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



Gambling with the Genetic Code

In the game of poker, players wager that their dealt hand will win the pot of cash. But the person with the best hand doesn’t always prevail: The player holding the worst hand can still seize control of their fate to win the pot.

In a way, our genes act as the dealt cards in the poker game called life. Many companies will provide a glimpse into the hand of cards by sequencing the genetic code. Based on the player’s genes, the companies provide advice – usually for a decent price – that purports to boost the person’s odds in winning.

Recently, the science writer Rebecca Robbins compared her genetic test results from five different companies (Robbins, 2016). The companies promised that the tests (geared towards athletes) would provide valuable insight and advice tailored to her genetic code, which would help maximize her performance.

Instead of clear insightful information, Rebecca received a mixed bag of molecular information (Robbins, 2016). The companies contradicted one another about how a particular genetic variant would affect her blood pressure, endurance, aerobic fitness, tendon health, and post exercise recovery time. The tests could not tell if her genes made her more suited for sports requiring endurance or short bursts of speed. Based on her genetic code, the “tailored” advice she received for optimizing her performance (e.g. stretching before exercising, eating a sensible/healthy diet, staying hydrated, etc.) was basic common sense that could benefit ANYONE in the general populace.

Aside from looking for a competitive edge in sports, many genetic tests will look for mutations that may determine the actionable course of treatment for a patient. However, the presence of the variant will not always change the course of standard medical treatment. Take the case of thrombophilia (a condition where a person can easily develop life threatening blood clots) as an example. Every year, Medicare spends $300 to $670 million dollars on genetic tests that determine if patients are likely to develop thrombophilia (Ross, 2016). Yet even if the tests came back positive, the regimen for treating a blood clot would not change (Ross, 2016). Why are patients and doctors willing to undergo the extra testing and spend the extra money?

Maybe it has something to with DNA sequencing becoming cheaper and the turnaround time for results becoming quicker? Maybe it has something to do with success stories about patients receiving treatment based on genetic information? Numerous cases exist in the literature about a patient (let’s say a cancer patient) getting a quick swab of the cheek, blood draw, or biopsy of the tumor. The results from the genetic testing reveal that a harbored mutation would make the patient more responsive to treatment X versus treatment Y. After undergoing the treatment suggested by the test results, the patient goes into remission. While this sounds like a cutting-edge breakthrough in modern medicine applicable to every ailment, it is a rare scenario in the grand scheme of things. It’s a good reminder that we do not necessarily need more information. We just need the right information.

Despite the genetic code appearing concrete, the dictated outcomes are malleable and heavily influenced by external factors, such as lifestyle choices, environmental factors, etc. In a recent study published in the New England Journal of Medicine, researchers observed that people with genetic predisposition for heart problems who lived a healthy lifestyle fared better than those with great genes who made poor lifestyle choices (Khera et al., 2016). In another study, people were identified harboring mutations guaranteed to cause serious ailments. Interestingly, these individuals were physically fine and showed no signs of the genetically caused ailment (Chen et al., 2016).

It is evident from these cases that getting a peek at our genetic playing cards is not going to give us the absolute certainty of winning. Something more than genetics is responsible for what is happening. One possible explanation could be proteins becoming activated or inhibited as a response to external cues, which might affect their measureable concentration within the body. Using SomaLogic’s SOMAscan technology, the changes in protein levels can be monitored as the body responds to external cues, such as diet, exercise, etc. By monitoring the protein levels, it is possible to gauge a patient’s chances of a cardiovascular event (Ganz et al., 2016), adjust drug dosage for maximum benefit (Park et al., 2013) or determine if a patient may have an adverse reaction to a drug (manuscript in preparation). This kind of information can be used more effectively by patients and their doctors to seize control and make more informed decisions towards a win in the poker game called life.

