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A High-Throughput Functional Complementation Assay for Classification of BRCA1 Missense Variants
(2013)
Peter Bouwman
; Hanneke van der Gulden
; Ingrid van der Heijden
; Rinske Drost
; Christiaan N. Klijn
; Pramudita Prasetyanti
; Mark Pieterse
; Ellen Wientjens
; Jost Seibler
; Frank B. L. Hogervorst
; Jos Jonkers
Ewa Malgorzata Michalak
; Karim Nacerddine
; Alexandra Pietersen
; Vincent Beuger
; Inka Pawlitzky
; Paulien Cornelissen-Steijger
; Ellen Wientjens
; Ellen Tanger
; Jost Seibler
; Maarten van Lohuizen
; Jos Jonkers
Christoph A. Gebeshuber
; Christoph Kornauth
; Lihua Dong
; Ralph Sierig
; Jost Seibler
; Martina Reiss
; Stefanie Tauber
; Martin Bilban
; Shijun Wang
; Renate Kain
; Georg A. Böhmig
; Marcus J. Moeller
; Hermann-Josef Gröne
; Christoph Englert
; Javier Martinez
; Dontscho Kerjaschki
Obesity-induced overexpression of miR-802 impairs glucose metabolism through silencing of Hnf1b
(2013)
Jan-Wilhelm Kornfeld
; Catherina Baitzel
; A. Christine Könner
; Hayley T. Nicholls
; Merly C. Vogt
; Karolin Herrmanns
; Ludger Scheja
; Cécile Haumaitre
; Anna M. Wolf
; Uwe Knippschild
; Jost Seibler
; Silvia Cereghini
; Joerg Heeren
; Markus Stoffel
; Jens C. Brüning
Sandra Wiegand
; Birgit Voigt
; Dirk Albrecht
; Johannes Bongaerts
; Stefan Evers
; Michael Hecker
; Rolf Daniel
; Heiko Liesegang
Commercial materials with polyvinylpolypyrrolidone and polymeric amberlites (XAD7HP, XAD16) are commonly used for the adsorptive downstream processing of polyphenols from renewable resources. In this study, beta-zeolite-based adsorbent systems were examined, and their properties were compared to organic resins. Batch adsorption experiments were conducted with synthetic solutions of major polyphenols. Adsorption isotherms and desorption characteristics of individual adsorbent were determined based on these results. Maximum adsorption capacities were calculated using the Langmuir model. For example, the zeolites had capacities up to 203.2 mg/g for ferulic acid. To extend these results to a complex system, additional experiments were performed on rapeseed meal and wheat seed extracts as representative renewable resources. HPLC analysis showed that with 7.5% w/v, which is regarded as the optimum amount of zeolites, zeolites A and B could bind 100% of the major polyphenols as well as release polyphenols at high yields. Additionally, regeneration experiments were performed with isopropyl alcohol at 99°C to evaluate how zeolites regenerate under mild conditions. The results showed only a negligible loss of adsorption capacity and no loss of desorption capacity. In summary, it was concluded that beta-zeolites were promising adsorbents for developing new processes to isolate polyphenols from renewable resources.
Biotechnological downstream processing is usually an elaborate procedure, requiring a multitude of unit operations to isolate the target component. Besides the disadvantageous space-time yield, the risks of cross-contaminations and product loss grow fast with the complexity of the isolation procedure. A significant reduction of unit operations can be achieved by application of magnetic particles, especially if these are functionalized with affinity ligands. As magnetic susceptible materials are highly uncommon in biotechnological processes, target binding and selective separation of such particles from fermentation or reactions broths can be done in a single step. Since the magnetizable particles can be produced from iron salts and low priced polymers, a single-use implementation of these systems is highly conceivable. In this article, the principles of magnetizable particles, their synthesis and functionalization are explained. Furthermore, applications in the area of reaction engineering, microfluidics and downstream processing are discussed focusing on established single-use technologies and development potential.
The invention relates to a system for the implementation of chemical, biological or physical reactions, consisting of - one or more magnetic micro-reactors, each comprising a shell made of hydrophobic magnetic nanoparticles encapsulating an aqueous core, - a plane platform comprising a surface to receive the micro-reactors, - a source that generates a magnetic field above or underneath the platform for manipulating the one or more hydrophobic magnetic micro-reactors, or for moving them along the surface of the platform from one position to another position, characterized in that the aqueous core of the one or more magnetic micro-reactors contains a reaction solution or buffer, and wherein the magnetic field generated by the source correlates to a defined position on the surface of the platform.
