Send in the Modifications

War movies are full of it. Bullets whizzing past the infantry. Supplies dwindling to the last scrap of shoe leather. The enemy advancing ever closer. Morale falling faster than the apple hitting Sir Isaac Newton’s head. Suddenly, a burst of brilliant light emerges from beyond the hills heralding the arrival of reinforcements. The battered infantry is reinvigorated to make that final push on the enemy’s line and claims victory.

Outside of such Hollywood moments, the arrival of reinforcements actually happens routinely and even in the most unlikely of places. For example, a test tube. Though an unlikely location, scientists at SomaLogic saw the benefits upon the arrival of reinforcements.

At the core of SomaLogic’s technology are SOMAmer® reagents. These reagents are “evolved” to bind a protein and are composed of varying amounts of four nucleic acid bases, one of which is modified with “protein-like” sidechains. These modified bases in turn enable the SOMAmer to tightly bind its target protein, even in a complicated mix of many different proteins.

This effect raises an interesting question: If modifying one of the four bases used to create SOMAmers yields such great protein binders, what happens when reinforcements are added (e.g., what if two of the four bases are modified)? To answer this question, SomaLogic scientists modified a second base, and found that the number of very strong binding SOMAmers significantly increased (as did the number of different binding sites on the target protein) (Gawande et al., 2017). They also found that they could make shorter SOMAmers with no apparent loss of binding capabilities.

These reinforcing modifications and enhanced traits expand the number of proteins that can be targeted by SOMAmers (and, by extension, the reach of the SOMAscan assay into the proteome). They also increase the already broad range of uses for SOMAmer reagents. For example, the use of SOMAmers with two modifications makes it easier to find pairs for sandwich assays (e.g., an assay in which one SOMAmer captures the protein and second SOMAmer detects the captured protein, making a “sandwich” around the protein). With the small size and great stability, the two modifications may make SOMAmers worthy therapeutic candidates or great tools for other applications, such as drug delivery. Clearly, the arrival of these modification reinforcements only strengthens the power of the SOMAmer technology.

Reference

Gawande, B. N., Rohloff, J. C., Carter, J. D., von Carlowitz, I., Zhang, C., Schneider, D. J., & Janjic, N. (2017). Selection of DNA aptamers with two modified bases. Proc Natl Acad Sci U S A. doi:10.1073/pnas.1615475114

Lions and Tigers and Diseases…Oh My!

A newborn fawn laying in a flowering meadow takes its first wobbly steps and soon gleefully frolics. Unbeknownst to the little fawn, a mountain lion intently watches the little morsel. Fortunately, the fawn’s mother knows the world is full of danger and guides her little one to safety.

Although we do not have to worry about being a mountain lion’s next meal (although it occasionally happens), the world still contains many dangers. If we do not want to share the would-be fate of the little fawn, we need our very own sentinels. This could not be truer when it comes to infectious diseases. Too recently, we observed how a small outbreak of a disease, such as Zika or Ebola, can quickly become an epidemic. If these are caught early, then fewer people suffer or lose their lives.

How can we enlist sentinels to stand watch? One way involves the creation of tests that can determine if a person is at risk of developing a serious illness, such as tuberculosis. A person possessing a latent tuberculosis infection (LTBI—i.e., with no obvious symptoms) could eventually develop an active tuberculosis infection that easily spreads. If these individuals can be identified and treated early, then the chance of transmission drops. The tuberculosis tests currently on the market are plagued by false positive results. A highly accurate test is crucial for preventing the global spread of this disease that affects 2 billion people.

Developing an improved assay to identify who is at risk of developing an active tuberculosis infection has been the work of a team of Colorado researchers (De Groote et al., 2017). The group focused on identifying biomarkers indicative of LTBI. The scientists used the SOMAscan® assay to identify biomarker candidates from people who either tested positive (confirmed LTBI) or negative (no LTBI) in three commercially available tuberculosis tests. The group identified several strong protein biomarker candidates and confirmed interferon gamma (IFN-g), a biomarker identified in previous studies. Using IFN-g alone, they could not definitively separate healthy people from those with LTBI (still had false positives). By including another biomarker (interleukin-2) in the search, they could accurately distinguish the LTBI individuals.

Although this work is preliminary, it is a significant step forward in the development of a reliable LTBI test. Thanks in part to the stability of SOMAmer® reagents, we can envision a test that could be readily deployed in remote villages to identify people with latent infections and get them the treatment they need. With this kind of sentinel, tuberculosis infections may become globally eradicated. Also, it will be one less mountain lion-esque danger that we must be concerned about as we frolic through the meadows of life.

