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Genomics-based determination of nanoparticle toxicity: structure-function analysis Alex Hadduck Biochemistry and Biophysics Dr. Alan Bakalinsky Food Science and Technology Slide 2 Fullerene Buckyballs -1985 discovery by Kroto, Curl, and Smalley. 1996 Nobel Slide 3 Oxygen Radical Scavenger Alzheimers Parkinsons Lou Gehrigs Slide 4 Drug Delivery Osteoporosis Tumor eradication Slide 5 And Much More Gas absorption/storage/purification Artificial muscles Superconductors Combat jackets Air pollution filters Bridge building Slide 6 Toxicity of Buckyballs Largemouth bass DNA deformation Eukaryotic cell growth inhibition Antimicrobial activity The how/why of fullerene toxicity is of great importance Slide 7 Experimental Overview S. cerevisiae submitted to many conditions in order to establish toxicity parameters, mimicking possible environmental exposure. Tests monitored either cell survival or growth rate. Once parameters have been established, gene- deletion libraries used to screen for fullerene- protective genes over 4800 non-essential genes. Insight into toxicity mechanisms. Expected human (and other) orthologs. Slide 8 Toxicity Variables Fullerene forms colloids small uniform aggregates in solution. Aggregate size, and therefore function, very sensitive to solution chemistry. pH, ionic strength (salts), preparation method, and organic matter (including cells) all play a role in how fullerene aggregates. Slide 9 Deletion Library Mutants of a single S. cerevisiae strain, each with a unique and non-vital gene missing. Significant increase in sensitivity in a mutant signifies missing gene plays a role in fullerene protection. Good way to quickly get to the mechanism of toxicity Slide 10 2007 The Summer of Toxicity Parameters Toxicity was not established early on We struggled with finding assays that best met our needs. Slide 11 No visible difference between test and control Slide 12 If only.. Slide 13 New Assays and Endpoints Without being able to reliably recreate results (either positive or negative), our first goal was to re-think how we gather our data. New assays had to be employed we chose to use optical density and plating We also added another possible route of toxicity growth rate inhibition. Slide 14 Plating Slide 15 Slide 16 Plate Counts Say we counted 100 cfu (colony forming units) in a plate after plating 100 microliters of a 10,000 fold dilution. Formula: (cfu/mL plated) x dilution factor = cells/mL So: (100/.1) x 10,000 = 1x10 7 cells/mL Slide 17 Optical Density Growth Rate UV spectrophotometer used to measure the light scattering of each test a direct correlation to cell count. Slide 18 What We Have Learned So Far Not toxic, but we arent finished Colloidal stability of fullerene even more sensitive than we thought. Crucial progress on proper procedures and assays for reproducible data Slide 19 The Future Continue to gather data either for or against fullerene toxicity in yeast. The hardiness of yeast may be a blessing in disguise. Slide 20 Thank You Howard Hughes Medical Institute College of Science Cripps Scholarship Fund Dr. Alan Bakalinsky Jeff Rowe Vihangi Hindogalla Dr. Qilin Li Bin Xie