Poxviruses such as virulent vaccinia virus (VACV) strain

MicroRNAs have recently been associated with cancer development by acting as tumor suppressors or oncogenes and could therefore be applied as molecular markers for early diagnosis of cancer. In this work, we established a rapid, selective, and sensitive gap hybridization assay for detection of mature microRNAs based on four components DNA/RNA hybridization and electrochemical detection using esterase 2-oligodeoxynucleotide conjugates. Complementary binding of microRNA to a gap built of capture and detector oligodeoxynucleotide, the reporter enzyme is brought to the vicinity of the electrode and produces enzymatically an electrochemical signal. In the absence of microRNA, the gap between capture and detector oligodeoxynucleotide is not filled, and missing base stacking energy destabilizes the hybridization complex. The gap hybridization assay demonstrates selective detection of miR-16 within a mixture of other miRNAs, including the feasibility of single mismatch discrimination. Applying the biosensor assay, a detection limit of 2 pM or 2 amol of miR-16 was obtained. Using isolated total RNA from human breast adenocarcinoma MCF-7 cells, the assay detected specifically miR-21 and miR-16 in parallel, and higher expression of oncogene miR-21 compared to miR-16 was demonstrated. Including RNA isolation, the gap hybridization assay was developed with a total assay time of 60 min and without the need for reverse transcription PCR amplification of the sample. The characteristics of the assay developed in this work could satisfy the need for rapid and easy methods for early cancer marker detection in clinical diagnostics.

Background: Separase, the trigger protease of eukaryotic anaphase, remains regulated in the absence of its inhibitor, securin. Results: Cdk1-cyclin B1 triggers precipitation of separase by phosphorylation but stabilizes it by inhibitory binding. Conclusion: Only separase that is first complexed by Cdk1-cyclin B1 can later be activated by cyclin B1 degradation. Significance: These minimal requirements of separase regulation could explain the faithful execution of anaphase in the absence of securin.

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Existing technologies for analysis of microbiological contaminants in food or clinical samples are often expensive and require laboratory settings and trained personnel. Here we present a lateral flow assay employing gold nanoparticle-oligodeoxynucleotide conjugates and four-component sandwich hybridisation for direct detection of specific sequences in bacterial 16S ribosomal RNA. Combined with rapid "one step" lysis the developed procedure allows detection of 5 × 10(4) colony forming units per mL Escherichia coli within less than 25 minutes. Several Escherichia coli strains were detected successfully, whereas non-related as well as closely related bacterial species produced no signal. The developed nucleic acid lateral flow assay is inexpensive, rapid to perform and requires no nucleic acid amplification step.