A simplified protocol for profiling heparin-contaminated circulating miRNAs: by microfluidic array.

Peripheral blood is a valuable, non-invasive source of biomarkers which include circulating miRNAs. Using microfluidic array-based techniques, miRNAs can be successfully measured in small amounts of blood plasma (< 0.5 mL) using cDNA pre-amplification. However, the use of heparin-based anticoagulants for blood collection hinders the detection of circulating miRNAs due to its inhibitory effect on PCR components. Although pre-treatment with heparinase have been shown to overcome heparin contamination in blood, its effect has not been described in array-based analyses or more sensitive applications with smaller sample volumes (i.e. 200 µL plasma) requiring pre-amplification. We show that the treatment of miRNA extracted from heparinised plasma with an optimised concentration of Bacteroides heparinase I prior to cDNA pre-amplification dramatically improves the number of detectable miRNA from 2 to 67 targets on the TaqMan® Array Human MicroRNA Cards. Furthermore, the titrated amount of heparinase (3 U) gave the best miRNA detection compared to those used in previous studies (6-24 U). This study provides novel data which demonstrates that heparinase treatment is compatible with protocols that involve pre-amplification of cDNA and microfluidic array-based techniques. This an improved methodology that permits miRNA-based biomarker analysis from small volume of heparinised plasma.

Expression profile and function of Wnt signaling mechanisms in malignant mesothelioma cells.

Malignant mesothelioma (MM) is an uncommon and particularly aggressive cancer associated with asbestos exposure, which currently presents an intractable clinical challenge. Wnt signaling has been reported to play a role in the neoplastic properties of mesothelioma cells but has not been investigated in detail in this cancer. We surveyed expression of Wnts, their receptors, and other key molecules in this pathway in well established in vitro mesothelioma models in comparison with primary mesothelial cultures. We also tested the biological response of MM cell lines to exogenous Wnt and secreted regulators, as well as targeting ß-catenin. We detected frequent expression of Wnt3 and Wnt5a, as well as Fzd 2, 4 and 6. The mRNA of Wnt4, Fzd3, sFRP4, APC and axin2 were downregulated in MM relative to mesothelial cells while LEF1 was overexpressed in MM. Functionally, we observed that Wnt3a stimulated MM proliferation while sFRP4 was inhibitory. Furthermore, directly targeting ß-catenin expression could sensitise MM cells to cytotoxic drugs. These results provide evidence for altered expression of a number of Wnt/Fzd signaling molecules in MM. Modulation of Wnt signaling in MM may prove a means of targeting proliferation and drug resistance in this cancer.

Synthesis and antimalarial evaluation of novel isocryptolepine derivatives.

A series of mono- and di-substituted analogues of isocryptolepine have been synthesized and evaluated for in vitro antimalarial activity against chloroquine sensitive (3D7) and resistant (W2mef) Plasmodium falciparum and for cytotoxicity (3T3 cells). Di-halogenated compounds were the most potent derivatives and 8-bromo-2-chloroisocryptolepine displayed the highest selectivity index (106; the ratio of cytotoxicity (IC(50)=9005 nM) to antimalarial activity (IC(50)=85 nM)). Our evaluation of novel isocryptolepine compounds has demonstrated that di-halogenated derivatives are promising antimalarial lead compounds.