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
  dont 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 worlds expert on the
  subject
• Your writing has to please convince both camps
• Dont 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/cb8500gr
  ants.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 Sogins 16s RNA
tree)
11
Alveolata
Ciliata
Apicomplexa
Dinoflagellata
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
  dont 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 worlds expert on the
  subject
• Your writing has to please convince both camps
• Dont 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/cb8500gr
  ants.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