Date post: | 31-Dec-2015 |
Category: |
Documents |
Upload: | acton-myers |
View: | 19 times |
Download: | 0 times |
Genomics-based determination of nanoparticle toxicity: structure-function analysis
Alex Hadduck – Biochemistry and Biophysics
Dr. Alan Bakalinsky – Food Science and Technology
…And Much More Gas absorption/storage/purification Artificial muscles Superconductors Combat jackets Air pollution filters Bridge building
Toxicity of Buckyballs Largemouth bass DNA deformation Eukaryotic cell growth inhibition Antimicrobial activity The how/why of fullerene toxicity is of great
importance
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.
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.
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
2007 – The Summer of Toxicity Parameters
Toxicity was not established early on We struggled with finding assays that best
met our needs.
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.
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 = 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
-2.5
-2
-1.5
-1
-0.5
0
0 2 4 6
Time (hours)
LN A600
Control
29 mg/L Fullerene
What We Have Learned So Far 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 Continue to gather data either for or against
fullerene toxicity in yeast. The hardiness of yeast may be a blessing in
disguise.