Feasibility studies for the measurement of time-like proton electromagnetic form factors from p¯ p→ μ+μ- at P ¯ ANDA at FAIR

This paper reports on Monte Carlo simulation results for future measurements of the moduli of time-like proton electromagnetic form factors, | GE| and | GM| , using the p¯ p→ μ+μ- reaction at P ¯ ANDA (FAIR). The electromagnetic form factors are fundamental quantities parameterizing the electric and magnetic structure of hadrons. This work estimates the statistical and total accuracy with which the form factors can be measured at P ¯ ANDA , using an analysis of simulated data within the PandaRoot software framework. The most crucial background channel is p¯ p→ π+π-, due to the very similar behavior of muons and pions in the detector. The suppression factors are evaluated for this and all other relevant background channels at different values of antiproton beam momentum. The signal/background separation is based on a multivariate analysis, using the Boosted Decision Trees method. An expected background subtraction is included in this study, based on realistic angular distributions of the background contribution. Systematic uncertainties are considered and the relative total uncertainties of the form factor measurements are presented

Cell-free systems based on CHO cell lysates: Optimization strategies, synthesis of "difficult-to-express" proteins and future perspectives

Nowadays, biotechnological processes play a pivotal role in target protein production. In this context, Chinese Hamster Ovary (CHO) cells are one of the most prominent cell lines for the expression of recombinant proteins and revealed as a safe host for nearly 40 years. Nevertheless, the major bottleneck of common in vivo protein expression platforms becomes obvious when looking at the production of so called “difficult-to-express” proteins. This class of proteins comprises in particular several ion channels and multipass membrane proteins as well as cytotoxic proteins. To enhance the production of “difficult-to-express” proteins, alternative technologies were developed, mainly based on translationally active cell lysates. These so called “cell-free” protein synthesis systems enable an efficient production of different classes of proteins. Eukaryotic cell-free systems harboring endogenous microsomal structures for the synthesis of functional membrane proteins and posttranslationally modified proteins are of particular interest for future applications. Therefore, we present current developments in cell-free protein synthesis based on translationally active CHO cell extracts, underlining the high potential of this platform. We present novel results highlighting the optimization of protein yields, the synthesis of various “difficult-to-express” proteins and the cotranslational incorporation of non-standard amino acids, which was exemplarily demonstrated by residue specific labeling of the glycoprotein Erythropoietin and the multimeric membrane protein KCSA

Eukaryotic cell-free systems: A novel platform technology for production and functional characterization of pore-forming toxins

Question: Bacterial infections still cause tremendous health risks worldwide. A variety of pathogenic bacteria in particular causes virulence by pore-forming toxins that insert themselves into membranes and cause cell lysis. Since highly resistant bacterial strains have evolved over the last decades, new methods for the characterization of bacterial toxins are needed in order to develop new and specific diagnostic tools as well as to improve present-day treatment regimens. Methods: In vivo production of toxins is rather difficult as toxins harm the cells" viability. Cell-free protein synthesis has emerged as a fast and cost-efficient method to synthesize and characterize a variety of toxins while using a cell lysate rather than viable cells. Here, we describe the cell-free synthesis and functional characterization of enterotoxins from Bacillus cereus as well as Staphylococcus aureus, Cytolysin from Vibrio vulnificus and Aerolysin from Aeromonas hydrophila.Cell-based toxicity assays, blood agar plates and electrophysiological measurements on planar lipid bilayers were used to determine the toxins" functionality. Results: In our studies, we showed the successful synthesis and characterization of various pore-forming toxins and the ability to use the cell-free system in versatile ways. Conclusion: This platform technology enables a fast and efficient characterization of a variety of toxins as a prerequisite to develop diagnostic methods and treatments

Dual versus triple therapy in eradication of Helicobacter pylori

Background/Aims: Duodenal ulcers should be treated by eradication of Helicobacter pylori. This study compared the efficacy of a proton pump inhibitor together with one or two antibiotics in eradication therapy, Methodology: 177 patients who were H. pylori positive were randomized to receive 14 days of either: lansoprazole 30mg bd and amoxicillin 1g bd (LA), omeprazole 20mg bd and amoxicillin 1g bd (OA) or lansoprazole 30mg bd, amoxicillin 1g bd and clarithromycin 500mg bd (LAC). The efficacy was assessed at four weeks and at six months after the end of treatment. Biopsies were taken for culture and bacterial sensitivity testing at inclusion and at four weeks after the end of treatment. Results: 149 patients were evaluated for efficacy. The eradication rate was significantly higher in LAC (96%) compared to LA (51%) and OA (64%) treatments (P<0.001). At baseline 17%, 21% and 19% of the patients in the LA, OA and LAC groups, respectively, were resistant to metronidazole and only one patient was resistant to clarithromycin. Post-treatment, four patients had acquired metronidazole resistance. Conclusions: LAC is more effective than LA and CA for eradication of H. pylori in duodenal ulcer disease

