The Mysterious Case of the Western Blot Tank

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This, my friends, is a gem I picked up a few months ago at surplus for $2. It’s a really old Western blot tank.

For those of you who don’t know, a Western blot is a way scientists have of looking for the presence of specific protein in a sample. The samples are put into this lovely box, and undergo something called gel electrophoresis. You load the samples into a gel tray, and then run a current across it in the box. Because the proteins will move through the gel based on how large they are (the smaller the protein, the faster it will move), you can look for the presence of a band in the same size as your protein of interest. A standardized “ladder” is used to help figure out the sizes of the protein(s) you have. There are a lot of other steps to the Western blot process, but I’ve explained what this tank does.

Now, I originally bought this tank basically to harvest its parts. I want the metal terminals sticking out of the top, and I want the platinum wire inside of it too. Why? So that I can use it for another technique called Clarity. I’ll do another post on this soon. Anyway, there I was at my desk, ready to tear the tank apart, and I read on the side the name “Krebs”. So I stop.

Dr. Edwin Krebs won the Nobel Prize in 1992 along with Dr. Edmond Fischer. They received this honor for their discovery of reversible phosphorylation. What is that and why is it important? Basically, phosphorylation occurs when a phosphate (pictured below) is added to a protein. Usually, adding the phosphate activates the protein, allowing it to perform a certain function. Dephosphorylation, taking away the phosphate, deactivates it. A lot of proteins in our cells undergo phosphorylation/dephosphorylation. And this action allows the proteins to play important roles in various chains of events that are vital to cell function.
phosphate

So, Krebs and Fischer made this discovery at my current school, the University of Washington, in the biochemistry department. Later, Krebs came back to UW and became chair of my department. It’s been a long time since that happened, though, so I’m surprised any of the equipment from Krebs’ lab even lasted this long. I plugged the sucker in and it’s still functional!

The Jobless Ph.D. Generation

My friend leaned forward over the table where we were having dinner. It was a loud, busy restaurant, but she lowered her voice conspiratorially and her eyes took on a sheen of excitement, tinged with fear. “I accepted a position working with a non-profit in the South American rainforest after I graduate, but I haven’t told my professor yet. If I already have a job lined up, he can’t stop me, right?”

At first I considered this apprehensive attitude to be unique and maybe unwarranted. Why wouldn’t a Ph.D. student want to tell her professor about such a unique job opportunity? However, over time, I saw this scenario as a recurring theme and not without reason. Classmates were told that they would not be allowed to pursue non-academic opportunities. Professors scorned the idea of being a “bench monkey” at a private biotech company. Internship programs advertised to us during our Ph.D. interviews were quickly retracted and made to disappear as soon as any real interest was shown.

After entering a Ph.D. program, it quickly became obvious that when in academia, the only respectable job is considered to be, you guessed it, academia. Becoming a research professor is typically considered the holy grail for Ph.Ds .in the sciences. Certainly, one can see how the position is an honorable one. To dedicate one’s life to the pursuit of science and discovery, for the sake of knowledge. And with tenure appointment comes the freedom to pursue the answers to the questions you care about, instead of the questions the stockholders of a company care about.

However, I have major issues with the view of academia as the be all end all of science careers. The drive by the U.S. to produce more scientists began around 1940-1950. Spurred by events such as the Manhattan Project and later the Space Race, increases in funding for science and technology drove a perceived need for more scientists. More recently, reports and testimony from the likes of Bill Gates have continued to encourage the production of more scientists (1, 2). Nevertheless, as numerous recent articles have warned, the population of science PhDs is steadily growing, while the number of available faculty jobs increases at a pace only a snail could envy (1, 2, 3, 4), .

New faculty positions versus new Ph.D.s.
Image courtesy of Nature Biotechnology (Reference 3).

As a result, the competition for faculty positions has become incredibly competitive, and Ph.D.s end up languishing in post-doctoral positions for upwards of 10 years, many never attaining professorship. Yet, instead of universities taking precautions and actively educating their students in a way that would provide them with the skills to be competitive in job markets outside of academia, these institutions continue to maintain a traditional instructional framework (5). While a Ph.D. program provides experience in skills such as project management, problem solving, and communication, students still come out with a very narrow window of extremely specialized knowledge and techniques that are often not transferable to the job market (6).

