Efficient splicing-based RNA regulators for tetracycline-inducible gene expression in human cell culture and C. elegans.

Synthetic riboswitches gain increasing interest for controlling transgene expression in diverse applications ranging from synthetic biology, functional genomics, and pharmaceutical target validation to potential therapeutic approaches. However, existing systems often lack the pharmaceutically suited ligands and dynamic responses needed for advanced applications. Here we present a series of synthetic riboswitches for controlling gene expression through the regulation of alternative splicing. Placing the 5'-splice site into a stem structure of a tetracycline-sensing aptamer allows us to regulate the accessibility of the splice site. In the presence of tetracycline, an exon with a premature termination codon is skipped and gene expression can occur, whereas in its absence the exon is included into the coding sequence, repressing functional protein expression. We were able to identify RNA switches controlling protein expression in human cells with high dynamic ranges and different levels of protein expression. We present minimalistic versions of this system that circumvent the need to insert an additional exon. Further, we demonstrate the robustness of our approach by transferring the devices into the important research model organism Caenorhabditis elegans, where high levels of functional protein with very low background expression could be achieved.

Development of an inverse low-temperature plasma ionization source for liquid chromatography/mass spectrometry.

RATIONALE: An argon inverse low-temperature plasma (iLTP) ionization source for liquid chromatography/tandem mass spectrometry was developed. The iLTP is constructed from simple chromatographic supply materials and is implemented into an atmospheric pressure chemical ionization (APCI) source replacing the APCI discharge needle electrode. The newly developed ion source was coupled to an ultra-high-performance liquid chromatography (UHPLC) system. METHODS: The argon iLTP was characterized by optical emission spectroscopy. The soft ionization of selected standards was also demonstrated by direct infusion experiments. In addition to the use of argon as the discharge gas, helium, synthetic air, and oxygen were used, which were tested for their performance using testosterone and vitamin D3 . RESULTS: Spectroscopic measurements of the argon plasma were conducted, demonstrating the main emission band of argon metastables with corresponding energies of 11.53 eV and 11.72 eV. Infusion experiments indicate a gentle ionization by iLTP, e.g. caffeine, testosterone, reserpine, vitamin D3 , and 25-hydroxyvitamin D3 , which resulted in the corresponding protonated molecules. The splitless coupling with UHPLC (possible flow rates >1000 µL min-1 ) shows promising results in interday repeatability (n = 10) for the substances with a relative standard deviation of less than 5% and limits of detection for caffeine, testosterone, reserpine, vitamin D3 , and 25-hydroxyvitamin D3 of 10 ng L-1 , 50 ng L-1 , 500 ng L-1 , 5 µg L-1 , and 5 µg L-1 , respectively. CONCLUSIONS: The argon iLTP ion source presented in this work shows promising approaches in the field of ionization of small organic molecules. The mechanism related to the discharge gas argon has not been elucidated so far and further investigations are needed. The iLTP ion source shows a very good performance with UHPLC coupling, even at increased flow rates. It could be shown that an argon iLTP can compete with the helium dielectric barrier discharge (DBD) preferred in the literature, making it a more economical choice.

Development of a fast-switching dual (ESI/APCI) ionization source for liquid chromatography/mass spectrometry.

RATIONALE: 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 high-throughput 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 D3 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 D3 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.

Analysis of volatile metabolites from in vitro biofilms of Pseudomonas aeruginosa with thin-film microextraction by thermal desorption gas chromatography-mass spectrometry.

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. Graphical abstract.

Analysis of Slow-Cycling Variants of the Light-Inducible Nuclear Protein Export System LEXY in Mammalian Cells.

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.

Genomic, Transcriptomic, and Functional Alterations in DNA Damage Response Pathways as Putative Biomarkers of Chemotherapy Response in Ovarian Cancer.

Defective DNA damage response (DDR) pathways are enabling characteristics of cancers that not only can be exploited to specifically target cancer cells but also can predict chemotherapy response. Defective Homologous Recombination Repair (HRR) function, e.g., due to BRCA1/2 loss, is a determinant of response to platinum agents and PARP inhibitors in ovarian cancers. Most chemotherapies function by either inducing DNA damage or impacting on its repair but are generally used in the clinic unselectively. The significance of HRR and other DDR pathways in determining response to several other chemotherapy drugs is not well understood. In this study, the genomic, transcriptomic and functional analysis of DDR pathways in a panel of 14 ovarian cancer cell lines identified that defects in DDR pathways could determine response to several chemotherapy drugs. Carboplatin, rucaparib, and topotecan sensitivity were associated with functional loss of HRR (validated in 10 patient-derived primary cultures) and mismatch repair. Two DDR gene expression clusters correlating with treatment response were identified, with PARP10 identified as a novel marker of platinum response, which was confirmed in The Cancer Genome Atlas (TCGA) ovarian cancer cohort. Reduced non-homologous end-joining function correlated with increased sensitivity to doxorubicin, while cells with high intrinsic oxidative stress showed sensitivity to gemcitabine. In this era of personalised medicine, molecular/functional characterisation of DDR pathways could guide chemotherapy choices in the clinic allowing specific targeting of ovarian cancers.

Thermogravimetry coupled to an atmospheric pressure photo ionization quadrupole mass spectrometry for the product control of pharmaceutical formulations and the analysis of plasticizers in polymers.

The development of a thermogravimetry coupled to an atmospheric pressure photoionization mass spectrometry (TG-APPI-MS) with a high temperature and flexible transfer line is presented. A method was developed to analyze plasticizers in solution which consist of a solvent evaporation step and subsequent evaporation of the analyte. These solutions of dibutyl phthalate (DBP) in hexane were used to investigate the repeatability (RSD: 3.6%) and linearity (R2: 0.9995) of the new developed system. With the new device the detection of different phthalates in a standardized PVC (polyvinyl chloride) polymer is shown. On the example of ASA, the degradation of a pharmaceutical drug is investigated. The dimerization and the possible trimerization of ASA during the thermal degradation is shown. Ten tablets of different ASA manufacturers were analyzed with the new developed analysis platform. The active substance was found in every tablet. Differences in mass spectral data as well as the studying of the pack insert were used to assign the tablets to companies and their subsidiaries. A unique formulation of ASA was found to have a different mass pattern when analyzed with TG-APPI-qMS. The developed device is a promising tool for the product control and the identification of falsified drugs.