1. Case-based Learning Using Protein and DNA Simulations to Analyze Cases Based on Infectious Diseases Mark Bergland and Karen Klyczek, University of Wisconsin-River Falls

2. Overview Description of Case It! ProjectDescription of Case It! Project Sample case study in human genetic diseaseSample case study in human genetic disease Hands-on demonstration of DNA analysis softwareHands-on demonstration of DNA analysis software Bacterial DNA analysis (new!) Bacterial DNA analysis (new!) Hands-on demonstration of protein moduleHands-on demonstration of protein module Sample HIV case study Sample HIV case study Other infectious disease case studies (if time permits) Other infectious disease case studies (if time permits)

3. Case It! Project URL for Case It! Home Page: http://caseit.uwrf.eduhttp://caseit.uwrf.edu (please register) http://caseit.uwrf.edu Contact: mark.s.bergland@uwrf.edu Supported by the CCLI program of the National Science Foundation Supported by the CCLI program of the National Science Foundation

4. Case It! Project Electronic framework for analyzing and discussing case studies in molecular biology Genetic and infectious diseases and associated ethical issuesGenetic and infectious diseases and associated ethical issues Students gather background information on casesStudents gather background information on cases Analyze DNA and/or protein sequences usingAnalyze DNA and/or protein sequences using Case It! Version 5.02 beta Case It! Version 5.02 beta Prepare web-page poster using automated systemPrepare web-page poster using automated system Poster session via Internet conferencingPoster session via Internet conferencing Role-playingRole-playing

5. Techniques for DNA analysis Features of Case It! Version 5.0 DNA and protein electrophoresisDNA and protein electrophoresis Restriction enzyme digestion and mappingRestriction enzyme digestion and mapping Southern blottingSouthern blotting Dot blottingDot blotting Polymerase Chain Reaction (single and 96-well)Polymerase Chain Reaction (single and 96-well) ELISAELISA Western blottingWestern blotting

6. Case It! web editor Features of the Case It! web editor Web page constructionWeb page construction Conferencing/discussion boardConferencing/discussion board Collaborate with classes at other institutionsCollaborate with classes at other institutions Instructors can add classesInstructors can add classes Upload class roster, organize teamsUpload class roster, organize teams

7. Sample case: Huntington’s Disease Susan is a 23-year-old whose father, age 55, and paternal aunt, age 61, have been diagnosed with Huntington’s chorea. A paternal uncle, age 66, appears to be unaffected by the disease. Susan wants to know if she inherited the mutated gene from her father so that she can prepare for that future if necessary. She arranges to undergo DNA testing for Huntington’s. Her 17-year old brother, John, also decides to be tested after talking with Susan.

8. Ethical issues raised by the case Should someone with a family history of HD be required to undergo testing? Should they have children if they test positive, or if they have not been tested? Should someone as young as John be tested? Should the results of such tests be made available to insurance companies? to potential employers? to potential mates?

9. Case analysis Case It! Investigator is used to gather background information on case Case It! Version 5.0 beta is used to analyze DNA sequences associated with case Case It! Launch Pad gives access to a web editor/conferencing system -electronic poster sessions -role playing Online tutorial gives detailed instructions (e.g. S. blot)

10. Bacterial DNA analysis Analysis of total genomic DNA Analysis of total genomic DNA »Restriction enzyme digestion (pulsed-field gel electrophoresis (PFGE)) »Southern blot »PCR Example: Salmonella Example: Salmonella »Food poisoning »Zoonotic transmission from pets (turtles, rodents)

11. PFGE to identify Salmonella strains (Nagano et al., Jpn. J. Infect. Disease 59:132, 2006)

12. Using PFGE to generate trees ( Woo and Lee, J. Microbiology, Feb 2006, p. 106)

13. Multiplex PCR to distinguish Salmonella strains (Alvarez et al., J. Clin. Microbiology, Apr 2004, p. 1734)

14. Run Case it simulation (v5.03 beta) to demonstrate these techinques – restriction enzyme digestion, DNA electrophoresis, Southern blot, and PCR Case It simulation runs gels horizontally, so previous two photos have been rotated for comparison… Note: V5.02 currently available for downloading Using Case It to simulate Salmonella studies

17. Protein module Case It! Version 5.0 ELISA (Enzyme immunoassay)ELISA (Enzyme immunoassay) Protein gel electrophoresis/ Western blotProtein gel electrophoresis/ Western blot Infectious disease casesInfectious disease cases »HIV »SARS »West Nile virus »Influenza »Ebola

18. ELISA test results

19. HIV viral proteins gp160 Precursor of envelope protein (env) gp120 Outer envelope protein (env) p55 Precursor of core proteins (gag) p41 Transmembrane envelope protein (env) p32 Integrase (pol) p24 Inner core protein (gag)

20. Western Blot Band pattern interpretation Lane 1, HIV+ serum (positive control) Lane 2, HIV- serum (negative control) Lane A, Patient A Lane B, Patient B Lane C, Patient C No bands Negative Bands for two of these three proteins: gp120, gp41, p24 Positive Any other band pattern Indeterminate Band interpretation: from www.biology.arizona.edu

21. HIV Case studies Case scenario - video and text (HIV – Anna), using Investigator v2 Case scenario - video and text (HIV – Anna), using Investigator v2 ELISA test - initial screening ELISA test - initial screening Western blot to follow up ELISA results Western blot to follow up ELISA results PCR to measure viral load PCR to measure viral load Upload graphical results to Case It web editor Upload graphical results to Case It web editor

22. Case it Web Editor URL: https://caseit.uwrf.edu URL: https://caseit.uwrf.eduhttps://caseit.uwrf.edu Username: guest.student@class.site Username: guest.student@class.siteguest.student@class.site Password: hidFavgi Password: hidFavgi

23. Comments from Zimbabwe students “To me it was an eye opener… Since Zimbabwe is hard hit by HIV it is important to have this project CASE IT since it affects the students’ lives at large. The videos shown are touching. These can help students feel it.” “It was also interesting to network with international students via conferencing. Indeed, the world is a global village.” “(I) was able to get an understanding of how American families react to their family members when they have tested HIV positive”

24. Comments from Zimbabwe students “I had no idea how I could do practicals on H.I.V testing given the limited apparatus and financial limitations in my school.” “I found the use of the computer very useful in simulations because it enables the carrying out of experiments which would otherwise be impossible due to the costs involved”

25. Comments from Zimbabwe students “ The programme was also very interesting because it had cases of real people that got infected in different ways and situations … and the counselling they were given and how some observed the advice and some ignored this advice got themselves into worse trouble. ”

26. Comments from Zimbabwe students “ The subject still remains every teacher’s concern. Of particular concern however, is the increase in the number of orphans. It is estimated that by the year 2005 approximately 910000 children will have lost their parents to AIDS. Poverty, depression, and stigma will haunt most of these children. They will most likely expose themselves to HIV infections in pursuit of basic need and will painfully erode their basic right to family, protection health and food. Our challenge is therefore to intensify awareness education to these children. ”

27. Invitation to participate We welcome participation in the Case It! ProjectWe welcome participation in the Case It! Project No cost to educatorsNo cost to educators High school and university educators are invitedHigh school and university educators are invited Contact mark.s.bergland@uwrf.eduContact mark.s.bergland@uwrf.edu