A visitor, the resulting hackathon, and a nice result.

So, I was sitting in a pub in Oxford (the head of the River - gorgeous place and one that Lewis frequents), and I met this guy +Artem Kaznatcheev. No, he wasn't having a pint at the table next to me. No, he wasn't there for an academic visit.  I met him on twitter, because of a tweet from +Steven Strogatz about mathematics in biology.  Here's how it all began:

In there, we can also see the first thoughts about making this blog - about 6 months before I actually did it. Better late than never?

Anyways, +Artem Kaznatcheev and +David Basanta and I (and some others) started what is now a 10 month long conversation, mostly on Google+, in a community Artem started and we co-moderate, called Evolutionary Game Theory.  This conversation has covered topics (subsequently blogged about) ranging from understanding vs. predicting (followed up nicely by +Philip Gerlee in his blog here), games bacteria play (and a recent +Jeff Gore paper in PLoS Biology), connectors in science and, more recently, the topic of our original connection: the use of game theory in cancer.

The conversations have been lots of fun, we all think a bit differently, but have many of the same ideals about science, understanding and the uses of mathematics.  Further, we are all hopeless nerds and *cough* workaholics.  So, when Artem noticed that the conference he was going to (Swarmfest 2013) was near us, he jumped at the chance to come meet us and get some work done.  On his way down, he gave a couple of David's papers a detailed read through to get the lay of the land (and blogged about it - clever way to annotate things for yourself as well as manage a post).

So, here's the hackathon part.  Artem arrived Wednesday night and he and David worked into the wee hours.  He then came in to #IMO and they spent the day doing a full analytic treatment of a game David and I published in the British Journal of Cancer.  They then worked again into the wee hours at David's house.  I arrived from an out of town trip the next day (Friday).  Artem came in to IMO and gave a talk (which we managed to broadcast on G+, something we hope to continue, but with better sound quality, any ideas on a bluetooth mic?).

Here's me and Chandler looking interested.  Also, you can see we had 4 or 5 others from all over, Germany, Oxford and I don't know where else... 

After his talk, we spent the afternoon identifying a tight question: in a growing tomour, what would change in a simple game or proliferative vs. motile cells between the middle and the edge, if anything?

One of the difficulties in EGT is that neighborhoods and population structure is not considered, indeed, it is assumed that the population is inviscid (well mixed).  A great paper from Martin Nowak at Harvard gave us a way to think about effective neighborhood sizes (formally, how to understand changing game dynamics on graphs of differing, but regular, degree).  This has some obvious applications to growing tumours - when they hit a basement membrane or an organ capsule they go from growing in 'free 3-d space' (neighbors on all sides) to growing almost in 2-d, against a wall (with neighbors only on one 'side').

So, we spent the rest of friday afternoon doing some analysis

Then, on Friday night, we celebrated by buying a bunch of redbulls and working 'till 2am at my house.  I dropped him off at his hotel, then picked him up for a late breakfast and we worked, using this great new on-line app we found +writeLaTeX (which is AWESOME) and started a manuscript.  At dinner time, we broke company...  Then, Saturday night, we really blew off some steam - and made the figures for the paper.  Dropped off at his hotel around 2am, he was picked up by David the next morning and they worked until his plane left.

So, that was the hackathon.  The result, we are proud to announce, is a paper, done and dusted, beginning to end, in 15 days, with the lion's share of the work done in the first 4 days (about 48 hours of which saw the three of us working full on).  To be fair, we thought hard about the question in conversations for several months, and the groundwork had been laid by previous papers, but really, this felt like doing theory the way you're meant to.  It felt inspired.  And, I think, this is the best paper I've been a part of so far.  But, don't take my word for it, check out the preprint, just released on the arXiv today:

http://arxiv.org/abs/1307.6914

We also have submitted it, and it is now under consideration at the Proceedings of the Royal Society, Series B.  While we were motivated by a cancer scenario, we feel the result applies more broadly to biology than many of our previous papers, and so have targeted a broader biological journal.  And, PRS B does publish theory, and has recently published some interesting work from +Arne Traulsen's group at Max Planck on evolution in structured populations, so it seemed like we have a chance...  we'll see.

After this experience, we hope to weave hackathons like this into our schedule more often.  It certainly isn't something I (or my family) could tolerate every week, but it was fun and highly productive and we'll try to do it again soon.