Resources

De Groote, M. A., Higgins, M., Hraha, T., Wall, K., Wilson, M. L., Sterling, D. G., . . . Belknap, R. (2017). Highly Multiplexed Proteomic Analysis of Quantiferon Supernatants To Identify Biomarkers of Latent Tuberculosis Infection. J Clin Microbiol, 55(2), 391-402. doi:10.1128/JCM.01646-16

Hot Proteins: The Prion Collective

Resistance is futile. These words have become the catch phrase of the Borg, an iconic alien race determined to assimilate all life into their collective. With no regard or any compassion, the aliens do what they want to achieve their objective. Being able to quickly adapt, defeating these foes becomes a herculean challenge for the protagonists of Star Trek. In these fictional scenarios, the writers can easily add a happy ending and give the heroes the means for conquering the infamous aliens until they meet again.

Life can imitate art. Very much like the aliens uttering the ominous resistance quote, a biological agent spreads throughout the environment and assimilates others into its collective. This biological agent is called a prion. Originating from the misfolding of the prion protein (PrPC) found in all mammals, prions can aggregate. If the aggregate encounters a normal PrPC, the normal protein misfolds and becomes assimilated into the aggregate. As the aggregate grows, havoc spreads through the infected mammal’s central nervous system until the unfortunate animals dies (Huang, Chen, & Zhang, 2015). The prime directive of the prion collective does not end with the animal’s death.

Prions are remarkably stable and can exist in the environment for several years. If they are released via the decomposition of an infected animal or waste (urine and feces), the prions can reside on the vegetation or be taken into the actual plant tissue as the plant grows in prion-contaminated soil. When another animal comes along and eats the infected plant material, the unsuspecting victim becomes infected with the prion collective (Pritzkow et al., 2015).

Can prions from one species infect a different species? The answer is yes. Should we start panicking? Yes and no. Bovine spongiform encephalopathy (mad cow disease), is well known to have crossed the species barrier to infect humans (Murdoch & Murdoch, 2015). Another transmissible spongiform encephalopathy, chronic wasting disease (CWD), is spreading throughout the herds of deer and other cervids in North America. In fact, one in four deer in Boulder, CO has CWD (Miller et al., 2008). Research indicates that while CWD can affect primates, it has been unable to assimilate human PrPC into the prion collective (Kurt & Sigurdson, 2016). Nevertheless, the Centers for Disease Control and Prevention provides information for hunters on how to handle deer or elk carcasses to minimize exposure to the potentially infectious agent (Centers for Disease Control and Prevention, 2017).

Like the alien race, CWD prions may one day adapt and become infectious in humans. If prions can be passed through animal feces and urine, will the food we consume (grown in areas of the country where a significant percentage of the deer are infected) continue to be safe?

A diagnostic test does exist for those concerned that they may have been infected (Groveman et al., 2017). The outlook is grim if the results are positive. It will be sometime before science can come up with a way to defeat this foe.

References

Centers for Disease Control and Prevention (2017). Chronic Wasting Disease Prevention https://www.cdc.gov/prions/cwd/prevention.html

Groveman, B. R., Orru, C. D., Hughson, A. G., Bongianni, M., Fiorini, M., Imperiale, D., . . . Caughey, B. (2017). Extended and direct evaluation of RT-QuIC assays for Creutzfeldt-Jakob disease diagnosis. Ann Clin Transl Neurol, 4(2), 139-144. doi:10.1002/acn3.378

Huang, W. J., Chen, W. W., & Zhang, X. (2015). Prions mediated neurodegenerative disorders. Eur Rev Med Pharmacol Sci, 19(21), 4028-4034.

Kurt, T. D., & Sigurdson, C. J. (2016). Cross-species transmission of CWD prions. Prion, 10(1), 83-91. doi:10.1080/19336896.2015.1118603

Miller, M. W., Swanson, H. M., Wolfe, L. L., Quartarone, F. G., Huwer, S. L., Southwick, C. H., & Lukacs, P. M. (2008). Lions and prions and deer demise. PLoS One, 3(12), e4019. doi:10.1371/journal.pone.0004019

Murdoch, B. M., & Murdoch, G. K. (2015). Genetics of Prion Disease in Cattle. Bioinform Biol Insights, 9(Suppl 4), 1-10. doi:10.4137/BBI.S29678

Pritzkow, S., Morales, R., Moda, F., Khan, U., Telling, G. C., Hoover, E., & Soto, C. (2015). Grass plants bind, retain, uptake, and transport infectious prions. Cell Rep, 11(8), 1168-1175. doi:10.1016/j.celrep.2015.04.036

Back in the Day

The feverish pace of technological evolution during the twentieth century must have been mind-boggling. My grandparents (one born in the nineteenth century and the others born slightly after the start of the twentieth century) had front row seats to witness the transition from horse carts to automobiles, from hot air balloons to trans-oceanic flights, from communicating via telegrams to watching television and using phones, and from cooking over a wood-burning stove to using microwaves. My grandparents and parents also witnessed the emergence of computers, and people walking on the moon. Compared to the last century, technology may not appear to be evolving as rapidly, but it does.

