Know your model.

In biomedical research, we are often trying to answer questions at different levels. By “level,” I mean from molecules, cells, tissue, organ, organ system, organism, community, and ecosystem. It is rare that biomedical research gets to the community and ecosystem level. There’s also a cost / benefit consideration to what “level” your particular research question is answering. Studying a problem at the level of the organism is much more expensive than studying a problem at the level of molecule — however one might argue that studying at the level of the organism is more biologically / clinically relevant.

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The cell model used in my lab, SH-SY5Y cells. Left: Non-differentiated cells, neuroblastoma, continue division. Right: Differentiated cells, stopped dividing, extended axons & dendrites, secrete neurotransmitter.

At each level, you have the thing in your lab that you are actually manipulating and observing and this thing is used to represent something greater, hopefully a more general concept. That’s known as a “model.” Continue reading

Soma to Germline Information Transfer via MicroRNA in Sperm

A few months ago, I posted about an article which  speculated that the evolution of the modern human brain was a result of Lamarckian evolution. To recap — this means that a trait is acquired during one’s life experience, and that trait is transferred to the offspring. The classic example is that giraffes have long necks because a precursor animal stretched its neck to reach high foliage, the next generation was born with longer necks because of its parents’ reaching. The next generation reached yet higher, and the next generation had yet longer necks. In humans, the example is something like a blacksmith has big muscles from working at a forge all day, he passes on his big-muscles to his son because of the use of his arms.

For something like brain development to be influenced by life experiences, I speculated that hormonal changes (e.g., corticotropin release hormone, ACTH, cortisol, norepinephrine) as a result of psychological stress or trauma would have to induce epigenetic changes in sperm.

Imagine my surprise when I directed my web browser to Nature News where researchers at the Brain Research Institute at the University of Zurich discovered that specific microRNAs were indeed increased in the sperm of male mice exposed to psychological trauma (being forced to be away from their mother during rearing, who was undergoing stressful experiences, i.e.: cold-stress or forced swimming)…. and these microRNA increases persisted for yet one more generation, and also was increased in their hypothalamus and cortex. So this is one interesting piece of the puzzle.  The Pubmed Link.

They do not yet know the mechanism of the microRNA-induced changes. But there are several steps that need to be taken to be sure that the observed effect is real, and a few grains of salt.

  1. The study used high-throughput analysis, and screening-type methodologies. These should always be followed up with low-throughput analyses because the reliability of any one target in a high throughput screen of RNA quantity is low. We did this with our PLOS One study. We used a different methodology to quantitate a panel of several microRNAs from our high throughput screen which quantitated 380. We chose some that were up, some that were down and some that were not different. We plotted the signal in the single-target method vs. the high-throughput method to validate that they matched. This, in my opinion, is a must-do, if you plan to make a claim about any one transcript.
  2. The mechanism is not understood. How do the microRNA’s change in the sperm? One good place to start would be to look at the promoter region for the candidate microRNAs that were altered — are these transcription factor sites or hormone response elements (e.g., that would respond to cortisol)?  Are there methylation sites?
  3. The next additional step is to show that changing the concentrations of those microRNAs has some effect on prenatal CNS development.
There are several variable sequences located the the 3` untranslated region (3`UTR) .... which is where the microRNA action takes place. We don't know yet if there are functional microRNA response elements here, but several are "predicted" by the power of having sequenced the human genome. One function of this gene is to feedback-inhibit signaling of hormones, including those responding to stress, but also involved in development & metabolism.
There are several variable sequences located the the 3` untranslated region (3`UTR) …. which is where the microRNA action takes place. We don’t know yet if there are functional microRNA response elements here, but several are “predicted” by the power of having sequenced the human genome. One function of this gene is to feedback-inhibit signaling of hormones, including those responding to stress, but also involved in development & metabolism.

This topic is very near and dear to my own research plans and goals. I’m going to recreate here a figure that I used to illustrate the point of the FKBP5 gene. This protein’s function is to modulate the glucocorticoid receptor nuclear translocation, it essentially inhibits it; so it serves as a feedback mechanism for particular cells to “turn off” cortisol signaling very quickly after it starts. But if FKBP5 is too high … no cortisol signaling was allowed in the first place. There are gene polymorphism in FKBP5 associated with major depression … and one of my future research plans is to determine the big how and why.  One potential way is by altering microRNA response element sequences.   …. aaaand it comes full circle. I would be interested to know of any of the microRNAs discovered to be changed in the sperm of traumatized male mice might also bind to FKBP5.  Cool stuff, huh?

 

Politics, Budgets, and NIH Funding for New Investigators

This post gets into the weeds discussing funding for biomedical research in the US. I want to discuss historical funding trends, changes in NIH policy, the relationship between politics and funding research, and ask whether “peer review” is truly unbiased.

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New R01s for Established (blue) and New (yellow) Investigators (top) over time. Fitted linear lines with 95% confident intervals are also plotted. Proportion of new R01 applications that are awarded to New Investigators (blue, bottom). Noted on the timeline are Government Shutdowns (green dashed), NIH New Investigator Policy changes (solid), NIH budget doubling period (magenta dashed), and the Budget Control Act (Sequestration).

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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

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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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

Proteins are Dynamic & Cool

These pictures are different renderings of the same molecule. It’s called the “BK Channel,” which is short for “Big Potassium (seriously) Channel.”  (K+ is the symbol for potassium ion). It’s called that because it’s the large-conductance potassium ion transporter and it’s located in the membranes of neurons — it’s job is to return the neuron to a resting potential by transporting positively charged K+ ions back into the cell that had been released during an axon “firing” an action potential. It’s essentially a component in a battery-recharge system that allows neurons to be in a state capable of doing one of its functions. The protein itself is just one of those molecules (different colors) and it self-assembles into these groups of 4 identical subunits.

kcnma1s
Different renderings of the BK Channel. It is composed of four identical subunits that assemble in the membranes of neurons. The region that is missing as a result of exposure to Methamphetamine and/or inflammatory factors is highlighted in yellow.

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