Anyways, we'd love feedback on the paper.  Artem has a more technical post today about the work and some future directions which you can read here.

Visit to Summer Science Program in Socorro, NM

About a year and a half ago, after my talk at TEDMED 2012, I got a call from a medical oncologist asking if I would come to visit a summer camp in the desert of New Mexico that featured a bunch of really smart kids and astrophysics. This sounded right up my alley (I love nerds, and I love stars), but the trip from Oxford to New Mexico seemed a little bit...  far.  So, I said I was interested, but maybe we could talk next year, when I was back in Tampa in IMO - which I promptly forgot about.  Thankfully, they didn't forget, and a year later, I got the call again.  So I packed up and headed for the Summer Science Program's, Socorro, New Mexico campus (on the campus of New Mexico tech, home of the Miners and well known for excellent work on explosives!).

As I left the rental car place in the Albuquerque airport, I asked the attendant how to get to 25 South and he said:

"You mean 25 North, there isn't anything to the south"

Which is how I knew it was the right direction...

After arriving, I was met at my hotel by the site director, Barb, and I asked to go up to the telescope to see the kids doing their observations.  This camp is set up in a really cool way, they spend 6 hours a day in the classroom doing intensive math/physics and computer science (Python) coursework.  They are split into teams of three, and at the very beginning they choose a near earth asteroid (the way they choose the asteroids is kind of a funny story, but maybe one of the students will comment with how that works...).  The teams then get a certain amount of telescope time, during which they take measurements of their asteroid's position.  They are then expected to do the math and write up some code to predict the orbit - which they then share with some astrophysicists at Harvard.  Really cool - not just taking courses and playing with telescopes, but DOING MEANINGFUL SCIENCE. Needless to say, I didn't fly all the way to NM to just give a talk, so at around midnight, I wandered up to the observatory.  It was DARK (perfect) and Barb got out a flashlight, which I asked her to extinguish so I could enjoy the darkness...  she demurred suggesting that we needed it to see any snakes in the path.

Me:  Snakes, pshaw...  wait, you mean like that one?

She ran away, and I snapped this pic - can anyone ID it?  Not the best pic - iPhone flash sucks...  its head was narrow, like a non-venomous snake, but I don't know my high desert fauna...  little help?

We finally got to the telescope and found the group observing.

They weren't able to see their asteroid this night, but they showed me a beautiful globular cluster (pictured) and a spiral galaxy that they found.  How cool. 

The TA who was there gave me a short tour and I found out that he is starting his DPhil in Oxford next year, at Summerville college (right next to the CMB, my home!) doing condensed matter physics.  Small world.

I finally crashed and awoke to take a short run and saw this really cool 'M' in the hills.  This delighted my daughter (Maren - of course the 'M' was for Maren!) who thought of the Thomas the tank engine episode about the Man in the hills.  It turns out it is for the NM tech Miners, but I like the M in the hills better.

Anyways, after my run, I went up to the campus and set up to give my talk,.which you can see here:



 Beyond an introduction into the uses of mathematics in cancer research (and theoretical biology in general) I focused on the need for taking risks in science, and how we ought not shy away from creative thinking.  Further, I talked a bit about my tortuous career path and how having done a ton of different things, and not just following a straight arrow course, had informed my science and my life in general. After the talk, they gave me a sweet green SSP engraved laser pointer (THANKS!) and I had lunch with the students and a chat afterwards...  it was at this point that I started ranting about open access science (I was overtired) and tried to convince them to put the findings from their summer research onto the arXiv (might need an endorsement for astrophysics...anyone willing to help?) and their asteroid tracking code onto github.  Why not!?

Here's the pic.  I was overjoyed to see a large cadre of girls at the camp - a good sign for our future!  I am certainly going to keep this place, a well-kept secret, in mind for when my kiddos are in high school.  I just wish I had had an opportunity like this - and I'm amazed that I had never heard of it.  My high school teacher - Bob Shurtz (LEGEND) - is the coach of the US Physics Olympiad team and is well plugged in in these matters, but had never heard of this camp.  Considering it has been around since Sputnik, this surprised me.  Further, there were kids from all over the world - India, Hungary and China in addition to the US.  Oh well.  In my next lifetime...

Yeah, that says 104F

Oh yeah... it was HOT.