In structural biology, the long time gold standards for obtaining high resolution structures were crystallography and nuclear magnetic resonance. Each technique had its strengths and weaknesses. When it came to studying large macromolecules, the size of the target threw a monkey wrench into the works for both techniques. To solve a high-resolution structure of a large and very important macromolecule, such as the ribosome, and accomplish what many thought was impossible instantly started speculations of Nobel prizes.

While the two techniques dominated the structural biology scene, another technique, cryo-electron microscopy (cryo-EM), started emerging as another viable imaging tool, with many advantages and a few disadvantages. Unlike crystallography, the macromolecules do not need to be coerced into a crystal. Also, cryo-EM eliminates the whole “phase” problem that involves taking diffraction spots and converting them into a meaningful structure. This technique, however, was limited to very large macromolecules, and the resulting structure typically looked like a balloon animal.

In what seems like a recent blink of an eye, the cryo-EM technology has improved considerably. No longer resembling blobs from a 50’s B-movie, the resulting structures now rival those generated from crystallography. Also, the technique is becoming more applicable to smaller molecules (about 5% the size of a eukaryotic ribosome), and it may be possible to go even smaller. If this happens, it is conceivable that cryo-EM could become the predominant means for determining the structure of proteins, RNA, and protein-nucleic acid complexes.

What changed?

The technology improved for both the equipment and the software to compensate for the poor contrast that occurs when the sample is irradiated while the image is taken. In capturing images of the molecules in a super-chilled solution, the process of taking images switched from static snapshots to movies. The new software compiles all the movie frames together to render a high-resolution structure.

As the technology continues to improve, it is possible cryo-EM could become the prevailing technique for solving structures. It’s exciting to realize we are potentially witnessing one of those momentous technological transitions. Before long, our descendants will be pondering our front row seats during this amazing technological age.

References

Kuhlbrandt, W. (2014). Cryo-EM enters a new era. Elife, 3, e03678. doi:10.7554/eLife.03678

Of Bunnies and Pipettes

From the window, I still note the presence of winter. I grab my blanket to avoid the cold draft’s frigid grasp. Low and behold, I start seeing small pops of color piercing the stark white blanket of snow. Crocuses of every shade imaginable herald the long-awaiting news of the winter’s ending reign of tyranny. The crocuses also foretell the ominous arrival of … bunnies!

Yes, bunnies. As adorable as they are, they can be a gardener’s greatest foe, decimating a lovely spring garden. Every year, the fluffy army grows larger and larger. How is it that these critters can reproduce with such great ease in exponential quantities?

Not everything reproduces as easily as bunnies. It may seem hard to believe, but scientific experiments can be difficult to replicate. But why? The reasons are as bountiful as the numbers in the fluffy horde waging war on spring flowers.

When trying to reproduce another lab’s research, the actual published paper can present hurdles. The often-overlooked methods section may omit key details crucial for the success of the project. These “tips and tricks” might include simple techniques, such as warming a solution to get a powder to readily dissolve. Other times, the source and catalog number of the reagents get omitted. This information might be key if different vendors have different manufacturing strategies or if slightly similar products are available.  In addition, authors write the methods section sparsely to meet the character/word limit set by a journal. A call to action has been raised for standardizing the writing of the methods section (Erdemir, 2013).

Sometimes, the reproducibility problem arises from the reagents themselves unbeknownst to the authors/researchers. Not too long ago, researchers learned that mice and rats presented different behaviors during an experiment depending on the gender of the person carrying out the experiment (Sorge et al., 2014). In another example, antibodies can be a source for data variability and headaches (see an earlier blog for expanded discussion on the matter).

To make things even more complex, the weather or location of the lab can affect the experimental outcome. In the crystallography field, stories exist where crystal quality could not be reproduced, with suspicions that the elevation difference or humidity level was responsible. Weather pitfalls are not limited to crystallography, but ordinary bench work as well. Problems can arise if the humidity is too high and causes the unexpectant breakdown of a chemical reagent.

So, how big of a problem is reproducibility in science? A pretty big one, actually. Amgen tried to reproduce the findings from fifty-three published papers, but only succeeded 11 % of the time (Begley & Ellis, 2012). Bayer HealthCare also encountered a similar problem and could only replicate 25% of the findings from their selected studies (Begley & Ellis, 2012). Instances such as these have motivated the creation of the Reproducibility Project: Cancer Biology. So far, the collaboration only could reproduce the findings from 2 of the 5 selected papers (Kaiser, 2017).

