The role of Cas8 in type I CRISPR interference

CRISPR (clustered regularly interspaced short palindromic repeat) systems provide bacteria and archaea with adapt- ive immunity to repel invasive genetic elements. Type I systems use ‘cascade’ [CRISPR-associated (Cas) complex for antiviral defence] ribonucleoprotein complexes to target invader DNA, by base pairing CRISPR RNA (crRNA) to protospacers. Cascade identifies PAMs (protospacer adjacent motifs) on invader DNA, triggering R-loop formation and subsequent DNA degradation by Cas3. Cas8 is a candidate PAM recognition factor in some cascades. We analysed Cas8 homologues from type IB CRISPR systems in archaea Haloferax volcanii (Hvo) and Methanothermobacter ther- mautotrophicus (Mth). Cas8 was essential for CRISPR interference in Hvo and purified Mth Cas8 protein responded to PAM sequence when binding to nucleic acids. Cas8 interacted physically with Cas5–Cas7–crRNA complex, stimulating binding to PAM containing substrates. Mutation of conserved Cas8 amino acid residues abolished interference in vivo and altered catalytic activity of Cas8 protein in vitro. This is experimental evidence that Cas8 is important for targeting Cascade to invader DNA

Targeting the “undruggable” RAS - new strategies - new hope?

K-RAS is the most frequently mutated oncogene in solid tumors, such as pancreatic, colon or lung cancer. The GTPase K-RAS can either be in an active (GTP-loaded) or inactive (GDP-loaded) form. In its active form K-RAS forwards signals from growth factors, cytokines or hormones to the nucleus, regulating essential pathways, such as cell proliferation and differentiation. In turn, activating somatic mutations of this proto-oncogene deregulate the complex interplay between GAP (GTPase-activating) - and GEF (Guanine nucleotide exchange factor) - proteins, driving neoplastic transformation. Due to a rather shallow surface, K-RAS lacks proper binding pockets for small molecules, hindering drug development over the past thirty years. This review summarizes recent progress in the development of low molecular antagonists and further shows insights of a newly described interaction between mutant K-RAS signaling and PD-L1 induced immunosuppression, giving new hope for future treatments of K-RAS mutated cancer

Complex interventional iliocaval recanalization due to plasmacytoma and cystic echinococcosis

Post-thrombotic syndrome is common after iliofemoral vein thrombosis. Conservative therapy, mainly limited to compression and anticoagulation therapy, might not be sufficient in controlling symptoms. Interventional recanalization of the chronically occluded iliac veins is an evolving method, promising rapid relief of symptoms. Here, we present two cases of complex interventions in one patient with preceding pelvic radiotherapy due to a plasmacytoma and in another patient in whom a cava wedge resection had been performed because of cystic echinococcosis in the liver

Microfluidic chip system for the selection and enrichment of cell binding aptamers

Aptamers are promising cell targeting ligands for several applications such as for the diagnosis, therapy, and drug delivery. Especially, in the field of regenerative medicine, stem cell specific aptamers have an enormous potential. Using the combinatorial chemistry process SELEX (Systematic Evolution of Ligands by Exponential enrichment), aptamers are selected from a huge oligonucleotide library consisting of approximately 10(15) different oligonucleotides. Here, we developed a microfluidic chip system that can be used for the selection of cell specific aptamers. The major drawbacks of common cell-SELEX methods are the inefficient elimination of the unspecifically bound oligonucleotides from the cell surface and the unspecific binding/uptake of oligonucleotides by dead cells. To overcome these obstacles, a microfluidic device, which enables the simultaneous performance of dielectrophoresis and electrophoresis in the same device, was designed. Using this system, viable cells can be selectively assembled by dielectrophoresis between the electrodes and then incubated with the oligonucleotides. To reduce the rate of unspecifically bound sequences, electrophoretic fields can be applied in order to draw loosely bound oligonucleotides away from the cells. Furthermore, by increasing the flow rate in the chip during the iterative rounds of SELEX, the selection pressure can be improved and aptamers with higher affinities and specificities can be obtained. This new microfluidic device has a tremendous capability to improve the cell-SELEX procedure and to select highly specific aptamers