Resources

Chen, R., Shi, L., Hakenberg, J., Naughton, B., Sklar, P., Zhang, J., . . . Friend, S. H. (2016). Analysis of 589,306 genomes identifies individuals resilient to severe Mendelian childhood diseases. Nat Biotechnol, 34(5), 531-538. doi:10.1038/nbt.3514

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

Khera, A. V., Emdin, C. A., Drake, I., Natarajan, P., Bick, A. G., Cook, N. R., . . . Kathiresan, S. (2016). Genetic Risk, Adherence to a Healthy Lifestyle, and Coronary Disease. N Engl J Med, 375(24), 2349-2358. doi:10.1056/NEJMoa1605086

Park, N. J., Wang, X., Diaz, A., Goos-Root, D. M., Bock, C., Vaught, J. D., . . . Strom, C. M. (2013). Measurement of cetuximab and panitumumab-unbound serum EGFR extracellular domain using an assay based on slow off-rate modified aptamer (SOMAmer) reagents. PLoS One, 8(8), e71703. doi:10.1371/journal.pone.0071703

Robbins, R. (2016). Genetic tests promised to help me achieve peak fitness. What I got was a fiasco.   Retrieved from https://www.statnews.com/2016/11/03/genetic-testing-fitness-nutrition/

Ross, C. (2016). Genetic test costs taxpayers $500 million a year, with little to show for it.   Retrieved from https://www.statnews.com/2016/11/02/genetic-test-medical-costs/



Hot Protein: FUS bucket

Remember the ice bucket challenge? Sure, it was a great way to rapidly refresh on a balmy summer day while producing a short video that could make you a short-lived Facebook trend. However, the challenge had the far more noble purpose of raising money and awareness for amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig’s disease. The hallmark of this disease is the continuous deterioration of one’s ability to move until death, which occurs 3-5 years after diagnosis. Genetics alone accounts for only about 10% of the cases. The causes of the other 90% of cases are unknown and the subject of many research projects.

Interestingly, 3 to 5% of the ALS cases known to be genetic in origin have mutations in the “Fused in sarcoma/ translocated in liposarcoma” (FUS/TLS or FUS) gene. With the ability to bind DNA, RNA and other proteins, the unique protein FUS may participate in many crucial cellular functions. It has been implicated in regulating RNA synthesis, repairing broken DNA strands, and regulating telomeres (DNA at end of chromosome that shortens with age). Although it is not completely understood how FUS binds to RNA, research demonstrates that FUS may contribute in messenger RNA (mRNA) processing, the localization of mRNA within the cell and be associated in the translation of mRNA into protein.

Recent research has shown that the mutant FUS protein localizes more to the cytoplasm and associates more with stress granules (cytoplasmic aggregates of RNA and protein that form in response to metabolic or environmental stresses). These stress granules act as holding facilities for mRNA that were actively being translated into protein, but halted. What the cell requires to return to homeostasis will largely affect the fate of the stalled translation complexes. The mutant FUS affects the dynamics, increases the size, and boosts the number of stress granules. In deceased ALS patients, markers for the granules have been observed. It has been postulated that the stress granule build up serves as the forerunner for the aggregates detected at the end-stage of ALS.

Aside from stress granules, dysfunctional FUS can influence the development of ALS in other ways.

Many of the FUS mutations correlated with ALS occur in FUS’s DNA-binding sites. These mutations could alter FUS’s role in DNA repair. It is also plausible that FUS’s role in telomere biology might be impaired by these mutations or by a different set of mutations. If the DNA damage builds up or telomeres erode too quickly, it can lead to cell death. This finding does not bode well for the majority neurons that do not replicate because it would imply that they will not last very long.

Although it may sound like we know almost everything that is needed to know about FUS, we still see only the very tip of the iceberg. Are there factors other than genetics that can cause FUS to go rogue and lead to a sporadic case of ALS? Only with more research will we get closure to fully understanding the underlying causes of this debilitating/ deadly disease, and douse it with an appropriate and effective cure.

Reference:

Sama, R. R., Ward, C. L., & Bosco, D. A. (2014). Functions of FUS/TLS from DNA repair to stress response: implications for ALS. ASN Neuro, 6(4). doi:10.1177/1759091414544472



I Can See You Clearly Now: Use of Aptamer-Based Therapeutics to Treat Age-Related Macular Degeneration

What do you enjoy looking at the most? A grinning baby? A beautiful sunset? A cherished loved one? What if you learned that you would no longer be able look at those ever again? For the nearly two million people who are diagnosed with some form of age-related macular degeneration (AMD) each year in the U.S., this is a brutally cold reality (Brown et al., 2005).

