Development of a matrix-based technology platform for the high throughput analysis of 3D cell cultures

The screening of large cell libraries is an important process in pharmaceutical discovery and R&D, e.g. to define drug targets or develop effective medicines. The goal of this project is the implementation of a screening platform based on 3D cultivation of primary human mesothelioma cells encapsulated in alginate hydrogels. To this end, new hydrogel compositions will be designed, tested and finally utilized in the Nanoliter Reactor (NLR) cultivation system that enables high throughput analysis of 3D cell cultures

Chemical Genetics Approach to Engineer Kinesins with Sensitivity towards a Small-Molecule Inhibitor of Eg5

Due to their fast and often reversible mode of action, small molecules are ideally suited to dissect biological processes. Yet, the validity of small-molecule studies is intimately tied to the specificity of the applied compounds, thus imposing a great challenge to screens for novel inhibitors. Here, we applied a chemical-genetics approach to render kinesin motor proteins sensitive to inhibition by the well-characterized small molecule S-Trityl-l-cysteine (STLC). STLC specifically inhibits the kinesin Eg5 through binding to a known allosteric site within the motor domain. Transfer of this allosteric binding site into the motor domain of the human kinesins Kif3A and Kif4A sensitizes them towards STLC. Single-molecule microscopy analyses confirmed that STLC inhibits the movement of chimeric but not wild-type Kif4A along microtubules. Thus, our proof-of-concept study revealed that this chemical-genetic approach provides a powerful strategy to specifically inhibit kinesins in vitro for which small-molecule inhibitors are not yet available.publishe

Development of a matrix-based technology platform for the high throughput analysis of 3D cell cultures

The screening of large cell libraries is an important process in pharmaceutical discovery and R&D, e.g. to define drug targets or develop effective medicines. The goal of this project is the implementation of a screening platform based on 3D cultivation of primary human mesothelioma cells encapsulated in alginate hydrogels. To this end, new hydrogel compositions will be designed, tested and finally utilized in the Nanoliter Reactor (NLR) cultivation system that enables high throughput analysis of 3D cell cultures

Choline Supplementation in Cystic Fibrosis—The Metabolic and Clinical Impact

Background: Choline is essential for the synthesis of liver phosphatidylcholine (PC), parenchymal maintenance, bile formation, and lipoprotein assembly to secrete triglycerides. In choline deficiency, the liver accretes choline/PC at the expense of lung tissue, thereby impairing pulmonary PC homoeostasis. In cystic fibrosis (CF), exocrine pancreas insufficiency results in impaired cleavage of bile PC and subsequent fecal choline loss. In these patients, the plasma choline concentration is low and correlates with lung function. We therefore investigated the effect of choline supplementation on plasma choline/PC concentration and metabolism, lung function, and liver fat. Methods: 10 adult male CF patients were recruited (11/2014–1/2016), and orally supplemented with 3 × 1 g choline chloride for 84 (84–91) days. Pre-/post-supplementation, patients were spiked with 3.6 mg/kg [methyl-D9]choline chloride to assess choline/PC metabolism. Mass spectrometry, spirometry, and hepatic nuclear resonance spectrometry served for analysis. Results: Supplementation increased plasma choline from 4.8 (4.1–6.2) µmol/L to 10.5 (8.5–15.5) µmol/L at d84 (p < 0.01). Whereas plasma PC concentration remained unchanged, D9-labeled PC was decreased (12.2 [10.5–18.3] µmol/L vs. 17.7 [15.5–22.4] µmol/L, p < 0.01), indicating D9-tracer dilution due to higher choline pools. Supplementation increased Forced Expiratory Volume in 1 second percent of predicted (ppFEV1) from 70.0 (50.9–74.8)% to 78.3 (60.1–83.9)% (p < 0.05), and decreased liver fat from 1.58 (0.37–8.82)% to 0.84 (0.56–1.17)% (p < 0.01). Plasma choline returned to baseline concentration within 60 h. Conclusions: Choline supplementation normalized plasma choline concentration and increased choline-containing PC precursor pools in adult CF patients. Improved lung function and decreased liver fat suggest that in CF correcting choline deficiency is clinically important. Choline supplementation of CF patients should be further investigated in randomized, placebo-controlled trials

Dynamic light-scattering measurement comparability of nanomaterial suspensions

Increased use of nanomaterials in everyday products leads to their environmental release and therefore, the information need on their fate and behaviour. Nanomaterials have to be suspended with high repeatability and comparability for studies on environmental effects. They also have to be well characterised with a focus on the state of agglomeration and particle size distribution. Dynamic lightscattering (DLS) is a common technique used for these measurements. If suspensions are prepared in different laboratories, then concern has risen about the comparability of the measured results, especially when different DLS instruments are used. Therefore, for quality assurance, a round-robin test was conducted to assess the comparability of different DLS instruments and a dispersion protocol in ten independent laboratories. Polystyrene and TiO2 were chosen as test (nano)materials. For the comparability of the DLS instruments, the average sizes of the PSL and a stabilised TiO2 suspension were measured. The measured average hydrodynamic diameter shows an overall good inter-laboratory comparability. For the PSL suspension, an average hydrodynamic diameter of 201 ± 13 nm and for the TiO2 suspension an average diameter of 224 ± 24 nm were detected. For the TiO2 suspension that was prepared at each laboratory following an established suspension preparation protocol, an average hydrodynamic diameter of 211 ± 11 nm was detected. The measured average particle size (mode) increased up to 284 nm with a high standard deviation of 119 nm if the preparation protocol could not established and different procedures or different equipment were employed. This study shows that no significant differences between the employed DLS instrument types were determined. It was also shown that comparable measurements and suspension preparation could be achieved if welldefined suspension preparation protocols and comparable equipment can be used