Corinne Byrdsong Spring 2014 Seminar

Relevance Regulation Effects of exposure Method of action Detection methods PCR analysis

1978 - Bulgarian dissident Georgi Markov assassinated in London April 2013 – Letters addressed to the US President March 2014 – Pennsylvania teen March 2014 – ricin in Georgetown dorm Both a criminal and a public health issue

Ricin is considered a biological and a chemical weapon Classified as Category B biological threat by the CDC

Prohibited by international treaties Biological Weapons Anti-Terrorism Act of 1989 Antiterrorism and Effective Death Penalty Act of 1996 USA PATRIOT Act of 2001 Public Health Security and Bioterrorism Preparedness Act of 2002

• Ricinus communis – Castor beans

• A major protein in castor beans

• Comes from the waste of castor oil production

Image from: http://www.printmag.com/design-inspiration/botany-blueprint-the-castor-bean/

Image from: http://dsonggonplant.blogspot.com/2012/02/ricinus-communis-usually-name-called.html

• Many uses for the oil: – Cosmetics – Paints, varnishes – Organic fertilizer – Nylon – Synthetic flavors – Potential biofuel

Image from: http://beautyeditor.ca/2010/11/15/my-brief-traumatic-foray-into-the-oil-cleansing-method-or-how-overreaching-can-sometimes-backfire-on-your-skin/

R. communis grows in most tropical and temperate regions throughout the world The Anarchist’s Cookbook The Poisoner’s Handbook The Internet Easily and inexpensively obtained

Image from: http://en.wikipedia.org/wiki/The_Anarchist_Cookbook

A plant toxin A lectin Ribosome inactivating protein (RIP)

Image from: http://www.wiley.com/college/boyer/0470003790/cutting_edge/molecular_recognition/ricin.gif

Inhibits protein synthesis This leads to the death of eukaryotic cells

Image from: http://ibbio.pbworks.com/w/page/28624940/Nucleic%20Acids%20and%20Proteins

Lethal dose depends on route of exposure Generally, 1 – 20 mg of ricin/kg body weight

Death takes place 36 – 72 hours after exposure, depending on the amount and route of exposure Accidental exposure unlikely

Depends on route of exposure Non-specific but severe

Will ultimately see respiratory, organ failure Ricin poisoning not contagious

No antidote Treat symptoms Try to get ricin out of body

Recent ingestion – flush stomach with activated charcoal

Know if a person has been exposed Decontamination measures Show ricin to be biologically active Presumptive screening

Rapid results Confirmatory analysis

Environmental Sample

Immunological assays

Mass spectrometry

Detection of ricin protein

Biological Assays

Detection of residual DNA Real-time PCR

Environmental Sample

Immunological assays

Mass spectrometry

Detection of ricin protein

Biological Assays

Detection of residual DNA Real-time PCR

Rapid screening Specific High sensitivity Quantitative

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Amplification of specific sequence Ricin-specific primers Ricin-specific probes ‘Real-time’ data collection

Point when amplification first detected during cycle

Important for: Risk assessment Medical treatment

Results in 4-5 hours

Detection of R. communis DNA Detect presence of DNA encoding the gene for ricin A-chain Specific nucleic acid sequences

• R. communis DNA as residual contaminant from ricin preparation

Small amounts detected High detection from various samples

Castor bean suspension Spiked powders

Amount of R. communis DNA Get idea of how much toxin present

Determined using exponential phase Doubling of products

Image from: http://www.biotechniques.com/BiotechniquesJournal/specialissues/2008/April/Twenty-five-years-of-quantitative-PCR-for-gene-expression-analysis/biotechniques-45217.html?pageNum=2

Direct investigators to next step Confirmatory assays

Agricultural plant-breeding programs Low levels of ricin

Ricin Biological and chemical agent Easily processed

Detection Methods Presumptive

PCR Analysis Confirmatory

Future studies

Dr. Staton Dr. Fenger Darcie Winkler Kel Daniel AJ Montañez KariAnna Baber

Bundowle, B., Schutzer, S. E., Breeze, R. G., Keim, P. S., & Morse, S. A. (Eds.). (2010). Microbial forensics. (2nd ed.). Burlington, MA: Elsevier/Academic Press. Hutchins, A. S., Astwood, M. J., Saah, J. R., Michel, P. A., Newton, B. R., & Dauphin, L. A. (2014). Evaluation of automated and manual dna purification methods for detecting ricinus communis dna during ricin investigations. Forensic Science International, 236, 10-15. doi: 10.1016/j.forsciint.2013.12.011. Melchior, W.B., Jr., & Tolleson, W.H. (2010). A functional quantitative polymerase chain reaction assay for ricin, Shiga toxin, and related ribosome-inactivating proteins. Analytical Biochemistry, 396, 204-211. doi: 10.1016/j.ab.2009.09.024 Puri, P., & Kumar, O. (2011). Integrating immunobased detection and identification methods for ricin analysis: An overview. Journal of Bioterrorism & Biodefense, S2:003. doi: Integrating Immunobased Detection and Identification Methods for Ricin Analysis: An Overview Schieltz, D.M., McGrath, S.C., McWilliams, L.G., Rees, J., Bowen, M.D., Kools, J.J., Dauphin, L.A., Gomez-Saladin, E., Newton, B.N., Stang, H.L., Vick, M.J., Thomas, J. Pirkle, J.L., & Barr, J.R. (2011). Analysis of active ricin and castor bean proteins in a ricin preparation castor bean extract, and surface swabs from a public health investigation. Forensic Science International, 209, 70-79. doi: 10.1016/j.forsciint.2010.12.013 Shea, D. A., & Gottron, F. (2010, December). Ricin: technical background and potential role in terrorism. Library of Congress Washington D.C. Congressional Research Service.

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