Also worth checking out - the students at SSP have a blog.  Good stuff.  Watch these kids - they are a bright crew.  And, with any luck, I interested one of two of them in theoretical biology!

Great experience as a lecturer, looks amazing as a student.  Spread the word.

The pre-metastatic niche is only half of the story of metastasis (it's the biological one)

Recently, Cancer Research UK posted an article on their blog in which they explain, in layman's terms, recent trends and ideas in research into metastatic spread. The focus of that article is on the concept of a 'pre-metastatic niche', the idea that the primary tumour emits signalling molecules that prime certain organs for the arrival of metastatic cells. We find this line of thought very interesting, as it could, at least in part, explain patterns of metastatic spread, but have strong opinions about how the ideas were presented and the lack of acknowledgment of the other factors that could be at play.  

First, the reader is given a condensed historical background, in which the surgeon Stephen Paget is given credit for having solved the riddle of metastatic patterns 150 years ago. His method of studying metastatic spread in breast cancer is briefly mentioned, however, as is often the case when the seed-soil hypothesis is mentioned, these old 'truths' do not seem to be carefully checked. For example, a much more recent study from by Dr. J Pickren (reported in The Principles of Metastasis by L. Weiss, p. 231, recently reviewed here), which reports a 4:1 ratio between splenic and hepatic metastases (compared to the 14:1 ratio that Paget observed). Another fact not accounted for by Paget in his analysis, is that the liver not only receives arterial blood, but also blood from the gut organs via the portal vein, thereby increasing the chance of it receiving circulating tumour cells (CTCs). If micro-metasases are present in the gut, then these secondary CTCs will most likely lodge in the liver increasing the risk of developing liver metastases. Lastly, Paget only studied a single location of primary tumours, making general conclusions difficult to draw - especially as the connectivity differs greatly between organs. These simple observations should make it clear that Paget's hypothesis is nothing more than an indication of what might be the case in certain circumstances, rather than a settled fact.

From reading the article one also gets the impression that CTCs are drawn to certain organs in the body (e.g. the caption of the 2nd figure reading "Tumour cells are selective about where they end up." or later in text "...which wandering tumour cells find irresistible."). This is not in agreement with what we know today (and have known for the last 30 years) about the dynamics of metastasis formation.

Figure 1: Human vascular system network topology schematic. It is evident by inspection of the network diagram that tumors originating in the gut and lung experience significantly different flow patterns and order in which they experience filtration at capillary beds than tumors originating in other parts of the ‘body’. The alternate pathways (purple) define the fraction of cells which evade arrest (filtration) at a given capillary bed. There are scant measurements of this in the literature, and none for clinical studies.

On the contrary CTCs have little influence over where they end up, instead the correct picture is that of a primary tumour releasing astronomical numbers of CTCs into the blood stream (roughly 100 million cells per day, of which most die in the blood stream), and that these cells are distributed according to physiology of the circulatory system. 

This means that each organ (except the lung and liver) receive a fraction of CTCs in direct relation to their relative blood supply, and only at this point, at which the cancer cells flow through the capillary bed of the organ, can organ specific mechanisms influence the fate of the cancer cell. This means that any explanation of why patterns of metastatic spread look as they do needs to first take into account the characteristics of the circulatory system, and only then the organ specific mechanisms such as the formation of a pre-metastatic niche.

These facts suggest (at least to us) that one should view the formation of the pre metastatic niche from a more passive point of view. The signals secreted by the primary tumour induce a systemic inflammatory response - which may or may not effect all organs. The evidence suggests that some distant sites respond in a way that makes them more hospitable to the CTCs that happen to pass though them and hence these cells are more likely to form overt metastases - but to present this as an active process is to stretch the data and to anthropomorphize to a dangerous extent.

When attempting to synthesize and communicate difficult scientific information to the public, it is always tempting to present a small slice of the story - and indeed, this is good practice as only so much can be communicated effectively at one time.  But when doing this, it is essential to point out where the limits of our understanding are, and not oversell current hypotheses as the 'truth'.  Science is, and always has been, a steady progression toward understanding, paved by models that are (we hope) less and less wrong.  The way we think today is not likely to be the same as the way we think in 10 years time.

