siRNA (small interfering RNA) and shRNA (small hairpin RNA) are powerful and commonly used tools in biomedical research. Currently, siRNAs are generally designed as two 21 nt strands of RNA that include a 19 nt completely complementary part and a 2 nt overhang. However, since the si/shRNAs use the endogenous miRNA machinery for gene silencing and the miRNAs are generally 22 nt in length and contain multiple internal mismatches, we tested if the functionality can be increased by designing the si/shRNAs to mimic a miRNA structure. We systematically investigated the effect of single or multiple mismatches introduced in the passenger strand at different positions on siRNA functionality. Mismatches at certain positions could significantly increase the functionality of siRNAs and also, in some cases decreased the unwanted passenger strand functionality. The same strategy could also be used to design shRNAs. Finally, we showed that both si and miRNA structured oligos (siRNA with or without mismatches in the passenger strand) can repress targets in all individual Ago containing cells, suggesting that the Ago proteins do not differentiate between si/miRNA-based structure for silencing activity.

Aims: Mycobacteria are a serious cause of infections in humans, with limited treatment options, as no new antibiotics have been developed against mycobacteria since the 1960s. In this study, the antimycobacterial activity of a small library of acetophenone (AP) compounds was analysed. Methods and Results: Twenty-three AP derivatives were examined for activity against mycobacteria using a microbroth assay. The compounds were bacteriostatic, with the most effective (cyclohexylacetophenone and piperidinoacetophenone) having minimal inhibitory concentrations of 246 lM M. Active compounds tended to be more hydrophobic, and may work by alkylation of as yet undetermined intracellular target protein(s). Cytotoxicity against eukaryotic cells was also determined and appears to be unrelated to the bacteriostatic activity. Significance and Impact of the Study: AP may serve as a novel group of useful therapeutics against the mycobacteria.

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In this brief review, we present recent results in the development of fluorescencebased assays for the detection of compounds with cytotoxic, anticancer and antimicrobial properties. As other reviews have explored various aspects related to these topics, this review will focus on the use of the Green Fluorescent Protein (GFP) for the detection of potentially toxic and/or therapeutic compounds. Since high-throughput screening of chemical compounds can be both expensive and laborious, development of low cost and efficient cell-based assays to determine biological activity should greatly enhance the early screening process. In our recent studies, we have developed a couple of GFP-based assays for the rapid screening of compounds with cytotoxic and bacteriocidal properties. As will be described in more detail in subsequent sections, a new 96-well assay has recently been developed that allows for the simultaneous screening of test compounds on gram positive and negative bacteria as well as mammalian (human cancer) cells. Our results demonstrate that both mammalian cells and bacteria can be analyzed in tandem to rapidly determine which compounds are specifically toxic to one of these cell types. The parallel screening of both eukaryotic and prokaryotic cells was found to be feasible, reproducible, and cost effective. BRIEF OVERVIEW ON THE PROPERTIES OF GFPFew methods exist that allow the visualization of living cells as they undergo apoptotic or necrotic modes of death. Since labeling of cells with dyes is not always efficient and may lead to interference of cellular functions, the preferred method for non-invasive detection is via the use of fluorescent markers. A fluorescent marker that has achieved prominence in visualization assays is GFP and its derivatives. These