Remember the most important rule of grant writing

1. Remember the most important rule of grant writing • Write with a purpose – not to fill 25 pages! • Everything you put into your grant should have a purpose and help to build the argument for funding • We already talked about the purpose and writing of the Specific Aims, Background & Significance, and Preliminary Data sections • Now you are ready to get into your Experimental Plan

2. Experimental Plan • This is the place to flesh out your specific aims with real experiments • Basically follow a more detailed version of the specific aim anatomy. • Essentially you write this like a paper, you just don’t have the data yet. • You still can construct arguments, weigh evidence etc. • Do not provide a boring technical run down of your experiments! • Make sure the rationale for doing an experiment is always clear, remember the ‘Biology First’ rule. Lead with the problem, then provide the solution. • Argument your way through the project guiding the reviewer through the logic and prioritization • Consider to summarize what you will learn at certain key points

3. Experimental Plan • You have to convince the reviewer that the methods are appropriate, that the experiments have a high likelihood of success and that you are well versed in these approaches • Make sure that your experiments test the hypothesis and that you provide a specific expectation towards the outcome • Discuss different possible outcomes and make clear how such results would impact your hypothesis and how that will change your plans. • What if your approach fails? Provide a discussion of potential pitfalls or problems and offer solutions to these problems or back up strategies • If your strategy is complicated a figure might help the reviewer to understand it.

4. How to handle technical detail (especially in the experimental plan)? • Be mindful of the diversity of reviewers • Some will hear about your area for the first time, while others are the world’s expert on the subject • Your writing has to please & convince both camps • Don’t loose the generalist, and let enough technical sparkle shine through to convince the specialist that you know your stuff • How can you have it all in one document?

5. How to handle technical detail (especially in the experimental plan)? • Ogres have layers! Try to write an onion. • Start the Aim/Subaim with a discussion of the rationale/question • Summarize your technical solution in a way everybody on the panel should understand (e.g. we will test importance by constructing and analyzing mutants) • Then dive into the nuts & bolts (how exactly will you make the mutants) • Wrap up with a discussion of what you will have learned that again is conceptual and not technical • The beginning and end is for everybody the center targets the specialist, make sure that the generalist reviewer can understand beginning and end without the center

6. The Finish line • Make sure you have sufficient time to finish • Proposals riddled with typos and grammatical errors come across as sloppy and annoy the reviewer • Make sure your references are complete and correct. • Have a copy editor!

7. Random thoughts on style • Obviously different folks write differently • Some simple things: • You do not “hope” you expect • Active can be more engaging than passive (phenotypes will be analyzed by … We will analyze the phenotypes) • Every time you want to write “make”, “do”, “look” … think if there might not be a more specific and polished term at your disposal • Let your enthusiasm shine through, find the level of hype you personally are comfortable with • Respond politely and constructively to reviewer criticism • If they did not understand something, do not point out that they are idiots, apologize for making it not clearer and then do a better job in constructing the argument • You can not fight the reviewers you have to win them over

8. Some web-resources: • http://webs.cb.uga.edu/~Estriepen/biopara/cb8500grants.html • http://www.hfsp.org/how/ArtOfGrants.htm • http://www.niaid.nih.gov/ncn/grants/default.htm

9. Toxoplasma & apicomplexan host cell invasion

10. The three kingdoms of life(Mitch Sogin’s 16s RNA tree)

11. Alveolata Dinoflagellata Ciliata Apicomplexa

12. Alveolata • Cortical alveoli or inner membrane complex (flattened membranous cisternae underlying the plasma membrane • Subpellicular microtubuli • Mitochondria with tubular cristae • Molecular phylogeny based on rRNA, tubulin and several other genes solidly supports this grouping PM IMC MT

13. Alveoli (IMC) and apical complex (nice figure by Marc-Jan Gubbels)

14. Apicomplexa • Cells contain an apical complex which is an assemblage of cytoskeletal elements and secretory organelles • No flagella or cilia except for the microgamete (sperm) • All members of the phylum are parasitic

15. Apicomplexa • Apicomplexans are haplonts and meiosis directly follows fertilization • All replication occurs inside of host cells (with the exception of the conclusion of meiosis in certain species) • There are several invasive zoite stages

16. Experimental Plan • This is the place to flesh out your specific aims with real experiments • Basically follow a more detailed version of the specific aim anatomy. • Essentially you write this like a paper, you just don’t have the data yet. • You still can construct arguments, weigh evidence etc. • Do not provide a boring technical run down of your experiments! • Make sure the rationale for doing an experiment is always clear, remember the ‘Biology First’ rule. Lead with the problem, then provide the solution.

17. Experimental Plan • You have to convince the reviewer that the methods are appropriate, that the experiments have a high likelihood of success and that you are well versed in these approaches • Make sure that your experiments test the hypothesis and that you provide a specific expectation towards the outcome • Discuss different possible outcomes and make clear how such results would impact your hypothesis and how that will change your plans. • What if your approach fails? Provide a discussion of potential pitfalls or problems and offer solutions to these problems or back up strategies • If your strategy is complicated a figure might help the reviewer to understand it.

