Casualties of the Budget Wars

Disparity Between 2001 R01 and Today's R01
Disparity Between 2001 R01 and Today’s R01

I am currently serving as a co-investigator on an R03 project. In NIH terms, this means a small, self-contained 2-year research project with an annual budget cap at $50,000 per year. As co-investigator, it provides me with 5% “Effort.”  That is — this project is budgeted in such a way that I am expected to spend 5% of my time working on it. This works out to be 0.6 months per year, or roughly 2 weeks and 3 days, or 13 days.  I was happy to help write my part of the project when the grant application was being submitted (“rising tides” and all), but I didn’t realize what I was getting into.

For this project, I am supposed to do sub-cellular fractionation followed by Western blotting on 3 regions from 50 mouse brains (25 per year).  Each sub-cellular fractionation generates 5 samples (total protein, crude synaptic densities, large synaptic plasma membranes, pre-synaptic vesicles, endosomal vesicles). Given the limits of the ultracentrifuge and time it takes to process the samples, I can do 6 per day just to generate the samples. (This is an 8-10 hour day, too). So that is roughly 5 days to process one region of one of the cohorts — or 15 days to process all 3 regions from one of the cohorts. In all, this generates 375 samples. We can run 4 Western blots per week (roughly; it’s a 3-day process with lots of incubation times, if you try to do more, it is easy to mess things up); let’s say it takes me 2 workdays to do 4 Westerns (this is generous). At 12.5 samples per Western (12 on one, 13 on t’other — making all 25 from a cohort’s region/fraction on two blots), that is about 16 days (of more or less non-stop benchwork) to complete the cohort. Not to mention data analysis, optimization, instrument preparation, supplies management, the emails, the meetings, the organization, storage, labeling (very important), note-taking, and record keeping. Continue reading

Basics of MicroRNA Biology

Last year, I gave a talk at the Psychoneuroimmunology Research Society (PNIRS) Annual Meeting.  Whatever the merits of the research presented at this meeting … I had a good time and I was tasked with giving a very basic talk as part of a short course. The short course was titled, “A Tutorial on 21st Century Molecular Biology.” I think the reason the conference organizers felt this course would be beneficial is that a large chunk of the researchers in this society are psychologists and psychiatrists; who do not have training in the latest and greatest in cell and molecular biology but they are trying (really, really hard!) to understand how the immune system interacts with the central nervous system, resulting in behavioral changes. In my opinion, they are trying to follow in the footsteps of Richard Sapolsky (who, it seems, has since moved on to other topics), who pioneered the study of stress & stress hormones (e.g., corticosterone), an immune suppressor, and their effects on brain signaling and brain structure, resulting in behavioral changes (e.g., depression). Though a lot of the research done now by adherents, has some serious issues on identifying causality and publish such things as “healing touch” for treatment of PTSD.

My lecture for this educational short course was titled, “The Basics of MicroRNA: A Newly Appreciated Genetic Modulator.”  I liked this presentation because it anthropomorphizes microRNA as a stick-figure with a sheriff’s badge.

 

Use LaTeX to Write Your Grants (and other complex documents)

I’m a big fan of automating tasks and finding efficient ways of doing things. Several years ago, I discovered the LaTeX, a typesetting and document prepping system that has entirely improved the aesthetics of the documents I’m able to produce but also increased the speed with which I can produce them.

It is not a word processing system in which you type on page and what you type appears how the document will be (“what you see is what you get” wysiwyg – wizzeewig). The files that you work in look like plain text, so you can focus on the content and less on how they look as your working. In the file that you’re working in, you designate environments, like section{My Section} … and then go with it … and go onto maybe twenty sections (or chapters, with a zillions subsections or subsubsections [and yes, subsubsubsections]). The same with figures. Why does this matter? Well, it takes away the worry of two key things in a grant document (and any other large document) — format & order.

This is a widely distributed plot of complexity and size vs. effort & time consumption. You'll see it on lots places on the web advocating for TeX.
This is a widely distributed plot of complexity and size vs. effort & time consumption. You’ll see it on lots places on the web advocating for TeX.

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Advanced Teaching Laboratory – Role play style

Several months ago, I posted that I would create an advanced undergraduate laboratory, for example in microbiology to teach experimental design and stats analysis principles to students. I made some progress on this project and wanted to share the outline / proposal. This work, though, is a labor of love. I can’t implement it in my current position, but would need to score an appointment with a teaching role. I am just a research assistant professor in a School of Medicine; writing grants and publishing papers. The concepts and principles could be adapted for any life sciences discipline like molecular biology, physiology, immunology (that would seriously be fun, honestly!), or entomology (even more fun but unlikely). I’m embedding the outline below and here is a link to the full working document, which is a lab manual. I’ll update it as the chapters fill out. Hopefully one day I’ll be able to implement my plans. Happy Autumn.

 

 

Towards an HIV Cure

In the past several months, there have been some highly publicized stories of individuals being “cured” of HIV. I put cured in quotes because these are case reports “functionally cured” … meaning after treatment has been withdrawn, the virus cannot be detected. There’s no way of knowing with 100% certainty that there isn’t at least one latent copy somewhere in the patients’ bodies. This is because of the nature of HIV and retroviruses in particular. The virus integrates its genome into the host’s genome. It can remain there dormant for years or the remainder of the host’s natural life without causing problems. While it is dormant, the host cells can themselves divide and replicate and expand what is known as the “viral reservoir.” At some point, many years down the line, the virus could be reactivated by some event (or from random chance) and what was just a single integration event …. could be thousands of cells producing new viral particles. So the only way to be 100% certain that someone is cured of a retrovirus is to check the genome of each and every susceptible cell and determine that it does not contain integrated latent virus. This isn’t really possible with a living person, so the best we can say is that someone continues to live “functionally cured.”

