The bifunctional dihydrofolate reductase thymidylate synthase of Tetrahymena thermophila provides a tool for molecular and biotechnology applications

BACKGROUND: Dihydrofolate reductase (DHFR) and thymidylate synthase (TS) are crucial enzymes in DNA synthesis. In alveolata both enzymes are expressed as one bifunctional enzyme. RESULTS: Loss of this essential enzyme activities after successful allelic assortment of knock out alleles yields an auxotrophic marker in ciliates. Here the cloning, characterisation and functional analysis of Tetrahymena thermophila's DHFR-TS is presented. A first aspect of the presented work relates to destruction of DHFR-TS enzyme function in an alveolate thereby causing an auxotrophy for thymidine. A second aspect is to knock in an expression cassette encoding for a foreign gene with subsequent expression of the target protein. CONCLUSION: This system avoids the use of antibiotics or other drugs and therefore is of high interest for biotechnological applications

Biochemical and molecular characterisation of Tetrahymena thermophila extracellular cysteine proteases

BACKGROUND: Over the last decades molecular biologic techniques have been developed to alter the genome and proteome of Tetrahymena thermophila thereby providing the basis for recombinant protein expression including functional human enzymes. The biotechnological potential of Tetrahymena has been proved in numerous publications, demonstrating fast growth, high biomass, fermentation in ordinary bacterial/yeast equipment, up-scalability, existence of cheap and chemical defined media. For these reasons Tetrahymena offers promising opportunities for the development of a high expression system. Yet optimised high yield strains with protease deficiency such as commonly used in yeast and bacterial systems are not available. RESULTS: This work presents the molecular identification of predominant proteases secreted into the medium by Tetrahymena thermophila. A one-step purification of the proteolytic enzymes is described. CONCLUSION: The information provided will allow silencing of protease activity by either knock out methods or by Tetrahymena specific antisense-ribosome-techniques. This will facilitate the next step in the advancement of this exciting organism for recombinant protein production

Metadata standards in the German funding program "Innovationswettbewerb INVITE". Which standards are used for which purpose? A contribution by INVITE Meta and the Digitalbegleitung (technological monitoring and research) within the framework of the German funding program INVITE

This paper is about the definition and recommendation of metadata standards for the field of continuing vocational education and training. The standards were discussed in a bottom-up approach and selected in several workshops attended by projects from the German funding program “INVITE” on behalf of the BMBF (German Federal Ministry of Education and Research). (DIPF/Orig.)In diesem Paper geht es um eine Darstellung und Empfehlung von Metadatenstandards für den Bereich der beruflichen Weiterbildung. Die Standards wurden bottom-up in mehreren Workshops durch Projekte aus dem Innovationswettbewerb INVITE des BMBF diskutiert und auf dieser Basis ausgewählt

Secretion of functional human enzymes by Tetrahymena thermophila

BACKGROUND: The non-pathogenic ciliate Tetrahymena thermophila is one of the best-characterized unicellular eucaryotes used in various research fields. Previous work has shown that this unicellular organism provides many biological features to become a high-quality expression system, like multiplying to high cell densities with short generation times in bioreactors. In addition, the expression of surface antigens from the malaria parasite Plasmodium falciparum and the ciliate Ichthyophthirius multifiliis suggests that T. thermophila might play an important role in vaccine development. However, the expression of functional mammalian or human enzymes remains so far to be seen. RESULTS: We have been able to express a human enzyme in T. thermophila using expression modules that encode a fusion protein consisting of the endogenous phospholipase A(1 )precursor and mature human DNaseI. The recombinant human enzyme is active, indicating that also disulfide bridges are correctly formed. Furthermore, a detailed N-glycan structure of the recombinant enzyme is presented, illustrating a very consistent glycosylation pattern. CONCLUSION: The ciliate expression system has the potential to become an excellent expression system. However, additional optimisation steps including host strain improvement as wells as measures to increase the yield of expression are necessary to be able to provide an alternative to the common E. coli and yeast-based systems as well as to transformed mammalian cell lines

Metadatenstandards im Innovationswettbewerb INVITE. Welche Standards werden für welchen Zweck eingesetzt? Ein Beitrag von INVITE Meta und der Digitalbegleitung im Rahmen des Innovationswettbewerbs INVITE

In diesem Paper geht es um eine Darstellung und Empfehlung von Metadatenstandards für den Bereich der beruflichen Weiterbildung. Die Standards wurden bottom-up in mehreren Workshops durch Projekte aus dem Innovationswettbewerb INVITE des BMBF diskutiert und auf dieser Basis ausgewählt. (DIPF/Orig.

Appropriateness to set a group health based guidance value for T2 and HT2 toxin and its modified forms

Acknowledgements: The Panel wishes to thank the members of the Working Group on HBGV for mycotoxins and their modified forms: Jan Alexander, Chiara Dall'Asta, Arno Gutleb, Manfred Metzler, Isabelle Oswald and Dominique Parent-Massin for the preparatory work on this scientific opinion, and the EFSA staff members: Marco Binaglia and Hans Steinkellner for the support provided to this scientific opinion.Peer reviewedPublisher PD

Appropriateness to set a group health based guidance value for nivalenol and its modified forms

The Panel wishes to thank the members of the Working Group on HBGV for mycotoxins and their modified forms: Jan Alexander, Chiara Dall'Asta, Arno Gutleb, Manfred Metzler, Isabelle P Oswald and Dominique Parent‐Massin for the preparatory work on this scientific output, and the EFSA staff members: Marco Binaglia and Hans Steinkellner for the support provided to this scientific output. Adopted: 15 March 2017Peer reviewedPublisher PD

Differential cross section measurements for the production of a W boson in association with jets in proton–proton collisions at √s = 7 TeV

Measurements are reported of differential cross sections for the production of a W boson, which decays into a muon and a neutrino, in association with jets, as a function of several variables, including the transverse momenta (pT) and pseudorapidities of the four leading jets, the scalar sum of jet transverse momenta (HT), and the difference in azimuthal angle between the directions of each jet and the muon. The data sample of pp collisions at a centre-of-mass energy of 7 TeV was collected with the CMS detector at the LHC and corresponds to an integrated luminosity of 5.0 fb[superscript −1]. The measured cross sections are compared to predictions from Monte Carlo generators, MadGraph + pythia and sherpa, and to next-to-leading-order calculations from BlackHat + sherpa. The differential cross sections are found to be in agreement with the predictions, apart from the pT distributions of the leading jets at high pT values, the distributions of the HT at high-HT and low jet multiplicity, and the distribution of the difference in azimuthal angle between the leading jet and the muon at low values.United States. Dept. of EnergyNational Science Foundation (U.S.)Alfred P. Sloan Foundatio