What can schools do to produce more well rounded Ph.D.s? Progress would be to offer courses that would train students in a wide variety of techniques. This would make them more attractive to potential employers. Additional courses in broader topics such as writing and business would also be beneficial (6). But classes can only provide limited experience; direct, hands-on training is also vital. Therefore, departments should also provide infrastructure and support that provides opportunities for internships and co-ops in a range of companies.

With plunges in federal funding for science research and increases in the number of people earning their Ph.D.s, advances in science may need to come from sources such as private research facilities like Seattle’s Allen Brain Institute. Other groups, such as Microryza and Sage Bionetworks, have started taking advantage of public interest and participation for funding and brainpower. It is becoming clear that traditional research is turning into a broken system.

I think it is time for a change of attitude towards acceptance of non-academic careers. Progress begins with the professors; they must become more open-minded to students’ pursuit of alternate career opportunities. This includes allowing them to devote some of their time to cultivating skills and relationships that will provide a solid foundation upon which to find the right job after graduating. Students, like my aforementioned friend, should not have reservations about discussing their job future with their professors.

In the dynamic job market for scientists where there is an increasing amount of competition for fewer academic positions, it is important for both professors and departments to provide support for their students. This includes changing the general attitude towards jobs that are not in academia, and providing programs that give students opportunities to gain skills and experience that will help them have a fulfilling career in science.

What is my next step towards finding my ideal job? Telling my professor I don’t want to be like him…

References
1. B.L. Benderly, Columbia Journalism Review, 17 January 2012.
2. B.L. Benderly, Scientific American, 22 February 2010.
3. M. Schillebeeckx, B. Maricque, C. Lewis, Nature Biotechnology 31, 938-941 (2013).
4. B. Vastag, Washington Post, 7 July 2012.
5. P. Fiske, Nature 472, 381(2011).
6. The Economist Newspaper Limited, The Economist, 16 December 2010.

Wherein I discover my personal mission statement

I recently entered the following article to a contest. I didn’t win, but it was a great exercise and it really forced me to think about why I want to be a science writer.

Within my group of friends, I am the only scientist. That seems to be unusual. Individuals in the same career field tend to gravitate towards each other. You can complain about the mundane trivialities of your job, and someone in your field can sympathize. Or you can geek out about a new topic or breakthrough. But in my group of friends, I have no co-conspirators. I can’t complain about spending hours on surgeries and behavioral testing, only to find out that my viral injection targeting was off. Nor can I grumble about that annoying primary antibody that just doesn’t work.

However, there is a scenario that occurs regularly when I do talk about science with my non-scientist friends. It transpires whenever they hear about a recently published finding that spreads virally among innumerable media and social networking sites.

Image courtesy of Wikipedia

Excitedly, they describe what was touted by a popular science website to be the big breakthrough of the year. Perhaps it’s a golden bullet cancer cure or definitive evidence that, once and for all, coffee is actually good for you (or not?. Also see here.)

This scenario is why I became interested in science writing. As I saw the reports on research with headlines reminiscent of a tabloid paper, I began to look more closely. I would read the feature, then go to the primary research article and see if the results matched it. More often than not, there were discrepancies. So, the next time I saw the person who triumphantly posted on Facebook about cancer being cured, I would try to explain to them the true meaning behind the research. On the other hand, not everyone gets so excited about science. Some individuals have a deep-seated mistrust in it, especially commercialized science; i.e., pharmaceutical companies. My university often hosts speakers who give talks about the difficulties of treating cancer, and I have lost count of the number of times I have been in the elevator and seen the graffiti on seminar posters yelling in all caps about the lies of big pharma. I believe the media is partially responsible for this attitude.

Often, media sensationalizes the findings of a single publication as being, more or less, a truth. Scientists know that one paper’s results cannot be taken independently. Results must be replicated, tested, criticized, and scrutinized. We also distinguish that they should not always be applied to a broad spectrum of conditions, but rather may only be relevant in a very specific, controlled scenario. We understand that a “cure” for one type of cancer doesn’t mean a cure for all cancers. And that the one “cure” probably doesn’t take into account practicality or effectiveness in human patients, but rather shows a decrease in tumor size in mice. We recognize that while mice and other animal models are invaluable for our research and provide the means to which we can try to better comprehend the workings of the human body, not all findings using animals can be directly applied to humans. Sadly, mainstream media rarely mentions these caveats, so the general public has an incomplete understanding of research science.