If findings cannot be replicated, how useful are they? What should the science community do?

The solutions may be as bountiful as the members of the fluffy brigade. We could follow the suggestion of the call to action for standardizing the methods section of papers. Also, we could upload well written lab notebooks (that include the “tips and tricks,” maybe atmospheric conditions and details about the experimenter for animal studies) to a website for all to see. Maybe the best thing to do, however, would be to reduce the emphasis of being the first to publish a novel finding. Instead, we should reward heavily the research groups that take the time to replicate previous papers, trouble shoot the reasons why a finding could not be reproduced, and publish their findings.

References

Begley, C. G., & Ellis, L. M. (2012). Drug development: Raise standards for preclinical cancer research. Nature, 483(7391), 531-533. doi:10.1038/483531a

Erdemir, F. (2013). How to write a materials and methods section of a scientific article? Turk J Urol, 39(Suppl 1), 10-15. doi:10.5152/tud.2013.047

Kaiser, J. (2017). Rigorous replication effort succeeds for just two of five cancer papers.   Retrieved from http://www.sciencemag.org/news/2017/01/rigorous-replication-effort-succeeds-just-two-five-cancer-papers

Sorge, R. E., Martin, L. J., Isbester, K. A., Sotocinal, S. G., Rosen, S., Tuttle, A. H., . . . Mogil, J. S. (2014). Olfactory exposure to males, including men, causes stress and related analgesia in rodents. Nat Methods, 11(6), 629-632. doi:10.1038/nmeth.2935

Hot Proteins: That Which Endures

What do redwoods, quahog clams, Greenland sharks, bowhead whales, and Galapagos tortoises have in common? Although this sounds like a riddle that could keep the greatest of philosophers up at night, the answer is quite simple: These organisms are among the longest-lived on the planet. While these organisms will outlive us by hundreds to thousands of years, we still have many things in common, including the need to regularly replenish the proteins we make (and which make us).

Not all proteins need to be replenished at the same rate. Some proteins quickly disappear almost the same moment they are created, but others have the potential to linger for centuries. Interest in these long-lasting proteins (LLPs) has spawned a new field of study (reviewed in (Truscott, Schey, & Friedrich, 2016)).

Where in the body do LLPs exist? One of the first LLPs that may pop into a person’s mind is keratin found in hair or fur. Even if removed from a fur/hair-bearing creature or if the creature dies, keratin can last for a millennia or more in the right conditions (mammoths and other ice age creatures have been discovered still bearing their fur). Aside from our exterior, LLPs can be found throughout our bodies. Research has shown that LLPs exist in the eyes, muscles, oocytes, bones, lungs, heart, liver and teeth. Surprisingly, the brain has the most diverse and most identified LLPs.

Just because LLPs can stick around for a long time does not mean that they maintain their original form. Just like automobiles are susceptible to rust and fading, LLPs too can succumb to changes as they age. These changes include a buildup of post-translational modifications (chemical compounds added or removed from the protein), enzymatic digestion, or spontaneous degradation. Though the LLPs are altered, these modified or truncated versions may be stable and detectable for a long time.

The build-up of modified LLPs may be related to many age-related diseases. In the eyes, LLPs have been implicated in many age-related problems, such as cataracts. For other instances, the field is still new, but evidence is mounting. Take amyotrophic lateral sclerosis (ALS) and Alzheimer’s disease as example cases. Both typically occur later in life. In the ALS field, it has been hypothesized that malfunctioning cellular components, which happen to be LLPs, are degrading with age and may contribute to the patients’ deterioration. In Alzheimer’s disease, the brain ravaging plaques contain LLPs that have been spontaneously modified over the years. This toxic build-up could be an underwriting cause for the manifesting symptoms.

Aside from the toxic build-up being a likely root cause of ailments, the build-up of modified LLPs could be the underpinning for other disorders too. For instance, modifications to proteins, such as LLPs, can elicit an immune response. This has been observed for proteins associated with rheumatoid arthritis, lupus erythematosis and potentially multiple sclerosis.

While it may seem that organisms are at the mercy of the changes that can befall LLPs, research shows that mechanisms do exist that can undo some of the age-related changes. However, the specific mechanisms for restoration of LLPs remains unknown. But it is clear the LLPs are bringing a new dimension to the discovery and understanding of disease biomarkers in addition to revealing yet another facet of the complexity of biology.

Resources

Truscott, R. J., Schey, K. L., & Friedrich, M. G. (2016). Old Proteins in Man: A Field in its Infancy. Trends Biochem Sci, 41(8), 654-664. doi:10.1016/j.tibs.2016.06.004