HIV Life Cycle. Current drugs prevent functioning of viral entry, reverse transcriptase, integrase, and protease. It does not prevent DNA replication via normal cell cycle of a latently-infected cell; which was what “eradication” therapy would target.

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Distance running with (distant) cousins

I like to run. I occasionally take Eli out on runs, which we thoroughly enjoy. There are some differences in our running styles that is a reminder that we are, in fact, different species and our most recent common ancestor lived a very long time ago and probably resembled some rodent-shrew hybrid. I often think about the anatomy of running and one of the key unusual features about human anatomy is how our ankle is arranged. Eli’s leg is, in fact, more representative of living mammals right now and my leg is the unusual one. If you look at his leg, you notice that his femur is relatively small compared to humans, and his tibia & fibula are also shorter than a humans. The major difference, which is what gives us the stability to balance on two legs while propelling is the angle that the tibia & fibula meet the calcaneus & talus. On him, they are elongated and give him torque and spring. On me, they are short, load and impact-bearing bones. For me to “walk” like Eli, it would be like walking on my toes. I tried it this morning and tripped after two steps (despite popular belief, I am not a ballerina). Some runners promote running on one’s toes, saying that it is optimal and less injury-prone, but I am not convinced. I think the loss of stability and reduction in efficiency by using so many different muscles to absorb impact rather than using our well-adapted heel is significantly less efficient.

running-dog-erick
Human skeleton and human specimen (left) with canine specimen and skeleton (right). Note the elongated femur in the human relative to the other bones in the lower limb compared to the canine femur relative to the lower limb. The canine calcineus/tarsus are longer and adapted for spring and torque, while in humans, they are shorter and load/impact bearing; meeting at a shorter angle than in canines.

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Optimally Designing Experiments in Academic Research

When we publish a paper, we usually just show the end result of an experiment that highlights the point we’re trying to make or the “discovery” that we have just made. Behind this one figure, there are typically many, many optimization experiments and titrations that narrowed down the BEST experimental parameters to illustrate this one point. These optimization experiments are often trial-and-error or experiments changing just one condition (or factor) at a time. For example, a simple Western blot has the following factors to consider: Primary antibody concentration, blocking buffer composition, blocking buffer concentration, antibody incubation time & temperature, amount of shaking, secondary antibody concentration, incubation time/temperature, and shaking; and washing conditions. That’s 11 factors for just one experiment and the optimal conditions for each experiment could be different from one experiment to the next. These factors often interact with each other, the optimal amount of antibody might be higher for shorter incubation times and everyone knows that the optimal temperature for short incubation times is higher than long incubation times. But with all these factors to consider, we could spend (waste?) months of time and thousands of dollars just figuring out how to do the experiment before actually testing the thing we’re interesting in. This is where the concept of Design of Experiments (DoE) comes in. This is using statistical principles (and software) to: (1) Consider the possible factors that could influence an outcome, (2) Build a model of how you think they might interact, (3) Design a set of conditions and measurements that covers the appropriate “experimental space,” (4) Measure the outcomes and model the effects of the factors. This is a principle used in biotech/pharma/agriculture and engineering to identify the optimal conditions for a particular process. It is sort of sad that biomedical research and undergraduate life science education ignores this tool, but we can change that. Continue reading

Out of the Box Treatment for Multiple Sclerosis

I came across an article that is an interesting illustration of the scientific process in medicine that’s a good example of : 1. Good science reporting, 2. A well-reported Phase 1 clinical trial, 3. Good prior plausibility, and 4. Out of the box thinking.

There’s been some discussion of  bad science reporting when it comes to medicine. If something is lauded as a miracle-cure and it turns out not to be, then confidence in biomedical research is eroded. Sometimes real harm is done when people forgo evidence-based treatment in search of miracle “natural” cures. This is what is happening with stem-cell research, (too many miracles promised). I think stem cells are a good laboratory model for studying cell biology, possibly of certain diseases — but there’s little plausability that injecting stem cells into the body will cure everything from arthritis to Multiple Sclerosis. Continue reading

Undergraduate Biology Should Teach More Evolution

I have been researching the literature in science education, specifically neuroscience, genetics, and microbiology, and I’m surprised to find that not much import is given to the theory of evolution (or natural selection). Given that “Nothing in biology makes sense except in the light of evolution” (a 1973 essay), I really expected to find a unit on evolution or at least this theme within the curricula of these disciplines.

Neuroscience

Kerchner, Hardwick, and Thornton surveyed faculty of undergraduate neuroscience programs to determine which “Core Competencies” were most important for undergraduate neuroscience majors (for the record, when I was an undergrad, very few colleges offered this field of study as a major, it was regarded to be a specialty reserved for graduate study — for example, you could major in biology, molecular genetics, biochemistry,  evolution-ecology-organismal biology, and microbiology, but not neuroscience at Ohio State).  The three most important core competencies for a neuroscience program:  1. Ability to engage in critical & integrative thinking, 2. Basic neuroscience knowledge, and 3. Scientific Inquiry / Research Skills (in that order). Interestingly, quantitative ability was ranked the least essential by the greatest proportion of faculty.

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