Barriers to use of preprint servers

A week or two ago, I wrote a guest post on my friend +Jonathan Eisen's blog where I outlined what I thought were some problems standing in the way of folks utilizing #openaccess #preprint servers.  I argued the micro-community building, like we're trying to do at Warburg's Lens, or that the folks at Haldane's Sieve have been doing, was a way forward.  In that same post, I put a small survey, from which I learned quite a bit, and I want to share those results with you here.

I've never used google speadsheets before, so I learned some lessons about coding answers (doesn't deal well with age ranges etc.) and I had to do a BIT of recoding.  There are lots of ways to look at the data, but I picked two to break out specifically.  First some summary statistics though:

 And...  the reason we did the whole survey.  WHY people don't utilize preprint servers.

I was really surprised by this one.  I thought that the 'afraid of being scooped' answer would win the day (this is the case from my anecdotal experience with asking colleagues).  I have a good answer to this one, to try to allay fears... but we don't even need it!  The number one reason was:

"Pre-print servers?  Meh."

And this is easy to change, it is just a culture shift, and one that is, I think in flux (in the right direction!).

You can see all the responses here, if you want, which includes all of the free text responses - where I think the real fodder for change lies.  These are hard (impossible) to display, except to copy and paste them all in.  But, I can tell you, having read them all, they fall into several categories:

- There is no benefit to me, so why spend the time
*** There is huge benefit, especially to early career folks, as you can show off your work to anyone, with just a link.  Further, with the growing popularity of pre-print discussion forums, you can get valuable feedback before submission, hopefully making the process smoother.

- I want to, but my supervisor/collaborator won't let me.
*** Point those folks to these blog posts!  Start the discussion.

- I would, if only some big fish in my field did it.
*** The Eisen brothers do it, so can you!

- I'd do it if tenure committees/funding agencies would give me credit.
*** This is a big issue, and I can only hope that the work we do here helps makes that change a reality.

- I'd do it if people could cite my preprints
***  THEY CAN!!!  And, google scholar picks up those citations and counts them.  Oh, brave new world, with such alt-metrics in it.

- I don't know the rules of the journals that I want to submit to, and I don't want to jeopardize my chances at "real" publication.
*** Just ask Wikipedia - here's the answer

-  There's too many choices!

-  There's too few choices!

Anyways, please do take a moment to read the responses, they are enlightening. I, for one, have learned that the barriers to getting pre-prints servers more utilized is simply one of outreach - so here I go.  If you are in favor of speeding up (and opening up) science, take a minute to engage a scientist in your lab on this issue.  It will pay dividends.  There are plenty of options for preprint servers now, nicely outlined by +Jonathan Eisen in a post he wrote here, and even more coming on line.  I am particularly excited about the bioRxiv coming out of Cold Springs Harbor, and am one of their affiliates in getting going.  Until then, I have personally used the physics arXiv for my last five papers as well as a test use of PeerJ which has nice alt-metrics.

Ah, the paradox of choice.

Whatever you choose, post your preprints before you submit.  Share your science.  Whatever you study, let everyone else see stand on your shoulders so they can see farther, and further the cause.

Institute for the Future: Health Horizons storification

I was an invited panelist at this year's Institute for the Future: Health Horizons meeting this past week.  I had NO IDEA what I was getting myself into when I accepted the invitation, and I have to say, I was skeptical.  My experience though, was without question, excellent.  Quality organization, quality program, great attendees and a top-notch location.  I'll write a proper post to describe the meeting in my own words, but until then, here is a storify version, in tweets.  Most are my own, but I used some tweets from others when I was on stage, and also to fill in some spots where I slacked off!

Thanks a million to +Miriam Avery for the invite, and to all my other new friends at the IFTF.  I hope to see you all again soon.

Cancer is a sine qua non for life as we know it.

There was a post today on National Geographic talking about a tumor that was found in a fossilized Neandertal's bone. It reminded me that I had written the piece below and hadn't found a home for it yet. The title, which is a big jarring, is:

Cancer is not a disease: there is no cure.

The way we describe things shapes and is shaped by the way that we think of them.  Cancer has been described as a disease for as long as we have written record of medicine.  It’s name comes from the greek word for crab, because the way it wedges itself into the host tissue is so like a crab wedges itself between rocks: inextricably.  

