Aya Murakami Background Transmissible Spongiform Encephalopathies and Prion Other for TSEs CWD Distribution in the US Symptoms and Diagnosis Biomarkers Research Goals Results Conclusion Transmissible spongiform encephalopathy (TSE) is a group of progressive diseases that affect the nervous systems and brains of many mammals, including human. Caused by accumulation of a misfolded isoform of the protein called prion protein (PrP) Prion is an infectious agent which forms aggregates in nervous tissues. Two protein conformations exist… PrPsen= native conformation of PrP present in all mammals and is sensitive to digestion with protease. It is a membrane associated protein but its function is unknown. PrPres= misfolded conformation and is resistant to digestion with protease Mad Cow Disease (Bovine Spongiform Encephalophathy; BSE) Scrapie Chronic Wasting Disease (CWD) Creutzfeldt- Jakob Disease (CJD) Cows and other mammals Sheep and goats Deer and elk humans Symptoms Weight loss Behavioral changes: Decreased interactions with other animals Listlessness Lowering of the head Blank facial expression Repetitive walking in set patterns A smell like meat starting to rot Diagnosis Post-mortem: detection of PrPres and vacuolization in nervous tissues. Ante-mortem: RAMALT (recto-anal mucosal associated lymphoid tissue) to detect PrPres. Biomarkers= indicators of diseases/ conditions Pregnancy Albumin in urine to measure the kidney functions ALT/AST level to evaluate the liver functions many other… Easy, fast, non-invasive testing: use of urine/ feces/ blood Biomarkers Possible Functions Dynamin 1 GTPase responsible for endocytosis Calpain Calcium dependent protease TCP-1 α A subunit of chaperonin involved in tubulin and actin folding And others… The Mission: To detect these biomarkers in feces using Western Blotting Why feces? -Easy to collect in the field. -Non- invasive For method development, we used feces samples from captive white tail deer that were inoculated with chronic wasting disease. Western blot is a common technique to detect a protein of interest in a sample using antibodies specific to the protein. Protein Separate proteins on SDS-PAGE Transfer proteins to nitrocellulose membrane Membrane is exposed to specific antibodies Protein bands detected by specific antibodies 250 150 100 75 50 Banshee Banshee Dundee Dundee Kool aid Kool Aid Red Red Niblet Niblet Loki Indy Swift Marker kD 1 21 2 1 2 1 21 2 This is very DIRTY!! We would like to see nice clear bands… Our Goals: 37 To make this better looking… By altering 20 15 extraction buffer dilution of the primary and secondary antibodies Extraction time/ methods antibody: decided 1:300 dilution was the best 1 21 2 1 2 1 21 2 kD Alteration of secondary antibody: decided 1:10,000 was the best This is a blot with 1:300 of 1°antbody and 1: 10,000 of 2°antibody 250 150 100 75 kD Banshee Banshee Dundee Dundee Kool aid Kool Aid Red Red Niblet Niblet Loki Indy Swift Marker Banshee Banshee Dundee Dundee Kool aid Kool Aid Red Red Niblet Niblet Loki Indy Swift Marker Alteration of primary 1 21 2 1 2 1 21 2 250 150 100 75 50 37 50 37 20 15 20 15 There is not much improvement. incubated the samples for 15 hrs to extract the mucus layers. In this experiment, 1, 3 and 5 hr extraction was performed. 1 hr extraction seemed to work the best. Dundee hr Banshee Dundee hr Banshee Dundee Dundee hr Red hr Dundee Kool Redaid hr Kool Aid hr Red RedRed (cont’l) IndyRed hr Niblet Indy hr Niblethr Indy Swift Loki hr Swift Indy hr Swift Swift hr Marker Swift (cont’l) Marker We previously 1 3 5 1 3 5 kD 250 150 100 250 150 100 75 75 50 50 37 37 20 20 15 10 15 1 3 5 1 3 5 1 21 2 1 2 1 21 2 1 hr extraction on the rotary shaker 10 15 10 Kool Aid 15 20 Red 20 50 37 Loki 50 37 250 150 100 75 CWD + CWD - 250 150 100 75 kD Indy extracted the feces 1 2 using tumbler shaker 1 2 1 2 1 2 1 2 kD (360°rotation). In this experiment, we used a rotary shaker instead. Marker Kool Aid Kool Aid Niblet Niblet Red Red Banshee Banshee Dundee Dundee Indy Swift Loki Marker extraction on the tumbler shaker 1 hr We previously 20 45 90 20 45 90 20 45 90 20 45 90 Tried our methods on kD 250 150 100 75 50 37 20 15 10 CWD+ CWD – Marker feces from deer of unknown CWD status. Blot was a little ugly. Decided to alter the buffer to have less salt and detergent. 1A 1B 2A 2B 3A 3B 4A 4B 5A 5B 6A 6B Alter the salt and NaCl: 130 mM detergent Detergent:concentration. 0.5% 1A 1B 2A 2B 3A0.5% 3B 4A 4B→0.05% 5A 5B 6A 6B Detergent: kD 250Addition of phenyl 150 100 75 50 37 20 15 10 methyl sulfonyl fluoride (protease inhibitor) kD 250 150 100 75 50 37 20 15 10 CWD+ CWD – Marker CWD+ CWD – Marker NaCl: 130 mM→10 mM NaCl: 10 mM Detergent: 0.05% 1A 1B 2A 2B 3A 3B 4A 4B 5A 5B 6A 6B Improved the method for protein extraction from deer feces for biomarker detection. Furthermore, these methods seemed to work for deer feces with unknown CWD status. This method has potential to serve as a non-invasive screen for CWD in the field. I would like to thank all the faculty members in Dr. Lewis’ Lab, especially Ted John for walking through the experiments with me and being a great mentor.