The discrepancy between the science reported to the public by the media and a more accurate interpretation of publications breeds a distrust of science and scientists. The relationship between society and science is further muddied when, a few years later, it is reported on the evening news that maybe coffee doesn’t have the health benefits previously broadcasted. It is made worse by the rare instance where a scientist commits the most grievous sin of data fabrication. Given these factors, it’s hard to blame the general public for their confusion and wariness. This disconnect is why I recently started this science blog, where my goal is to examine recently published findings that have been highlighted by the media and explain the true meaning behind the data.

While I have decided to contribute to this endeavor through writing, I believe that it is the responsibility of all scientists to ensure that the public understands the methodology behind what we do. Only then can we gain the support and trust that is vital to the survival of science.

Glow Fish

Now let me clarify my title for a moment here. When I say glow fish, I don’t mean GloFish. Although interestingly enough, there are similarities between GloFish and the fish I’m going to talk about. GloFish have a green fluorescent protein (taken from this jellyfish) or other colored fluorescent proteins, inserted into its genome so that it glows all over – pretty cool.

Image courtesy of Tumblr

Today’s featured article attracted a lot of attention after various popular news sites touted it as showing the ability to visualize fish thoughts. I’ll explain later why I think that’s a bit misleading. This study uses green fluorescent protein (GFP), like the one in GloFish, but it’s modified a bit. This GFP is changed so that it is only activated when calcium binds to another part of it (something called calmodulin). This results in a change in calmodulin, allowing it to bind to something called M13. This binding changes the GFP and allows it to increase its brightness. But why make the GFP this way? Well, neurons, when the are active, let more calcium inside of them, and the calcium activates this GFP. So this modified GFP, called GCaMP, lets you visualize the activity of the neurons. I made the graphic below to illustrate how GCaMP works.

Graphic of mechanism behind GCaMP activation.

There are various ways to actually see the fluorescence. After expressing the GCaMP in an animal like a mouse or rat, you can make the top of their skull really thin and take images through it. But this only lets you look at the top layers of the brain. You can also lower a reeeeally small confocal microscope into wherever-you-please in the brain to look at deeper areas.

Zebrafish embryo. Image courtesy of ucsd.edu

This is extremely difficult to do and very expensive. What the researchers in this article did was put a more sensitive form of GCaMP into a zebrafish – the very same species of fish that the GloFish are. The reason this type of fish is used is because they’re pretty much transparent as embryos. So it should make any fluorescence much easier to see. They are also relatively easy to genetically alter.

So, to the paper! They bred fish that expressed the GCaMP only in the neurons involved with vision. Then they put a display with a moving dot in front of the zebrafish larva. Depending on the direction the dot moved, the neurons that glowed exhibited a corresponding trail of activation. In other words, if the dot moved from left to right, there was a row of neurons that was activated sequentially from left to right. Additionally, separate areas of these “direction-selective” visual neurons were activated by different directions of movement. So the neurons that were turned on by the dot moving horizontally would be different from those turned on by the dot moving vertically. A similar result was seen when the researchers put a mobile paramecium (something the zebrafish naturally eat) near the fish.

So do you think these findings show that we can now visualize fish thoughts? Do fish even think? How do we even define thinking? Does it require consciousness? What I think this article actually showed was the ability to easily visualize the activity of neurons in zebrafish. It’s pretty straightforward, and more of a proof-of-concept technique paper. The tools they used already existed, they just improved upon them. A laudable effort by the researchers made this article accessible, interesting, and media-friendly. Notably, they made multiple videos. This includes a video abstract and clips of the fish in action where you can actually see the GCaMP neurons being activated. When possible, articles should try to do something similar – it makes the scientists reading it excited, and it makes the experiments more accessible to the lay reader.