The advent of the microscopic age at the turn of the 20th century brought with it an unprecedented view of the cellular level; anatomy and pathologies came into a new focus and a new science was born: microbiology.  As physicians, we were offered a new opportunity to study diseases at the cellular level, to describe the panoply of new patterns that we saw under the microscope much like a team of explorers coming into undiscovered jungle filled with undescribed flora and fauna.  We found that the cancers in each organ were not necessarily the same.  Indeed, we found a rich diversity of cancers that could be reliably classified and whose prognoses and patterns of progression correlated.  This richness of classification gave rise to the opportunity for disease specific treatment trials, and indeed accounted for most of our progress against these individual entities, and for the standard of care for most cancer types even today.  

The dawn of the genomic age, first with the human genome project, and then with the cancer genome atlas, promised and delivered another wave of discovery and deeper, more detailed classification.  Just as we can now tell how, and when two species of finch, or cave fish, diverged in their evolutionary history, so too can we tell when and how a tumor diverged from its tissue of origin.  Early on in this story, we were tantalized by the discovery of specific genomic errors (mutations) that seemed to explain a cancer’s growth, and with the discovery of imatinib, a targeted ‘cure’ for a specific cancer (CML), and cancer seemed to be on its knees, ready to be cured.  

The final cure, however, has continued to elude us.  As we continue to discover more and more specific mutations, drug companies continue to develop specific drugs to target their action.  Each of these drugs seems to work well in a subset of patients, for a time, but never provides the silver bullet that we have been promised, and ultimately fails in almost every case.  The problem is that we are stuck in a paradigm where each disease has a cause, and each cause has a remedy.  This linear thinking has dominated medicine, and indeed much of science, for most of human history, and has served us well.  But continuing to think of cancer as a disease in this paradigm is not going to get us any closer to a cure - we have to shift our thinking and expectations, and embrace the reality that cancer is the result of a non-linear, highly degenerate process, and therefore has no 'cure'.

We have to shift our focus in the study of the cancer genome and stop trying to develop a comprehensive list of errors in the code that cause cancer, but instead learn the guiding principles behind the process - the equations of motion, if you will.  Cancer is not a disease to be cured, but a pathologic condition of normal tissue evolving according to the very rules which allowed us to emerge from the primordial ooze.  It is an inconvenient sine qua non for existence in our universe, as evolving, living organisms.  

This reclassification is not intended to take anything away from those living with cancer, or who have suffered from it.  Within any single patient, this pathologic condition has the capacity to cause as much, or more, suffering than does any other disease.  And, as oncologists, our calling is to minimize this suffering, and when we can, cure our patient.  But until we stop thinking about cancer as a disease that is the product of a linear process, and realize that it is a pathologic condition that is produced by any number of trajectories across an evolutionary landscape; until we stop looking for a single, silver bullet cure for all patients that doesn’t exist, we will continue to waste time that we could be using to understand the evolutionary dynamics of cancer so we can develop strategies to cure each patient.


Some further reading:

Exploiting ecological principles to better understand cancer progression and treatment
arXiv preprint
Basanta and Anderson

Cancer attractors: a systems view of tumors from a gene network dynamics and developmental perspective.

Huang et al. Semin Cell Dev Biol. 2009 Sep;20(7):869-76. doi: 10.1016/j.semcdb.2009.07.003

Oxidants, antioxidants and the current incurability of metastatic cancers.

Jim Watson, Open Biol. 2013 Jan 8;3(1):120144. doi: 10.1098/rsob.120144.

NIH Loan Repayment Program

When I was trying to decide what to do with my life after the Navy, it came down to two possibilities: medicine or physics.  One, medicine, was wholly new to me but seemed like a good fit for my personality and it was a pretty sure bet, career wise.  The other, physics, had been my passion since I was introduced to it in 1992 by Bob Shurtz, my high school AP Physics teacher (and the greatest physics teacher of all time).  I had a really hard time picking between the two and ended up choosing medicine for pragmatic reasons, but always hoped to be able to incorporate my scientific inclincations with my new career.  I found out pretty late in the game about the MD/PhD programs funded by the NIH through the Medical Scientist Training Program, but applied to a few anyways and was rejected.  So, I started medical school as Case Western and was happy.