18. How to handle technical detail (especially in the experimental plan)? • Be mindful of the diversity of reviewers • Some will hear about your area for the first time, while others are the world’s expert on the subject • Your writing has to please & convince both camps • Don’t loose the generalist, and let enough technical sparkle shine through to convince the specialist that you know your stuff • How can you have it all in one document?

19. How to handle technical detail (especially in the experimental plan)? • Ogres have layers! Try to write an onion. • Start the Aim/Subaim with a discussion of the rationale/question • Summarize your technical solution in a way everybody on the panel should understand (e.g. we will test importance by constructing and analyzing mutants) • Then dive into the nuts & bolts (how exactly will you make the mutants) • Wrap up with a discussion of what you will have learned that again is conceptual and not technical • The beginning and end is for everybody the center targets the specialist, make sure that the generalist reviewer can understand beginning and end without the center

20. The Finish line • Make sure you have sufficient time to finish • Proposals riddled with typos and grammatical errors come across as sloppy and annoy the reviewer • Make sure your references are complete and correct. • Have a copy editor!

21. Random thoughts on style • Obviously different folks write differently • Some simple things: • You do not “hope” you expect • Active can be more engaging than passive (phenotypes will be analyzed by … We will analyze the phenotypes) • Every time you want to write “make”, “do”, “look” … think if there might not be a more specific and polished term at your disposal • Let your enthusiasm shine through, find the level of hype you personally are comfortable with • Respond politely and constructively to reviewer criticism • If they did not understand something, do not point out that they are idiots, apologize for making it not clearer and then do a better job in constructing the argument • You can not fight the reviewers you have to win them over

22. Some web-resources: • http://webs.cb.uga.edu/~Estriepen/biopara/cb8500grants.html • http://www.hfsp.org/how/ArtOfGrants.htm • http://www.niaid.nih.gov/ncn/grants/default.htm

23. Apicomplexa are intracellular parasites • As almost all apicomplexa T. gondii only replicates within cells • Good experimental system for cell biological and genetic approaches

24. Three modes of intracellular development & replication

25. T. gondii and host cell invasion • Toxoplasma is an opportunistic pathogen • Toxoplasma does not enter the host cell by phagocytosis • Invasion results in the formation of a specialized compartment the parasitophorous vacuole • Parasite motility is needed for invasion (the gliding machinery) • Protein secretion from several secretory organelles is involved in invasion and manipulation of the host cell

26. The Toxoplasma life cycle From Chidoni, Moody & Manser, 2001

27. Toxoplasma is an opportunistic pathogen • 15-70% of the adult population is chronically infected (current rate in the US is 21.5%) • Most people show no or only benign symptoms (head ache, sore throat, lymphadenitis, fever) • In rare case ocular involvement • Two situations can lead to severe disease: loss of a functional immune system and primordial infection during pregnancy

28. Congenital toxoplasmosis is a problem in 1/1000 pregnancies • Both the probability and severity of the disease depend on when the infection takes place during pregnancy (early: low transmission, but severe disease, late: high transmission, more benign symptoms) • Children who are asymptomatic at birth often can develop disease later on

29. Treatment is available • Treatment against parasites as well as to alleviate the symptoms are quite successful • Despite calcification throughout the brain this 10 month old child developed completely normal

30. Do you have to get rid of your cat when you are pregnant?

31. T. gondii is a major pathogen in late stages of AIDS • 25% of all seropositive AIDS patients develop severe Toxoplasma encephalitis • TE can be treated with pyrimethamine and sulfa but not all patients tolerate side effects • In the majority of cases this is due to reactivation of the chronic infection rather than new infection

32. Life cycle of T. gondii

33. Latent bradyzoite cysts confer life-long infection • Cysts form in brain and skeletal muscle • Bradyzoite cyst persist in the immune host • Bradyzoites are resistant to all currently available drugs

34. Bradyzoite cysts are highly infective if ingested • Bradyzoites (not tachyzoites) are resistant to low pH and digestive enzymes during stomach passage • Protective cyst wall is finally dissolved and bradyzoites infect tissue and transform into tachys • Tachyzoites: pathogenesis, Bradyzoites: epidemiology

35. Intracellular parasitism • Macrophages are important “microbe killers”, however several pathogens have found ways to escape killing • Trypansoma cruzi -- induces phagocytosis and escapes into the cytoplasm • Mycobacterium tuberculosis -- induce phagocytosis and block lysosomal maturation • Leishmania appears to thrive in a fully matured lysosome • Toxoplasma was equally thought to induce phagocytosis and then some how block fusion - however, an active invasion model has gained wide acceptance

36. Host cell invasion Dr. Gary Ward University of Vermont