism of Co(II) by Birnessite

International audiencePhyllomanganates of the birnessite family are the most abundant manganese oxides on Earth and the strongest inorganic oxidants in the environment. Birnessite controls the oxidative scavenging of cobalt in soils, lake and marine sediments, and ferromanganese crusts and nodules, leading to enrichments of the order of one billion times the concentration in solution. However, a detailed mechanistic understanding of the enrichment processes is lacking. Here, we perform density functional theory (DFT) calculations to explore the mechanisms of Co(II) to Co(III) oxidation on the layer edge and surface of birnessite nanoparticles. We show that Co(II) sorption on a layer edge is an unlikely oxidation pathway. In contrast, Co(II) sorbed on a Mn(IV) vacancy site exposed on the layer surface as an octahedral triple-corner sharing (TCS) complex enters the vacancy where it is oxidized to Co(III) by a layer Mn(IV) cation, which is reduced to Mn(III). The stepwise reaction proceeds as follows. The octahedral TCS complex is transformed to a smaller tetrahedral TCS complex, allowing Co(II) to cross the surface oxygen layer and to fill the empty octahedral Mn(IV) site. When in the octahedral vacancy, Co(II) is converted from the high-spin (t2g 5 eg 2) to the low-spin (t2g 6 eg 1) state and the Co(II) octahedron becomes strongly distorted by the Jahn-Teller effect. Afterward, the electron exchange reaction between Mn(IV) (t2g 3 eg 0) and Co(II) (t2g 6 eg 1) takes place, resulting in the formation of a regular lowspin Co(III) (t2g 6 eg 0) octahedron and a Jahn-Teller distorted high-spin Mn(III) (t2g 3 eg 1) octahedron. These findings refine previously proposed mechanisms of Co(II) oxidation by birnessite and fill gaps in our understanding of global Co sequestration in natural systems

Density Functional Theory Modeling of the Oxidation Mechanism of Tl(I) by Birnessite

This study establishes a theoretical foundation for the oxidation pathway of monovalent thallium (Tl(I)) to trivalent thallium (Tl(III)) on birnessite, which is responsible for the over million times enrichment of Tl in marine ferromanganese deposits over seawater concentration. Tl(I) oxidation occurs on vacant Mn(IV) sites located on basal planes of the birnessite layers and on the edge sites, in agreement with experiment. Two Mn(IV) atoms are reduced to Mn(III) when Tl(I) gives up two electrons in two one-electron steps with formation of an intermediate Tl(II) inner-sphere complex. Tl(I) oxidation is facilitated at pH > 4-5 by the partial hydrolysis of the Tl(III) inner-sphere product on the reactive basal and edge sites. Oxidation by O2 is thermodynamically unfavorable. Although density functional theory has predictive power for intermediate Tl(II) complex, it would be difficult to characterize as Tl(II) is highly reactive, and therefore probably short-lived. These findings provide the first atomic-scale description of the oxidation of Tl(I) by a manganese oxide and fill gaps in our understanding of global thallium sequestration in natural systems

Size and curvature regulate pattern selection in the mammalian brain

Mammalian brains display a wide variety of shapes and surface morphologies. Their characteristically folded surface is closely correlated to neuronal activity and serves as a clinical indicator for physiological and pathological conditions. Yet, the regulators of pattern formation in evolution and development remain poorly understood. Here we show how brain size and curvature affect the folding pattern in the developing mammalian brain. We model cortical folding as the instability problem of a bilayered system subjected to growth-induced compression. Using analytical estimates and continuum models for finite growth, we systematically explore the effects of geometric factors on the evolution of surface shape. We demonstrate that extrinsic geometric features–including brain size, cortical thickness, and cortical curvature–tightly regulate pattern selection: The mammalian brain is extremely soft and even small environmental changes can create extremely large alterations in its surface morphology. Our simulations explain why gyrification increases with brain size and why longer brains tend to fold more longitudinally than radially. Our results suggest that brain folding is driven, at least in part, by extreme mechanics, rather than by phylogeny alone

Allergological and toxicological risk assessment of genetically modified (GM) plants - Development of a model system

Technical advances in biotechnology lead to a rising number of genetically modified plants and derived food and feed. Therefore, the international interest in adequate and standardized analytical methods increases to assess health risks in a rapid and low cost manner.
To address this issue a decision support system (DSS), which will be based on baseline data and thresholds of key parameters, is going to be established in the framework of a joint project (BioOK). Determination of the potential allergenicity or toxicity of novel proteins caused by the genetic modification compared to near isogenic variants (NIV) is a key issue in this context. For the development of DSS the model plant “potato” was used to compare values of relevant parameters of GM plants with those of NIV using different allergological and toxicological methods which are shown in the present poster. No significant differences in comparison of the results of GM plants and NIV were found so far.
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