Transcript PowerPoint - Oregon State University

Genomics-based determination of nanoparticle
toxicity: structure-function analysis
Alex Hadduck – Biochemistry and Biophysics
Dr. Alan Bakalinsky – Food Science and Technology
Fullerene
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Buckyballs -1985 discovery by Kroto, Curl,
and Smalley. 1996 Nobel
Oxygen Radical Scavenger
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Alzheimer’s
Parkinson’s
Lou Gehrig’s
Drug Delivery
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Osteoporosis
Tumor eradication
…And Much More
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Gas absorption/storage/purification
Artificial muscles
Superconductors
Combat jackets
Air pollution filters
Bridge building
Toxicity of Buckyballs
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Largemouth bass
DNA deformation
Eukaryotic cell growth inhibition
Antimicrobial activity
The how/why of fullerene toxicity is of great
importance
Experimental Overview
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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, genedeletion libraries used to screen for fullereneprotective genes – over 4800 non-essential genes.
Insight into toxicity mechanisms.
Expected human (and other) orthologs.
Toxicity Variables
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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.
Deletion Library
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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
2007 – The Summer of Toxicity
Parameters
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Toxicity was not established early on
We struggled with finding assays that best met
our needs.
No visible difference between test and
control
If only…..
New Assays and Endpoints
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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.
Plating
Plating
Plate Counts
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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 = 1x107 cells/mL
Optical Density – Growth Rate
UV spectrophotometer
used to measure the
light scattering of each
test – a direct
correlation to cell
count.
BY4742 Growth 1
0
0
2
4
6
-0.5
LN A600
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-1
Control
29 mg/L Fullerene
-1.5
-2
-2.5
Time (hours)
What We Have Learned So Far
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Not toxic, but we aren’t finished
Colloidal stability of fullerene even more
sensitive than we thought.
Crucial progress on proper procedures and
assays for reproducible data
The Future
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Continue to gather data either for or against
fullerene toxicity in yeast.
The hardiness of yeast may be a blessing in
disguise.
Thank You
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Howard Hughes Medical Institute
College of Science Cripps Scholarship Fund
Dr. Alan Bakalinsky
Jeff Rowe
Vihangi Hindogalla
Dr. Qilin Li
Bin Xie