Cystic fibrosis (CF) is an autosomal recessive inherited disease which leads to a production of thickened mucus in the airways. These conditions are conducive to poly-microbial infections, like chronic lung infection, in which Pseudomonas aeruginosa (P. aeruginosa) is the major pathogenic bacterium colonizing CF lungs at the end of the lifetime of CF patients. This in vitro study uses a P. aeruginosa biofilm model under partly cystic fibrosis conditions, with a sampling of volatile extracellular metabolites. The gas sampling was done with thin-film microextraction (TFME) and commercial polydimethylsiloxane (PDMS) films, whereas the analysis of loaded films was done by gas chromatography coupled to quadrupole mass spectrometry and thermodesorption (TD-GC-qMS). For this purpose, two commercially available films were characterized by means of thermogravimetry coupled to a qMS with atmospheric pressure photo ionization (TG-APPI-qMS), regarding homogeneity and temperature stability. The selected film was cleaned using a method developed in this study. The TD-GC-qMS method was successfully used for standards of volatile metabolites which were known to be produced by P. aeruginosa. Limits of detection and quantification of the method for middle and less polar compounds in low nanomolar range (0.5 nM and 1.5 nM) were achieved. The developed method was finally applied to investigate the extracellular volatile metabolites produced by biofilms of the strain P. aeruginosa DSM 50071 under aerobic and anaerobic conditions. In sum, eleven metabolites could be found under both conditions. Furthermore, it was shown in this study that different oxygen conditions (aerobic and anaerobic) resulted in emitting different extracellular volatile metabolites. Specific metabolites, like 1-undecene (aerobic) and 2-undecanone (anaerobic), could be identified. The results are promising, in that the biofilm model may be applicable for the identification of P. aeruginosa under clinical conditions. Furthermore, the model could be the basis for studying extracellular volatile metabolites from different mono-or co-cultures of various bacteria, as well as the implementation of pulmonary conditions, like these in CF lungs. This possibility allows the development of a non-invasive "at-bedside" breath analysis method for CF patients in focus of various bacterial infections.

High-throughput liquid chromatography/mass spectrometry (LC/MS) is an increasing topic in analytical chemistry. Especially the idle time of a mass spectrometer should be reduced for an efficient and cost-saving use. Therefore, a fast-switching dual ion source was developed, which uses the most important ionization techniques at atmospheric pressure, electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI), with one or more LC systems. Methods:The performance of the developed ion source is shown by infusion experiments and chromatographic analyses of different standard substances. A highthroughput method is demonstrated by coupling two UHPLC systems to the dual ion source with a triple quadrupole mass spectrometer. Results:No decrease in the ion abundance and a stable performance of the mass spectrometer are presented while using the dual ion source. Instrumental limits of detection are 30 ng L −1 for testosterone using ESI and 1 μg L −1 for vitamin D 3 using APCI. A fast switching between two UHPLC systems and the dual ion source leads to a high sample throughput of 50 samples in 75 min with relative standard deviations for testosterone and vitamin D 3 of 1.5% and 3.8%, respectively.Conclusions: This work presents the development of a dual ESI and APCI ion source operating simultaneously or in switched mode. The results show sensitive and reliable performance as well as the hyphenation to one or more HPLC systems.

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The optogenetic tool LEXY consists of the second light oxygen voltage (LOV) domain of Avena sativa phototropin 1 mutated to contain a nuclear export signal. It allows exporting from the nucleus with blue light proteins of interest (POIs) genetically fused to it. Mutations slowing the dark recovery rate of the LOV domain within LEXY were recently shown to allow for better depletion of some POIs from the nucleus in Drosophila embryos and for the usage of low light illumination regimes. We investigated these variants in mammalian cells and found they increase the cytoplasmic localization of the proteins we tested after illumination, but also during the dark phases, which corresponds to higher leakiness of the system. These data suggest that, when aiming to sequester into the nucleus a protein with a cytoplasmic function, the original LEXY is preferable. The iLEXY variants are, instead, advantageous when wanting to deplete the nucleus of the POI as much as possible.