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Adam_Krahn_REU2016_Final Presentation


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Adam_Krahn_REU2016_Final Presentation

  1. 1. A Characterization of Toxin Properties in the Venom of the Wandering Spider Ctenus hibernalis By Adam Krahn Mentor: Dr. Brad C. Bennett
  2. 2. Spider Venom Characteristics • Functions: Paralysis, Lethality, Digestion, and Bacterial Protection • Composition: Proteins/peptides, salts, and other organic components Ctenus hibernalis Dr. Hataway
  3. 3. Study Questions and Objectives Objectives: • Characterize C. hibernalis venom • Assess its biological activity • Express and analyze individual venom proteins Question: • Why is C. hibnernalis venom likely less toxic than Phoneutria nigriventer venom? Phoneutria nigriventer
  4. 4. Methodological Overview • Spider Collection and Venom Extraction • Characterization via SDS-PAGE and BCA Assays • Growth Inhibition Assays for Antimicrobial Activity • Mammalian Cell Assays for Toxicity and Viability • Recombinant Expression of C. hibernalis Proteins
  5. 5. Spider Collection and Venom Extraction Collection: • Site: Homewood Preserve • Method: By hand at night Extraction: • Anesthetized with CO2 • Milked via electrostimulation of the venom gland Venom Extraction Apparatus
  6. 6. SDS-PAGE Showed Most Components Between 50 and 75 kDa and less than 15 kDa Average Venom Concentration: 47.7 mg/mL
  7. 7. Growth Inhibition Assays Revealed No Antimicrobial Effect on 8 Species of Bacteria
  8. 8. Time Course Growth Inhibition Assays Showed No Evidence of Immediate yet Temporary Antimicrobial Activity
  9. 9. C. hibernalis Venom Markedly Reduces C2C12 Myocyte Viability Average Median Lethal Dose Concentration: 0.035 ug/uL Median Lethal Dose Concentrations: 1) 0.030 ug/uL 2) 0.039 ug/uL MTT Assay 96-well plate
  10. 10. Bright Field Microscopy Shows Dose Dependent Morphological Changes Gewies. 2003
  11. 11. Recombinant Expression Was Unsuccessful, Likely Due to Primer Imprecision 6000bp 2072bp 600bp 100bp PCR Amplification (genes 1-5) Vector Digest PCR Test for DNA Template Viability
  12. 12. Conclusions • Venom potency appears selective toward eukaryotic cells • Toxin mechanism of action is likely not purely cytolytic (appears to be necrotic) • A transcriptome of the venom gland is needed to express recombinant venom components
  13. 13. Acknowledgements • Dr. Bennett • Dr. Hataway and Dr. Sahawneh • Dr. D. Johnson and Antonio Bradley • Jonathan Berkuta • Shannon Gilstrap and Kris Martinez • Marissa Garrett • REU Students who assisted with spider catching • Samford University • NSF
  14. 14. References • Casewell, N. R. et al., (2013) • De Paula Le Sueur, L. et al., (2005) • Diniz, M. R. V. et al., (2006) • Haeberli, S. et al., (2000) • Herzig, V. et al., (2002) • Kang, C. et al., (2009) • Kim, M. et al., (2014) • Mattiello-Sverzut, A. C. et al., (1998) • Silva, A. O. et al., (2012) • Vassilevski, A. A. et al., (2009) • Zhong, Y. et al., (2014)
  15. 15. Agar Plate Assays Confirmed the Absence of Growth Inhibition