Charlotte Ayn Cialek

Developing sustainable production schemes by recycling industrial waste streams can avoid the pollution of traditional methods and reduce our dependency on fossil fuel. To achieve this, scientists have engineered microorganisms to convert waste, such as whey from the cheese industry, cellulose from agriculture, and wastewater from the brewing industry, into valuable products such as nutritional protein, nutraceuticals, and biofuels ( 1–3). But food and fuel are not the only products we can dream of making from waste: some microorganisms have the astounding natural ability to generate electricity from their metabolism. If scaled and customized, this capability could be harnessed to generate electricity from waste. However, natural electrogenic microorganisms are not easily cultivated on waste streams or genetically engineered in a laboratory setting.

Bacteria use a bag of tricks to thwart attempted assault. One strategy involves forming a barrier called a biofilm that can both deflect antibiotics and block the human immune defense ( 1, 2). A bacterial colony shielded by a biofilm can persist and drastically increase the severity of an infection. Not all bacteria produce biofilms, but the ones that do cause >65% of human infections ( 1, 2).

A major challenge to our understanding of translational control has been deconvolving the individual impact specific regulatory factors have on the complex dynamics of mRNA translation. MicroRNAs (miRNAs), for example, guide Argonaute and associated proteins to target mRNAs, where they direct gene silencing in multiple ways that are not well understood. To better deconvolve these dynamics, we have developed technology to directly visualize and quantify the impact of human Argonaute2 (Ago2) on the translation and subcellular localization of individual reporter mRNAs in living cells. We show that our combined translation and Ago2 tethering system reflects endogenous miRNA-mediated gene silencing. Using the system, we find that Ago2 association leads to progressive silencing of translation at individual mRNA. The timescale of silencing was similar to that of translation, consistent with a role for Ago2 in blocking translation initiation, leading to ribosome runoff. At early time points, we observed occasional brief bursts of translational activity at Ago2-tethered mRNAs undergoing silencing, suggesting that translational repression may initially be reversible. At later time points, Ago2-tethered mRNAs cluster and coalesce with endogenous P-bodies, where a translationally silent state is maintained. These results provide a framework for exploring miRNA-mediated gene regulation in live cells at the single-molecule level. Furthermore, our tethering-based, single-molecule reporter system will likely have wide-ranging application in studying general RNA-protein interactions.

Bone metastasis is a painful complication of advanced prostate cancer. Endothelin-1 is a tumor-secreted factor that plays a central role in osteoblast activation and the osteosclerotic response of prostate cancer metastatic to bone. Antagonists that block the activation of the endothelin A receptor (ET_AR), located on osteoblasts, reduce osteoblastic bone lesions in animal models of bone metastasis. However, ET_AR antagonists demonstrated limited efficacy in clinical trials of men with advanced prostate cancer who also received standard androgen deprivation therapy (ADT). Previous data from our group suggested that, in a mouse model, ET_AR antagonists might only be efficacious when androgen signaling in the osteoblast is lowered beyond the ability of standard ADT. This notion was tested in a mouse model of prostate cancer bone metastasis. Castrated and sham-operated male athymic nude mice underwent intracardiac inoculation of the ARCaP_M castration-resistant prostate cancer cell line. The mice were then treated with either the ET_AR antagonist zibotentan or a vehicle control to generate four experimental groups: vehicle+sham (Veh+Sham), vehicle+castrate (Veh+Castr), zibotentan+sham (Zibo+Sham), and zibotentan+castrate (Zibo+Castr). The mice were monitored radiographically for the development of skeletal lesions. The Zibo+Castr group had significantly longer survival and a single incidental lesion. Mice in the Zibo+Sham group had the shortest survival and the largest number of skeletal lesions. Survival and skeletal lesions of the Veh+Sham and Veh+Castr groups were intermediate compared with the zibotentan-treated groups. We report a complex interaction between ET_AR and androgen signaling, whereby ET_AR blockade was most efficacious when combined with complete androgen deprivation.