Micropatterning of Plasma Membrane Proteins to Analyze Raft Localization in Living Cells

International audienc

Temporal resolution of protein–protein interactions in the live-cell plasma membrane

We have recently devised a method to quantify interactions between a membrane protein (“bait”) and a fluorophore-labeled protein (“prey”) directly in the live-cell plasma membrane (Schwarzenbacher et al. Nature Methods 5:1053–1060 2008). The idea is to seed cells on surfaces containing micro-patterned antibodies against the exoplasmic domain of the bait, and monitor the co-patterning of the fluorescent prey via fluorescence microscopy. Here, we characterized the time course of bait and prey micropattern formation upon seeding the cells onto the micro-biochip. Patterns were formed immediately after contact of the cells with the surface. Cells were able to migrate over the chip surface without affecting the micropattern contrast, which remained constant over hours. On single cells, bait contrast may be subject to fluctuations, indicating that the bait can be released from and recaptured on the micropatterns. We conclude that interaction studies can be performed at any time-point ranging from 5 min to several hours post seeding. Monitoring interactions with time opens up the possibility for new assays, which are briefly sketched in the discussion section

Molecular Pathogenesis of Post-Transplant Acute Kidney Injury: Assessment of Whole-Genome mRNA and MiRNA Profiles.

Acute kidney injury (AKI) affects roughly 25% of all recipients of deceased donor organs. The prevention of post-transplant AKI is still an unmet clinical need. We prospectively collected zero-hour, indication as well as protocol kidney biopsies from 166 allografts between 2011 and 2013. In this cohort eight cases with AKI and ten matched allografts without pathology serving as control group were identified with a follow-up biopsy within the first twelve days after engraftment. For this set the zero-hour and follow-up biopsies were subjected to genome wide microRNA and mRNA profiling and analysis, followed by validation in independent expression profiles of 42 AKI and 21 protocol biopsies for strictly controlling the false discovery rate. Follow-up biopsies of AKI allografts compared to time-matched protocol biopsies, further baseline adjustment for zero-hour biopsy expression level and validation in independent datasets, revealed a molecular AKI signature holding 20 mRNAs and two miRNAs (miR-182-5p and miR-21-3p). Next to several established biomarkers such as lipocalin-2 also novel candidates of interest were identified in the signature. In further experimental evaluation the elevated transcript expression level of the secretory leukocyte peptidase inhibitor (SLPI) in AKI allografts was confirmed in plasma and urine on the protein level (p<0.001 and p = 0.003, respectively). miR-182-5p was identified as a molecular regulator of post-transplant AKI, strongly correlated with global gene expression changes during AKI. In summary, we identified an AKI-specific molecular signature providing the ground for novel biomarkers and target candidates such as SLPI and miR-182-5p in addressing AKI

A novel class of bidentate amine ligands: Development of a synthetic strategy and application in coordination chemistry

I herein report the comprehensive study of a novel class of reduced bis(aryl)acenaphthenequinonediimines (ergo bis(aryl)aminodihydroacenaphthenes) as ligands for catalytic applications. A new synthetic approach towards bis(aryl)acenaphthenequinonediimines and a method for the transformation of the imine to the corresponding amine was developed. The described methods represent a quicker way to obtain the pertinent substances and have a higher scope than traditional syntheses. The elaborated reaction strategy might also serve as a versatile protocol for the preparation of other rigid 1,2- diamine ligands from diketones. The reaction conditions applied in the reduction of the diimines resulted in a significantly increased yield and were applied to the vast majority of the synthesized bis(aryl)acenaphthenequinonediimines. Some of the novel diamines were crystallized and structurally characterized through X-ray diffraction studies. Unfortunately, the attempted total hydrogenation of not only the imine, but also the aromatic functionalities employing catalytic strategies did not succeed in the desired alkylamine scaffold. Despite the fact that the copper mediated oxidation of alcohols to aldehydes showed only little activity when using the diamines as ligands, it nevertheless serves as an example that the reduced bis(aryl)acenaphthenequinonediimines function as ligands, with their catalytic application being subject to future investigations, however. The bis(aryl)acenaphthenequinonediimines prepared by the described method have been employed as ligands for manganese(I) complexes. The spectroscopic properties of these complexes are herein discussed, along with a thorough analytical characterization.submitted by Paul Sunzenauer BSc.Universität Linz, Masterarbeit, 2018(VLID)329930

Molecular models.

<p>Schematic representation of the constructed DN molecular model and the tacrolimus MoA molecular model with the list of significantly enriched molecular pathways based on the two molecular model features sets. Molecular pathways in bold font within the red border were significantly enriched in molecular features of both molecular model sets. Each box in the molecular model representations depicts one process unit, characterized by highly inter-connected proteins, with individual nodes representing protein coding genes.</p