I distinctly remember one day during MS-1 when I was shadowing a hand surgeon in the OR at the Louis Stoke VA (our teaching VA in Cleveland), and the surgery resident, a PGY-2 in general surgery, described the NIH Loan Repayment plan to me.  He was going to take a few years off after his PGY-3 year to pursue a PhD in tissue engineering.  The benefit of doing this, he said, was that he could pick his clinical interest first, then pursue a related research area, rather than the MSTP folks who have to pick a scientific discipline before they know what sort of doctor they'll be.  This has come into sharper focus for me as my best friend from medical school did his PhD in cardiac stem cells, assuming he would do cardiology, and fell in love with urologic surgery.  It isn't so much that his training was wasted, it certainly wasn't, but he lost the momentum that he could have had had his clinical interests been aligned with his doctoral studies.  So, in this sense, I'm glad that I wasn't accepted into the MSTP programs, as by going straight through medical school, I found my 'calling' as an oncologist, and then subsequently found how research fits into that - and this is an important distinction.

So, to continue the story, I chose radiation oncology, and now, between PGY-4 and 5, I have been taking time off to do dedicated translational research, and because of the NIH extramural loan repayment program, I am paying off a year of medical school for each year of qualified research!  (Considering the opportunity cost of not practicing radiation oncology, I am still a bit 'behind' each year I postpone taking a faculty job, but this dulls the pain significantly.)

While I was disappointed that I didn't get in to the MSTP programs, I truly feel that, at least for me, this path is one that will better suit me for a rewarding career.  Obviously, each person is different - had I had a burning passion about a certain kind of research early on, then following that path when I was younger might have been easier.  I certainly face a whole slew of personal difficulties as a 37 year old PhD student with 2 young kids that I wouldn't have had I pursued this earlier.  On the other side of the coin, with nearly ten years of medical experience, I bring a lot more to the table for my research than I would have after MS-2.  At the end of the day, I am happy that the NIH funds both pathways, and we are in dire need of more physicians doing science.  Like I said in my TEDMED talk, I truly feel that the physician-scientist plays a unique role in science. Indeed we are specifically trained to connect seemingly disparate pieces of data to build a theory, like a constellation of symptoms to build a diagnosis.  As science gets more and more specialised, and knowledge silos get deeper and deeper, I feel this role will grow in importance as well - highlighting the need to keep these programs going.

While I love science and research, I came out of medical school with ~$200,000 in debt.  I could not have afforded to take this time off if it weren't for this program.  So, thanks NIH.

OK - so some nuts and bolts.  You get a health related doctoral degree (specifics on eligibility here).  Graduate.  Get a job doing research at a non-profit, preferably NIH funded (post-doc sort of thing).  They pay off your loans!  Each quarter I get notified that they sent $8,750 off to Sallie Mae.  The only continuing work, besides the research, is that your supervisor has to fill out a form saying you are working hard (thanks +Alexander Anderson) and you have to verify that the money is going where it is supposed to.  Easy.

Here are some links that the folks at NIHLRP provided me to supply with data about who has gotten funded.

http://www.lrp.nih.gov/pdf/LRPFY2012DataBook.pdf

http://www.lrp.nih.gov/pdf/1211.1_trends_report.pdf

The application process was similar to any other grant writing endeavor.  There was a 6 page project description as well as a biosketch and some institution-specific paperwork.  Importantly, a major component is the training program that you and your mentor create, which must be described in detail in a letter.  For me, this included a PhD training program, but this is not necessary.  Here is my successful grant application:

LRP research plan final from University of Oxford, Moffitt Cancer Center

You can then, competitively, renew this grant as well.  I don't know how that process goes since I missed the renewal application cycle (whoops!).  It turns out, they don't remind you...  But, I'll be submitting a renewal application this year, as long as there isn't a 5 year gap, you can renew.  This is important, as when I go back to finish my clinical training, I will not qualify, as I won't be able to dedicate at least 50% of my time to the research.  However, once I am faculty again, as long as I have educational debt and am spending 50% of my time doing qualifying research, I can continue to apply.

Long winded, sorry.  Short story: great program. Give it a shot!  We need more health professionals doing research, and this is a great way to defray some of the opportunity cost of not practicing.

At press time - I realize I've forgotten a few key points.  They are:

Application cycle runs September 1-Nov 15th.  Funding level is nearly 50%, so your odds are good, and the initial contract period is 2 years.  Go apply!

Also - here is a 'tip sheet' on best practices for applications:  http://www.lrp.nih.gov/pdf/0310_1_application_tips.pdf