Post on 15-Jan-2016
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