Versatile cell-free protein synthesis systems based on chinese hamster ovary cells

We present an alternative production platform for the synthesis of complex proteins. Apart from conventionally applied protein production using engineered mammalian cell lines, this protocol describes the preparation and principle of cell-free protein synthesis systems based on CHO cell lysates. The CHO cell-free system contains endogenous microsomes derived from the endoplasmic reticulum, which enables a direct integration of membrane proteins into a nature like milieu and the introduction of posttranslational modifications. Different steps of system development are described including the cultivation of CHO cells, cell harvesting and cell disruption to prepare translationally active CHO cell lysates. The requirements for DNA templates and the generation of linear DNA templates suitable for the CHO cell-free reaction is further depicted to underline the opportunity to produce different protein variants in a short period. This experimental setup provides a basis for hig h-throughput applications. The productivity of the CHO cell-free systems is further increased by using a non-canonical translation initiation due to the attachment of an internal ribosomal entry site of the Cricket paralysis virus (CRPV IRES) to the 5´ UTR of the desired gene. In this way, a direct interaction of the IRES structure with the ribosome facilitates a translation factor independent initiation of translation. Cell-free reactions were performed in fast and efficient batch reactions leading to protein yields up to 40 μg/mL. The reaction format was further adjusted to a continuous exchange CHO cell-free reaction (CHO CECF) to prolong reaction time and thereby increase the productivity of the cell-free systems. Finally, protein yields up to 1 g/L were obtained. The CHO CECF system represents a sophisticated resource to address structural and functional aspects of difficult-to-express proteins in fundamental and applied research

Linear DNA templates in cell-free protein synthesis based on CHO cell lysate.

<p>Linear IRES-luciferase and IRES-Mel-EPO templates tested during cell-free protein synthesis reactions in the presence of ¹⁴C leucine. Different concentrations of linear DNA product and T7 RNA polymerase (Pol) were added to the individual reactions. Protein yield was quantified by hot TCA precipitation followed by scintillation measurement. Error bars show standard deviations calculated from triplicates.</p

Quantitative analysis of cell-free protein synthesis: A comparison of different luciferase encoding vector backbones probed in CHO cell lysates.

<p><b>A.</b> Total protein yields were determined by incorporation of radioactive ¹⁴C leucine into <i>de novo</i> synthesized proteins followed by hot TCA precipitation and scintillation measurement. Yields of active luciferase were quantified by standard luciferase assay. <b>B.</b> Western blot analysis of cell-free produced luciferase using primary Anti-Luc antibody (concentration 1:1000) and secondary Anti-rabbit-HRP conjugate antibody (1:2000). Analysis of luciferase bands was performed by using ECL reagent and detection of corresponding light emission. Error bars show standard deviations calculated from triplicate analysis. NTC sample contains translation mixture without synthesized protein.</p

“Difficult-to-express” proteins synthesized in the CHO lysate based cell-free system.

<p>“Difficult-to-express” proteins synthesized in the CHO lysate based cell-free system.</p

Residue specific fluorescence labeling of Mel-EPO and KCSA in cell-free systems based on translationally active CHO cell lysate.

<p>Cell-free protein synthesis based on CHO cell extracts was carried out in the presence of BODIPY-TMR-tRNA(Phe) to allow the fluorescence labeling of <i>de novo</i> synthesized Mel-EPO and KCSA. Produced glycosylated and transmembrane proteins were separated by SDS-PAGE. In-gel fluorescence was detected using a variable mode imager (Typhoon Trio Plus, GE Healthcare).</p

Protein yields using different luciferase encoding plasmids in cell-free synthesis based on CHO cell lysates.

<p>Protein yields using different luciferase encoding plasmids in cell-free synthesis based on CHO cell lysates.</p