Development, testing and validation of a targeted NGS-panel for the detection of actionable mutations in lung cancer (NSCLC) using anchored multiplex PCR technology in a multicentric setting.

Lung cancer is a paradigm for a genetically driven tumor. A variety of drugs were developed targeting specific biomarkers requiring testing for tumor genetic alterations in relevant biomarkers. Different next-generation sequencing technologies are available for library generation: 1) anchored multiplex-, 2) amplicon based- and 3) hybrid capture-based-PCR. Anchored multiplex PCR-based sequencing was investigated for routine molecular testing within the national Network Genomic Medicine Lung Cancer (nNGM). Four centers applied the anchored multiplex ArcherDX-Variantplex nNGMv2 panel to re-analyze samples pre-tested during routine diagnostics. Data analyses were performed by each center and compiled centrally according to study design. Pre-defined standards were utilized, and panel sensitivity was determined by dilution experiments. nNGMv2 panel sequencing was successful in 98.9% of the samples (N = 90). With default filter settings, all but two potential MET exon 14 skipping variants were identified at similar allele frequencies. Both MET variants were found with an adapted calling filter. Three additional variants (KEAP1, STK11, TP53) were called that were not identified in pre-testing analyses. Only total DNA amount but not a qPCR-based DNA quality score correlated with average coverage. Analysis was successful with a DNA input as low as 6.25 ng. Anchored multiplex PCR-based sequencing (nNGMv2) and a sophisticated user-friendly Archer-Analysis pipeline is a robust and specific technology to detect tumor genetic mutations for precision medicine of lung cancer patients.

Size dependent targeted delivery of gold nanoparticles modified with the IL-6R-specific aptamer AIR-3A to IL-6R-carrying cells.

The delivery of gold nanoparticles (AuNPs) to specific cells strongly depends on the properties e.g. the size of the particles and is of great interest for a large variety of biomedical applications. Here we investigated the size dependence of the receptor-ligand mediated AuNP delivery to cells by comparing very small "molecular" Au-clusters of only 2 nm to larger 7 nm and 36 nm AuNPs with a distinct surface plasmon resonance. Since the molecular weight in this range changes by almost three orders of magnitude, we show how the amount of gold relates to the number of delivered AuNPs. We attached small interleukin-6 receptor (IL-6R) specific aptamer molecules (AIR-3A) in different amounts to the particles and investigated the specificity of the delivery to IL-6R-carrying cells. To reduce unspecific interaction the particles were additionally covered with polyethylene glycol (PEG). Besides particle size and concentration we varied additional parameters such as aptamer surface coverage as well as incubation time and temperature. We found that in particular, small particles with diameters of less than 2 nm show an up to six times higher delivery rate for the aptamer-conjugated AuNPs compared to untargeted PEG-coated AuNPs. The specificity reduces with a decreasing aptamer/PEG ratio, and also with an increase in particle size where the unspecific uptake is much higher. In addition we also compared the delivery efficiency of this aptamer-mediated delivery system with an antibody-mediated system targeting the same receptor to validate the performance of this approach.

Novel macrolide resistance module carried by the IncP-1beta resistance plasmid pRSB111, isolated from a wastewater treatment plant.

The macrolide resistance plasmid pRSB111 was isolated from bacteria residing in the final effluents of a wastewater treatment plant. The 47-kb plasmid confers resistance to azithromycin, clarithromycin, erythromycin, roxithromycin, and tylosin when it is carried by Pseudomonas sp. strain B13 and is very similar to prototype IncP-1beta plasmid pB3, which was previously isolated from an activated-sludge bacterial community of a wastewater treatment plant. The two plasmids differ in their accessory regions, located downstream of the conjugative transfer module gene traC. Nucleotide sequence analysis of the pRSB111 accessory region revealed that it contains a new macrolide resistance module composed of the genes mphR(E), mph(E), and mrx(E), which putatively encode a transcriptional regulator, a macrolide phosphotransferase, and a transmembrane transport protein, respectively. Analysis of the contributions of the individual genes of the macrolide resistance module revealed that mph(E) and mrx(E) are required for high-level macrolide resistance. The resistance genes are flanked by two insertion sequences, namely, ISPa15 and ISRSB111. Two truncated transposable elements, IS6100 and remnants of a Tn3-like transposon, were identified in the vicinity of ISRSB111. The accessory element of pRSB111 apparently replaced the Tn402-like element present on the sister plasmid, pB3, as suggested by the conservation of Tn402-specific terminal inverted repeats on pRSB111.