1. Elucidating Acute-phase cancer responsive proteins from Horseshoe crabs (Carsinocorpius rotundicauda) Group Members: Foo Chuan Hui Joshua (4s2-05) Wong Tuck Wing Ryan (4s2-31) Anu Venkatachalam (AOS) Estelle Gong (AOS)

2. Carcinoscorpius rotundicauda

3. Background “Cancer” refers to a class of diseases with no single cure Current methods demonstrate variable effectiveness May cause harm to other body parts

4. Rationale Survived two mass extinction events over the past 400 million years Have been known to benefit cancer research

5. Rationale Limulus Amebocyte Lysate (LAL) Detects endotoxins, forms clot Innate immune system Rich network of proteins Respond to a variety of Pathogen-associated molecular patterns (PAMPs)

6. Rationale Infection studies on the Singapore horseshoe crab, demonstrated that 10 6 cfu of Pseudomonas aeruginosa was rapidly suppressed Lethal to mice Horseshoe crabs completely cleared the infection within 3 days

7. Rationale Proteins found in the blood of horseshoe crabs potentially provides a more effective way of treating cancer No damage and irradiation to adjacent cells Chemotherapy – toxicity Radiation therapy – damage from radiation

8. Purpose Elucidate specific proteins in Horseshoe crab blood that recognize and bind surface antigens or PAMPs of cancer cells To propose potential peptide-based drugs for cancer detection & treatment.

9. Hypothesis Proteins present in horseshoe crab blood recognize and bind to PAMPs of cancer cells.

10. Variables Independent PAMPs of cancer cells Dependent Responsive proteins Constant Temperature Time of binding Elution buffer used

11. Materials Horseshoe crab blood Human colorectal cancer cell lysate Hydrophobic column SDS-PAGE Sodium Dodecyl Sulphate Polyscrylamide Gel Buffer solutions Urea solution Micropipettes Centrifuge 15mL centrifuge tubes 70% ethanol Autoclave Refrigerator

12. Methodology Identification MALDI-TOF Seperation SDS-PAGE Extraction Hydrophobic Column

13. Method – Collection of blood Horseshoe crabs were collected from the estuary of the Kranji River

14. Method Washed to remove mud and debris Acclimatized Stress might affect composition of blood Washed the carapace around the vicinity of the cardiac chamber with water and swabbed with 70 % ethanol Removes bacteria Prevent clotting of blood

15. Method The crabs partially bled by inserting a sterile needle (18 gauge; Becton Dickinson ™ ), puncturing the cardiac chamber Pressure differences caused blood to be ejected About 10 mL collected for each crab

16. Method Prosoma Opisthosoma

17. Method Needle inserted at hinge

18. Method Hemolymph was collected into pre-chilled, pyrogen-free centrifuge tubes Clarified from hemocytes Centrifugation at 150 x g for 15 min at 4 ºC Cell debris, contaminants and excess hemocyanin were removed Further centrifugation at 9,000 x g for 10 min at 4°C The hemolymph was then quick-frozen in liquid nitrogen and stored at -80 °C.

19. Method – Hydrophobic Column Hemolymph will be passed through an hydrophobic column pre-loaded with the membrane extract of human colorectal cancer cell membranes. Proteins that recognise PAMPs associated with these cancer cells will bind to the column. These proteins will be eluted with increasing concentrations of urea solution.

21. Method – Separation of proteins Collected proteins will be analysed by Sodium Dodecyl Sulphate Polyscrylamide Gel Electrophoresis (SDS- PAGE). Proteins from the SDS-PAGE profile will then be extracted and digested by trypsin.

22. Method SDS-PAGE An electric field is applied across the gel, causing the negatively- charged proteins to migrate across the gel towards the anode Proteins are separated according to electrophoretic mobility Molecular mass

23. Method – Identification via mass spectrometry Lastly, Matrix Assisted Laser Desorption Ionization - Time of Flight (MALDI-TOF) analysis will be conducted to identify proteins or peptides of interest

24. Application Identified proteins can serve as an alternative method of curing cancer, without harmful side effects on the patient.

25. References Ng P M L, Jin Z, Tan S S H, Ho B & Ding J L. 2004.C-reactive protein: a predominant LPS- binding protein responsive to Pseudomonas infection. J Endotoxin Res. 10 (3): 163-74. Medzhitov R & Janeway C Jr. 2000. Innate Immune Recognition: mechanisms and pathways. Immunol Rev. 173: 89-97. Iwanaga S.2002. The molecular basis of innate immunity in the horseshoe crab. Curr Opin Immunol. 14 : 87-95 Stormer L. 1952. Phylogeny and taxomony of fossil horseshoe crabs. J Paleontol. 26: 630-39. ERDG (2003-2009). The Horseshoe Crab. Available online at: http://horseshoecrab.org/med/med.html http://horseshoecrab.org/med/med.html Sharon Rorem (2001). Horseshoe Crabs: True Blue Bloods. Available online at: http://www.suite101.com/article.cfm/aquatic_animals/79177 http://www.suite101.com/article.cfm/aquatic_animals/79177 Maryland Horseshoe Crabs. Available online at: http://www.dnr.state.md.us/fisheries/general/hscpix/hscbiol.html http://www.dnr.state.md.us/fisheries/general/hscpix/hscbiol.html Maryland Department of Natural Resources (2005). Medical Uses. Available online at: http://www.dnr.state.md.us/education/horseshoecrab/other.html http://www.dnr.state.md.us/education/horseshoecrab/other.html Radiation Therapy. Available online at: http://en.wikipedia.org/wiki/Cancer#Radiation_therapy http://en.wikipedia.org/wiki/Cancer#Radiation_therapy

26. Acknowledgements Mentor SRC lab technicians