On Assignment: Non-Dilutive Funding for Biotech Companies

Earlier this month I was asked to be a recap writer for an event held by the Washington Biotechnology and Biomedical Association. The event consisted of a series of panels discussing non-dilutive funding sources, tips for applying, and success stories from CEOs of local biotech companies. Here’s my recap:

Non-Dilutive Funding: Finding It, Getting It, and Using It Strategically to Move Your Company Forward

For small biotech companies, finding sources of funding that don’t require giving up shares has many benefits. This WBBA event consisted of several keynote and panel sessions with speakers who covered topics ranging from non-dilutive funding sources to programs that assist with commercialization of products to stories from CEOs of successful companies.

A panel composed of experts representing organizations that offer sources of non-dilutive funding conversed about specific details they look for in applications:

  • Make sure to involve an expert in the field that your product is tied to.
  • Consider the feasibility and relevance of the study.
  • Have a measurable, hard-line goal to mark the end of your proposed project.
  • Your application should have an adequate business rational/proposition.
  • Know what your product is!
  • Tell a story – detail a clear path in your application.
  • Include a balance of commercialization and scientific details.

Sources of non-dilutive funding include:

The second panel about commercialization programs offered the following advice:

  • Sometimes things might not go according to plan, so be sure to maintain an open line of communication with your funders from the beginning. Have measurable milestones along the way to mark your progress and don’t be afraid to give good and bad news.
  • Programs such as Innovate Washington and WBBA can help entrepreneurs with consulting on how to sell their product. However, it is important to be coachable and open-minded.

Resources for companies looking for assistance in reaching the commercialization stage:

The last panel consisted of CEOs who shared their company’s success stories and gave suggestions based on their experiences:

  • Consider your audience – think about who cares about your product and what they will get out of it.
  • Grants can help show private funders you have a credible and cohesive idea and that you may already have something that works.
  • Personal credibility and networking is important.
  • Expect that funding from a grant may not always arrive on time, so have backup funding sources.
  • Other sources of non-dilutive funding exist besides grants, including your own money, cost-cutting, and revenue from customers who are already paying for your product.

Seminar Short: Dr. Darwin Berg

I thought I’d try my hand at writing short summaries of seminars that I attend. These are aimed a bit more towards readers with some background in biology, as I won’t spend as much time explaining the more basic concepts.

Last week Dr. Darwin Berg from the University of California – San Diego was invited to come speak by my department. The title of his talk was “Nicotinic Control of Neural Nets.” I have to admit, the ‘neural nets’ part caught my eye. I didn’t know what it referred to.

Dr. Berg’s talk focused on acetylcholine receptors. There are two types of acetylcholine receptors – muscarinic and nicotinic. This seminar focused on the nicotinic type (so named due to the ability of nicotine to also bind to them). These receptors directly activate ion channels that are selective for positively charged ions (cations) after acetylcholine binds to them.

The really fascinating thing about these nicotinic acetylcholine receptors is that early in development, nicotinic activity helps convert GABAergic signaling from excitatory to inhibitory (which is their normal signaling function throughout your life). There are two subtypes of nicotinic receptors. One is heteromeric, meaning the five units that make it can be various combinations of α and β types. The other is homomeric and contains only α7 subunits. Dr. Berg’s lab showed that If you knock out this homomeric receptor, you get a delayed switch in GABA function in the hippocampus. Conversely, if you knock out the heteromeric receptor, you do not see this delay.

What is even more interesting about this result is the effect this delay has. You might think that more excitatory activity for longer might be a good thing. Maybe the neurons have more connections because of this initial activity, or help accelerate the innervation between brain areas. Instead, they found that lack of the homomeric α7 receptor decreased innervation as well as the number of glutamatergic synapses. So you need the nicotinic acetylcholine α7 receptor to promote glutamatergic synapse formation during development.

Pretty cool, right? So during development nicotinic signaling is important for converting GABA signaling as well as the maturation of connections and circuitry. Dr. Berg never did mention what he meant by neural nets though….

Thoughts on Cancer “Cure”: Nanoparticles

Conjuring up images of artificially designed and created nanostructures floating through our bodies and delivering drugs to tumors seems futuristic, to say the least.  The visualizations crossing my mind are reminiscent of the cartoonish and ultra-stylized clips distributed in a House episode when they finally figure out what extremely unlikely combination of diseases a person has (lupus + Legionnaires’ disease for the win!); something like this.  The reality about nanoparticles is this: they have a diverse set of uses and hold much promise for therapeutic strategies.

