Structural and biochemical studies have revealed the importance of a conserved, mobile domain of RNA Polymerase II (Pol II), the Trigger Loop (TL), in substrate selection and catalysis. The relative contributions of different residues within the TL to Pol II function and how Pol II activity defects correlate with gene expression alteration in vivo are unknown. Using Saccharomyces cerevisiae Pol II as a model, we uncover complex genetic relationships between mutated TL residues by combinatorial analysis of multiply substituted TL variants. We show that in vitro biochemical activity is highly predictive of in vivo transcription phenotypes, suggesting direct relationships between phenotypes and Pol II activity. Interestingly, while multiple TL residues function together to promote proper transcription, individual residues can be separated into distinct functional classes likely relevant to the TL mechanism. In vivo , Pol II activity defects disrupt regulation of the GTP-sensitive IMD2 gene, explaining sensitivities to GTP-production inhibitors, but contrasting with commonly cited models for this sensitivity in the literature. Our data provide support for an existing model whereby Pol II transcriptional activity provides a proxy for direct sensing of NTP levels in vivo leading to IMD2 activation. Finally, we connect Pol II activity to transcription start site selection in vivo , implicating the Pol II active site and transcription itself as a driver for start site scanning, contravening current models for this process.

Cancer chemotherapies suffer from multi drug resistance, high non-specific toxicity and heterogeneity of tumors. We report a method of plasmonic nanobubble-enhanced endosomal escape (PNBEE) for the selective, fast and guided intracellular delivery of drugs through a self-assembly by cancer cells of separately targeted gold nanoparticles and encapsulated drug (Doxil). The co-localized with Doxil plasmonic nanobubbles optically generated in cancer cells released the drug into the cytoplasm thus increasing the therapeutic efficacy against these drug-resistant cells by 31-fold, reducing drug dose by 20-fold, the treatment time by 3-fold and the non-specific toxicity by 10-fold compared to standard treatment. Thus the PNBEE mechanism provided selective, safe and efficient intracellular drug delivery in heterogeneous environment opening new opportunities for drug therapies.

BackgroundSulfur metabolism is required for initiation of cell division, but whether or not it can actively promote cell division remains unknown.Methodology/Principal FindingsHere we show that yeast cells with more mtDNA have an expanded reductive phase of their metabolic cycle and an increased sulfur metabolic flux. We also show that in wild type cells manipulations of sulfur metabolic flux phenocopy the enhanced growth rate of cells with more mtDNA. Furthermore, introduction of a hyperactive cystathionine-β-synthase (CBS) allele in wild type cells accelerates initiation of DNA replication.Conclusions/SignificanceOur results reveal a novel connection between a key sulfur metabolic enzyme, CBS, and the cell cycle. Since the analogous hyperactive CBS allele in human CBS suppresses other disease-causing CBS mutations, our findings may be relevant for human pathology. Taken together, our results demonstrate the importance of sulfur metabolism in actively promoting initiation of cell division.

Chemotherapies are often impeded by drug resistance of cancer cells, high non-specific toxicity and low selectivity and efficacy of drug delivery. We developed a platform for the selective, fast, guided intracellular delivery of drugs into cancer cells with new cell-specific agents, plasmonic nanobubbles (PNBs). PNBs are not particles but transient events, vapor nanobubbles, induced by a short laser pulse around gold nanoparticles. Therapeutic effects of PNBs have a localized mechanical, non-thermal, nature and can be dynamically tuned in cancer cells to support non-invasive imaging, disruption of endosomes and drug carriers, injection of drug and mechanical cell ablation with single cell selectivity in a heterogeneous tissue. Simultaneous treatment of a bulk tissue activates those functions only in cancer cells while leaving adjacent normal cells intact. Optical and acoustical properties of PNBs provide a real time guidance of therapeutic effect. PNB mechanisms were evaluated for intracellular delivery of free and encapsulated doxorubicin into drug-resistant oral cavity squamous cell carcinoma (OCSCC) cells mixed in a co-culture with normal cells. Gold nanoshells were conjugated with Panitumumab antibody to target EGFR that is overexpressed by OCSCC. Receptor-endocytotic targeting resulted in cancer cell-specific clusters of nanoshells. These clusters selectively generated PNBs only in cancer cells while normal cells did not produce PNBs under simultaneous treatment with a single, short near-infrared laser pulse of low, biologically safe, energy. PNBs selectively delivered free extracellular drug only to cancer cells by creating transient nano-holes in cellular membranes and inbound nanojets that brought the extracellular drug into the cytoplasm. Thus PNBs worked as nano-injector of extracellular drug. Compared to a standard treatment with doxorubicin alone, PNB injection improved therapeutic efficacy by 12-fold and at the same time reduced drug dose by 10-fold and non-specific toxicity by 3-fold. Plasmonic nanobubble-enhanced endosomal escape (PNBEE) provided intracellular delivery of the liposome-encapsulated drug, Doxil, that was administered separately with gold nanoshells through a self-assembly of mixed nanoshell-Doxil nanoclusters by cancer cells. Small PNBs generated in these nanoclusters disrupted both liposomes and endosomes and ejected the released doxorubicin into cytoplasm. Cancer cell-specific generation of PNBs in a mixed co-culture of OCSCC and normal cells increased the therapeutic efficacy of Doxil by 31-fold and at the same time reduced drug dose by 20-fold, treatment time by 3-fold and non-specific toxicity by 10-fold. The described mechanisms of nano-injection and PNBEE are universal, can be applied to various cancers and provide a guided intracellular drug delivery in heterogeneous tissues through the application of plasmonic nanobubbles. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5693. doi:1538-7445.AM2012-5693