Monitoring of Extracellular TCA Cycle Intermediates in Mammalian Cell Culture

In recent years, monitoring of metabolites such as glucose, lactate, ammonia, glutamine, glutamate, and other amino acids has been introduced for many cell cultivations. Assaying intracellular metabolites might reveal further insights into metabolism. However, some of the mainly intracellular or even mitochondrial appearing metabolites can also be found in the medium supernatant at micromolar concentrations. Since no active transport mechanisms for excretion of these intermediates are known for most mammalian production cell lines, they might leak out of the cells and thus could be correlated to intracellular concentrations. In this work, we are investigating extracellular concentrations of five organic acids (succinic, malic, fumaric, citric, isocitric acid) during growth looking at different media and cell lines. Moreover, clear influences on concentrations were also expected to occur after viral infection of MDCK cells. The analysis is performed using an anion-exchange chromatography system (DX-320, Dionex, Idstein, Germany) with a conductivity detector. Due to high protein content of the medium, sample preparation posed some problems. Several strategies have been investigated for an optimal recovery of the abovementioned organic acids. Experiments showed that certain amounts of these organic acids already occurred in fresh medium, coming from both serum and peptone being supplements for the culture medium. Nevertheless, first results indicate significant changes of concentrations during the course of cultivation, probably not correlated to dead cells. In batch cultivation, malic, fumaric and citric acid show an almost linear increase after the lag phase, whereas isocitric and succinic acid seem to reach a constant level during stationary phase

A system for production of defective interfering particles in the absence of infectious influenza A virus

<div><p>Influenza A virus (IAV) infection poses a serious health threat and novel antiviral strategies are needed. Defective interfering particles (DIPs) can be generated in IAV infected cells due to errors of the viral polymerase and may suppress spread of wild type (wt) virus. The antiviral activity of DIPs is exerted by a DI genomic RNA segment that usually contains a large deletion and suppresses amplification of wt segments, potentially by competing for cellular and viral resources. DI-244 is a naturally occurring prototypic segment 1-derived DI RNA in which most of the PB2 open reading frame has been deleted and which is currently developed for antiviral therapy. At present, coinfection with wt virus is required for production of DI-244 particles which raises concerns regarding biosafety and may complicate interpretation of research results. Here, we show that cocultures of 293T and MDCK cell lines stably expressing codon optimized PB2 allow production of DI-244 particles solely from plasmids and in the absence of helper virus. Moreover, we demonstrate that infectivity of these particles can be quantified using MDCK-PB2 cells. Finally, we report that the DI-244 particles produced in this novel system exert potent antiviral activity against H1N1 and H3N2 IAV but not against the unrelated vesicular stomatitis virus. This is the first report of DIP production in the absence of infectious IAV and may spur efforts to develop DIPs for antiviral therapy.</p></div

Linear magnetoresistance in a quasi-free two dimensional electron gas in an ultra-high mobility GaAs quantum well

We report a magnetotransport study of an ultra-high mobility ($\bar{\mu}\approx 25\times 10^6$\,cm$^2$\,V$^{-1}$\,s$^{-1}$) $n$-type GaAs quantum well up to 33 T. A strong linear magnetoresistance (LMR) of the order of 10$^5$ % is observed in a wide temperature range between 0.3 K and 60 K. The simplicity of our material system with a single sub-band occupation and free electron dispersion rules out most complicated mechanisms that could give rise to the observed LMR. At low temperature, quantum oscillations are superimposed onto the LMR. Both, the featureless LMR at high $T$ and the quantum oscillations at low $T$ follow the empirical resistance rule which states that the longitudinal conductance is directly related to the derivative of the transversal (Hall) conductance multiplied by the magnetic field and a constant factor $\alpha$ that remains unchanged over the entire temperature range. Only at low temperatures, small deviations from this resistance rule are observed beyond $\nu=1$ that likely originate from a different transport mechanism for the composite fermions

Metaproteomics of complex microbial communities in biogas plants

Production of biogas from agricultural biomass or organic wastes is an important source of renewable energy. Although thousands of biogas plants (BGPs) are operating in Germany, there is still a significant potential to improve yields, e.g. from fibrous substrates. In addition, process stability should be optimized. Besides evaluating technical measures, improving our understanding of microbial communities involved into the biogas process is considered as key issue to achieve both goals. Microscopic and genetic approaches to analyse community composition provide valuable experimental data, but fail to detect presence of enzymes and overall metabolic activity of microbial communities. Therefore, metaproteomics can significantly contribute to elucidate critical steps in the conversion of biomass to methane as it delivers combined functional and phylogenetic data. Although metaproteomics analyses are challenged by sample impurities, sample complexity and redundant protein identification, and are still limited by the availability of genome sequences, recent studies have shown promising results. In the following, the workflow and potential pitfalls for metaproteomics of samples from full-scale BGP are discussed. In addition, the value of metaproteomics to contribute to the further advancement of microbial ecology is evaluated. Finally, synergistic effects expected when metaproteomics is combined with advanced imaging techniques, metagenomics, metatranscriptomics and metabolomics are addressed