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

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

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.

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?

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

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!

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

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

Toxoplasma & apicomplexan host cell invasion

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

Alveolata

Ciliata ApicomplexaDinoflagellata

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

MTIMC

PM

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

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

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

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.

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.

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?

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

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!

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

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

Apicomplexa are intracellular parasites

As almost all apicomplexa T. gondii only replicates within cells

Good experimental system for cell biological and genetic approaches

Three modes of intracellular development & replication

T. gondii and host cell invasion

Toxoplasma is an opportunistic pathogenToxoplasma does not enter the host cell by

phagocytosis Invasion results in the formation of a specialized

compartment the parasitophorous vacuoleParasite motility is needed for invasion (the gliding

machinery)Protein secretion from several secretory organelles is

involved in invasion and manipulation of the host cell

The Toxoplasma life cycle

From Chidoni, Moody & Manser, 2001

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 involvementTwo situations can lead to severe disease: loss

of a functional immune system and primordial infection during pregnancy

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

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

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

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

Life cycle of T. gondii

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

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

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

Host cell invasion

Dr. Gary WardUniversity of Vermont