Goodbye Poke, Hello Pee: Alternatives to Plasma for Heart Disease Diagnosis

I admit it: The sight of needles gives me the shivers. The moment the doc requests a blood sample, I want to hightail it to the next state over. And I am not alone in this viewpoint. Many people despise needles. This apprehension can be bad enough that people forgo medical treatment just to avoid the poke.

How nice would it be if lab samples could be collected without involving pain? Happily, this idea is becoming a reality.

Researchers at SomaLogic and the University of California San Francisco have sought to minimize the use of the dreaded needles by utilizing urine instead of plasma to assess heart health (Hraha et al., 2016). When arteries start to harden (arteriosclerosis), changes occur throughout the body, even in kidneys. And certain changes in the kidneys could potentially be observed through fluctuations in protein signatures in urine.

With access to urine and plasma samples from people with stable coronary heart disease, the researchers began their quest. To identify potential biomarkers, they used a version of the SOMAscan® assay that measured 4316 protein concentrations simultaneously. These measurements were compiled with the patients’ medical histories to yield a panel of markers that could predict an oncoming cardiovascular event. The panel performance for urine samples was comparable to a panel developed for plasma, which itself already performed better than standard prediction methods (Ganz et al., 2016).

Jubilation! It is foreseeable that urine collection can replace blood collection in the near future, helping ease people’s minds. It may make them more inclined to go to the doc and not forgo medical treatment.

References

Ganz, P., Heidecker, B., Hveem, K., Jonasson, C., Kato, S., Segal, M. R., . . . Williams, S. A. (2016). Development and Validation of a Protein-Based Risk Score for Cardiovascular Outcomes Among Patients With Stable Coronary Heart Disease. JAMA, 315(23), 2532-2541. doi:10.1001/jama.2016.5951

Hraha, T., DeLisle R., Ash, J., Ostroff R., Williams S., Ganz P. (2016, November). Urinary Proteome and Its Application to Predict Cardiovascular Risk in Patients with Stable Coronary Heart Disease. Poster session presented at American Heart Association Scientific Sessions, New Orleans, LA.



When to Trust Reagents: Gotta Have Faith… or Do We?

Have you experienced the following scenario? You are at a rock concert (or a team-building exercise at work) and lean back into a mosh pit (or group of your co-workers) with the expectation that they will catch you. If you have faith in people, then you would probably lean back and fall. If they catch you, great. If they let you fall, then an enormous “ouch” awaits you.

In research, many times we exercise a blind trust with our reagents and too often experience a huge ouch due to a lack of validation. Sometimes, the ouch can be tolerable. Other times, it truly is a matter of life or death.

Strict quality control is crucial for the development of safe and potent vaccines. A failed vaccination opens the door for the recipient to develop the disease and potentially die, even though the person thinks he or she is immune. Pharmaceutical companies realize this possibility and take great care to ensure the vaccines will work every time, year after year. Antibodies are needed to test vaccine potency, but antibody performance can be questionable at times. Recently, Merck began to explore alternatives to antibodies.

In Merck’s investigation of antibody alternatives, it assessed SomaLogic’s SOMAmer® reagents (Trausch, Shank-Retzlaff, & Verch, 2017). Thanks in part to SomaLogic’s proprietary technology, SOMAmers exhibit the same tight specific interaction with their target proteins as is observed in high quality antibodies. However, unlike antibodies, SOMAmers are made synthetically. This reduces the batch-to-batch variability, increases purity, speeds development, and reduces cost.

The Merck scientists conducting the work remarked on the virtues of the SOMAmers. They noted that SomaLogic delivered SOMAmers possessing the desired specificity. In the vaccine potency assay, the SOMAmers performed well. The use of the SOMAmers allowed the scientists to develop a new version of the assay that required fewer materials, fewer steps and required less time.

As trust in antibodies can falter, it’s good to know that reliable alternatives exist. Ready to lean back?

Reference

Trausch, J. J., Shank-Retzlaff, M., & Verch, T. (2017). Development and Characterization of an HPV Type-16 Specific Modified DNA Aptamer for the Improvement of Potency Assays. Anal Chem, 89(6), 3554-3561. doi:10.1021/acs.analchem.6b04852



I Got You: The Strong Bond Between a SOMAmer and Its Protein

What thoughts give you a warm fuzzy feeling? A basket of golden retriever puppies or wriggly mewing kittens? Maybe loved ones or friends? We humans evolved to be social creatures. Without a social network, we could potentially face a lonely existence that wreaks havoc on our mental and physical health (Perissinotto, Stijacic Cenzer, & Covinsky, 2012). We are not alone in our need of social contact: Many creatures from the humble bee to the giants of the sea need connections to thrive.

In a sense, one can imagine that the tiny nucleic acids called “aptamers” are also “evolved” to share a strong connection with a target, such as proteins. The modified aptamers (SOMAmers®), created out of SomaLogic’s proprietary technology, share some of the strongest bonds with their partners that are difficult to break on their own.

In a recent review, researchers at SomaLogic analyzed the binding interaction of SOMAmers with their targets and compared them to the ones shared between unmodified aptamers and their targets (Gelinas, Davies, & Janjic, 2016). The analysis revealed that the SOMAmer’s unique modifications are critical to the strength of the binding event. This type of binding tends to be stronger than interactions between aptamer and protein, which involve base stacking (an arrangement of nucleic acid bases and amino acids that resembles a stack of pancakes), hydrogen bonding, and electrostatic interactions (positively charged molecules binding to negatively charged molecules). The strong interaction shared between a SOMAmer and its target is more reminiscent of the interaction between antibodies and their proteins than of DNA-protein interactions.

Aside from analyzing the binding interactions, the researchers also compared the structures of both aptamers and SOMAmers. In their analysis, they observed that both aptamers and SOMAmers shared some structural elements that are abundant in the nucleic acid structure field. With their modifications, however, SOMAmers go on to adopt many new and novel structures.  It is increasingly clear that SOMAmer modifications provide the glue that can both hold the SOMAmer together and drive the strong bond with its specific target protein partner.

References

Gelinas, A. D., Davies, D. R., & Janjic, N. (2016). Embracing proteins: structural themes in aptamer-protein complexes. Curr Opin Struct Biol, 36, 122-132. doi:10.1016/j.sbi.2016.01.009

Perissinotto, C. M., Stijacic Cenzer, I., & Covinsky, K. E. (2012). Loneliness in older persons: a predictor of functional decline and death. Arch Intern Med, 172(14), 1078-1083. doi:10.1001/archinternmed.2012.1993



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