Paul A. Pellegrini
; Nicholas R. Howell
; Rachael K. Shepherd
; Nigel A. Lengkeek
; Elisabeth Paulßen
; Andrew G. Katsifis
; Ivan Greguric
Elisabeth Paulßen
; Van So Le
; Nigel Lengkeek
; Paul Pellegrini
; Tim Jackson
; Ivan Greguric
; Ron Weiner
Christina Huck
; Johannes Schiffels
; Cony N. Herrera
; Maximilian Schelden
; Thorsten Selmer
; Arshak Poghossian
; Marcus Baumann
; Patrick Wagner
; Michael Josef Schöning
Living cells are complex biological systems transforming metabolites taken up from the surrounding medium. Monitoring the responses of such cells to certain substrate concentrations is a challenging task and offers possibilities to gain insight into the vitality of a community influenced by the growth environment. Cell-based sensors represent a promising platform for monitoring the metabolic activity and thus, the “welfare” of relevant organisms. In the present study, metabolic responses of the model bacterium Escherichia coli in suspension, layered onto a capacitive field-effect structure, were examined to pulses of glucose in the concentration range between 0.05 and 2 mM. It was found that acidification of the surrounding medium takes place immediately after glucose addition and follows Michaelis–Menten kinetic behavior as a function of the glucose concentration. In future, the presented setup can, therefore, be used to study substrate specificities on the enzymatic level and may as well be used to perform investigations of more complex metabolic responses. Conclusions and perspectives highlighting this system are discussed.
Matthias Bäcker
; D. Rakowski
; Arshak Poghossian
; Manfred Biselli
; Patrick Wagner
; Michael Josef Schöning
A microfluidic chip integrating amperometric enzyme sensors for the detection of glucose, glutamate and glutamine in cell-culture fermentation processes has been developed. The enzymes glucose oxidase, glutamate oxidase and glutaminase were immobilized by means of cross-linking with glutaraldehyde on platinum thin-film electrodes integrated within a microfluidic channel. The biosensor chip was coupled to a flow-injection analysis system for electrochemical characterization of the sensors. The sensors have been characterized in terms of sensitivity, linear working range and detection limit. The sensitivity evaluated from the respective peak areas was 1.47, 3.68 and 0.28 μAs/mM for the glucose, glutamate and glutamine sensor, respectively. The calibration curves were linear up to a concentration of 20 mM glucose and glutamine and up to 10 mM for glutamate. The lower detection limit amounted to be 0.05 mM for the glucose and glutamate sensor, respectively, and 0.1 mM for the glutamine sensor. Experiments in cell-culture medium have demonstrated a good correlation between the glutamate, glutamine and glucose concentrations measured with the chip-based biosensors in a differential-mode and the commercially available instrumentation. The obtained results demonstrate the feasibility of the realized microfluidic biosensor chip for monitoring of bioprocesses.
Biopharmaceuticals such as antibodies are produced in cultivated mammalian cells, which must be monitored to comply with good manufacturing practice. We, therefore, developed a fully automated system comprising a specific exhaust gas analyzer, inline analytics and a corresponding algorithm to precisely determine the oxygen uptake rate, carbon dioxide evolution rate, carbon dioxide transfer rate, transfer quotient and respiratory quotient without interrupting the ongoing cultivation, in order to assess its reproducibility. The system was verified using chemical simulation experiments and was able to measure the respiratory activity of hybridoma cells and DG44 cells (derived from Chinese hamster ovary cells) with satisfactory results at a minimum viable cell density of ~2.0 × 10⁵ cells ml⁻¹. The system was suitable for both batch and fed-batch cultivations in bubble-aerated and membrane-aerated reactors, with and without the control of pH and dissolved oxygen.
Poly(vinyl acetate), PVAc, with a degree of polymerization Xn = 10 was prepared by chain-transfer radical polymerization using carbon tetrachloride and used as oligomeric plasticizer for commercial PVAc. However, the chlorinated chain ends cause a low thermal stability requiring mild Cl/H substitution. The product exhibits high thermal stability and excellent melt-compounding properties. Blends of oligomeric and commercial PVAc show single glass transition temperatures which decrease with higher oligomer content and exhibit small negative deviations from Fox' linear additivity rule. This indicates plasticization and miscibility being mainly due to entropic effects. Injection-moulded thick specimens show ductile behaviour at oligomer contents >10 wt %, while sheets with a thickness of 0.2–0.5 mm appear flexible already at 7.5 wt %. The oxygen permeability coefficients are an order of magnitude lower than those of low-density polyethylene. Due to the sum of their properties, the plasticized sheets present a promising alternative in the preparation of barrier materials.