Multiplex genotyping of KRAS point mutations in tumor cell DNA by allele-specific real-time PCR on a centrifugal microfluidic disk segment

Point Mutations on the Kirsten rat sarcoma viral oncogene homolog (KRAS) have been identified as an important predictive biomarker for response to cancer therapy targeting the epidermal growth factor receptor. KRAS mutations are prevalent in up to 40 % of all colorectal carcinomas, and routinely conducted KRAS genotyping is becoming mandatory to predict therapy success and to reduce therapy costs. We report a low-cost, disposable and ready-to-use centrifugal microfluidic cartridge (termed GeneSlice) containing preloaded primers and probes. The GeneSlice cartridge enables the parallel detection of the seven most relevant KRAS point mutations by allele-specific real-time PCR. It represents a cost effective alternative to dideoxy-sequencing with a faster time-to-result (~ 2 h versus up to 20 h in case of dd-sequencing). Microfluidic processing of the GeneSlice along with allele-specific amplification and real-time detection are conducted in a slightly modified, commercially available PCR thermocycler. Intra-chip standard deviation of C_q values on the GeneSlices is negligible (GeneSlice 1: C_q,std.dev. = 0.13; GeneSlice 2: C_q,std.dev?=?0.26). In 23 of 24 experiments, the data for genotyping 6 cancer cell lines (n?=?4 per cell line) agreed with dd-sequencing. Additionally, DNA derived from microdissected formalin-fixed and paraffin embedded colorectal carcinomas of two cases was genotyped correctly and reproducibly (n?=?3 per patient; one GeneSlice excluded from evaluation). The GeneSlice therefore clearly demonstrated the potential to become a valuable tool for routine diagnostics of KRAS mutations by reducing costs and hands-on time. Figure

Photograph of a centrifugal microfluidic cartridge “GeneSlice” for multiplex genotyping of KRAS point mutations from tumor cell DNA by allele-specific real-time PCR. Information about the mutation status is required to predict success of state-of-the-art cancer therapy with antibodies     

Weyl-flow and the conformally symplectic structure of thermostatted billiards: The problem of the hyperbolicity

The highly complex nature of the transport in thermostatted billiards has been of interest in the last few decades because of industrial and medical applications. The onset of hyperbolic dynamics (deterministic chaos) in such a billiard has evidenced an interesting stabilization of the transport properties, especially in microporous media. Recently, different mathematical methods have been developed for establishing hyperbolicity in thermostatted billiards, among these, the Weyl-flow and the conformally symplectic structure techniques.This paper deals with analytical investigations on the possible hyperbolic nature of two thermostatted billiards: The nonequilibrium Ehrenfest gas (NEEG) and the pump model (PM). Despite numerical investigations supporting the idea of their dissipative dynamics, the hyperbolicity of these billiards has not been yet established. The analysis developed in this paper shows how the Weyl-flow technique has failed for NEEG, revealing the necessity to develop new strategies in order to obtain hyperbolicity. On the contrary, we prove that the PM has a conformally symplectic structure, which is the basis for establishing the hyperbolicity of such a hybrid dynamical system.     

Synthesis and Properties of the Weakly Coordinating Anion [Me3NB12Cl11]−

The weakly coordinating anion [Me₃NB₁₂Cl₁₁]^? has been prepared by a simple two‐step procedure. The anion [Me₃NB₁₂Cl₁₁]^? is easily obtained in batches of up to 20 g by chlorination of the known [H₃NB₁₂H₁₁]^? anion with SbCl₅ at about 190 °C and subsequent N‐methylation with methyl iodide. Starting from Na[Me₃NB₁₂Cl₁₁], several synthetically useful salts with reactive cations ([NO]⁺, [Ph₃C]⁺, and [(Et₃Si)₂H]⁺) were prepared. Full spectroscopic (NMR, IR, Raman, TGA, MS) characterization and single‐crystal X‐ray diffraction studies confirmed the identity and purity of the products. The thermal, chemical, and electrochemical stability as well as the basicity of the [Me₃NB₁₂Cl₁₁]^? anion is similar to that of the structurally related weakly coordinating 1‐carba‐closo‐dodecaborate and closo‐dodecaborate anions. The facile preparation of the [Me₃NB₁₂Cl₁₁]^? anion and its ideal chemical and physical properties make it a cheap alternative to other classes of weakly coordinating anions.     

Reaction of α,β-poly(N-hydroxyethyl)-DL-aspartamide with derivatives of carboxylic acids

Benzoic acid, acetylsalicylic acid, and 4-acetamidobenzoic acid have been covalently linked by ester bonds to α,β-poly(N-hydroxyethyl)-DL -aspartamide (PHEA), a water soluble and nontoxic polymer, in order to study PHEA as a drug carrier.