It’s possible that you, like myself, first heard of nanoparticles thanks to this teenager: Angela Zhang.  Angela won the Siemens national science contest for high school students about a year ago for creating a possible cancer cure.  What was this cure?  Nanoparticles.  I didn’t know much about nanoparticles before Angela’s headline flooded my Facebook page, except for hearing about the concept of delivering drugs to specific areas of the body via microscaffolds.  So I dug a little deeper.  What I found is that idea of using nanoparticles for cancer therapy has been around for years.

I know I’m neglecting many notable researchers who have been working with nanoparticles but so that this post doesn’t turn into a review article, I am choosing to feature a particular researcher: Naomi Halas from Rice University in Houston, Texas.  Structures composed of colloidal gold, which is synthesized through a chemical reduction reaction, had been synthesized into nanoparticles before, but Halas’ lab was the first to create gold conjugated nanoshells in the 1990s.  In these assemblies, the gold particles form a coating around a silica core.

So how can these nanoparticles be used therapeutically?

Imaging: If you add iron oxide to the inner silica scaffold of a gold nanoparticle, you find yourself with a powerful imaging tool.  The nanoparticle structure allows for additional conjugation of specific selective antibodies.  These antibodies can be designed to recognize things like a particular type of cancer.  Once they recognize the cancer they bind to it, thereby also binding the nanoparticles to it.  The iron oxide in the nanoparticle is paramagnetic – impressive word, right?  What it means is that the four unpaired electrons in the iron oxide molecule, making it highly magnetic.  This can be useful when imaging using a MRI machine.  The MRI machine is basically a huge magnet, which is able to force the proton of the hydrogen atoms in your body to spin in one direction. The machine then sends out a radio frequency pulse which reverses this spin.  When the hydrogens then revert the spin back to align with the direction of the magnet, they send out a signal that can be detected by the scanner.  Since the iron oxide particles are paramagnetic, they can decrease the amount of time it takes for the protons to realign with the MRI magnet.  Because the hydrogens in your body’s tissue without iron oxide realign slower than those with, this difference increases the contrast of the scanner.  The gold nanoshells themselves also increase imaging capabilities due to their ability to absorb and scatter light at a specific wavelength.  This can enhance the ability of fluorescent molecules to emit light, increasing the intensity of the signal it emits.  Short version: iron oxide nanoparticles gold nanoparticles amplify imaging quality of cancerous areas.

Photothermal Therapy: As I previously mentioned, these gold nanoshells are able to absorb and scatter light.  These properties can result in a temperature increase of the gold, thereby also heating up the surrounding tissue.  You might imagine that if the nanoparticles were not conjugated to an antibody targeting a specific cancer, this thermal activity could result in deleterious effects to your cells.  But if the antibodies are extremely specific, the increased temperature would be confined to areas containing cancerous cells.  This heat results in cellular damage and often death of the cells.

Combination therapy:  Individually, these two therapeutic properties of gold nanoshells are impressive.  But why restrict yourself to one or the other?  The particles can be used for increasing imaging quality if the intensity of light used to excite them is low.  At a higher intensity, the photothermal properties can be employed.

Of course, there are concerns with using this type of tool in humans.  As a pharmacology researcher, my first thoughts go to uptake, retention, and clearance.  If the particle is eliminated from the body too quickly, it doesn’t have enough time to circulate through the body and find the target it needs to bind to.  If it hangs around for too long, it could start to have negative side effects.  This is partly dependent on the size of the particle, but properties such as circulation to/from the target can also come into play.  Stability of the particle itself is also a concern that has already been addressed through the addition of polyethylene glycol, which increases its longevity in the body.

Overall, gold nanoparticles seem to be an extremely useful tool in the treatment of cancer (not to mention its other applications that I didn’t cover here).  What bothers me greatly is the headlines that appeared concerning Angela Zhang’s accomplishments (which I am not trying to negate): they proclaimed that she had found a cure for cancer.  Is this correct?  I don’t believe so.