Two forms of AMD exist (American Macular Degeneration Foundation, 2016). The first form, known as the “dry” version, constitutes the majority of AMD cases. The dry version is characterized by the thinning of the macula (the portion of the retina responsible for the fine details we can see) and by the buildup of drusen (yellow clumps). The second form, known as the “wet” version, only makes up 10% of AMD cases. The growth of new blood vessels that grow into the layer between the white of the eye and the retina causes the wet version. Being very weak, the blood vessels tend to leak behind the macula, which causes the macula to distort and impair vision. In both types, the peripheral vision remains intact, but the central vision becomes compromised and blurry.

Although the wet version is not as prevalent as the dry form, several treatments do exist. One subset of these vision-saving drugs are based on aptamers (Drolet, Green, Gold, & Janjic, 2016), similar to molecules that underlie SomaLogic’s unique protein measurement technology.

Several decades ago, vascular endothelial growth factor (VEGF) emerged as a key player for eye disorders, such as AMD. VEGF was responsible for not only the leakage of blood vessels, but also inducing the growth of new ones, which had been observed in the wet form of AMD. Treatment of AMD with aptamer-based drugs became attractive for several reasons. For administering a drug into the eye, only a small amount of the aptamer would be required.  The addition of the drug to the eye has a reduced chance of triggering an immune response. Also, the retention of the drug in the eye typically is much longer than elsewhere in the body. With an aging population with deteriorating eyesight, demand for an effective AMD drug definitely existed.

As evidence mounted for VEGF being an attractive target for AMD, concern also grew that inhibiting all the different forms of VEGF could lead to undesirable side effects. Therefore, a group of researchers at a biotech company called NeXagen (which later became NeXstar, and was subsequently acquired by Gilead) focused on a short and relatively abundant version of VEGF, so-called VEGF-165, which seemed to be a target for inhibition without the same range of side effects.

After a decade involving development, optimization, and clinical trials, the aptamer inhibitor for VEGF-165 (referred to now as Macugen) became the first aptamer-based therapeutic to receive FDA approval. This drug indeed improved the vision of many wet AMD sufferers. In its first year, the sales of Macugen were ~$185 million dollars (U.S). But, as often happened, Macugen soon had competition from compounds that targeted all versions of VEGF, and which outperformed Macugen.

During the time that Macugen was being developed, the scientists also began development of an inhibitor towards another protein of interest, platelet-derived growth factor (PDGF). They had learned that the new blood vessels in the eye become less reactive to VEGF inhibition as they mature. Thus, inhibiting both VEGF and PDGF may reduce the spread of slightly more mature blood vessels. In AMD animal models, combining Macugen and a PDGF inhibitor worked better than the use of either one alone.

The Ophthotech Corporation took this new PDGF inhibitor (now called Fovista) through several phases of clinical testing. In a Phase 1 study, the combined use of Fovista and Lucentis (a Macugen competitor drug aimed at VEGF) significantly increased the number of participants whose vision improved compared to individuals who received only Lucentis. This study also yielded a first for AMD treatment: The new problematic blood vessels began to disappear for all the participants who received the combination therapy. Further positive results in Phase 2 studies prompted Ophthotech  to move forward with Phase 3 clinical studies.

PDGF and VEGF are not the only targets that have had new aptamers inhibitors created. A potent aptamer-based inhibitor has been made to Complement component 5 (C5), another protein implicated in AMD. This aptamer (called Zimura by Ophthotech) is showing promise in early clinical trials.

Decades ago, AMD patients did not have a lot of options for how to preserve their sight. With more drugs coming onto the market and new technology being developed, the potential of maintaining clearer vision becomes greater. Also, the reality for AMD patients becomes rosier. They have a better chance of prolonging their ability to clearly witness the beauty around them and see the smiles of happy loved ones.

References

American Macular Degeneration Foundation. (2016). About Macular Degeneration. Retrieved on August 19, 2016 from https://www.macular.org/about-macular-degeneration.

Brown, G. C., Brown, M. M., Sharma, S., Stein, J. D., Roth, Z., Campanella, J., & Beauchamp, G. R. (2005). The burden of age-related macular degeneration: a value-based medicine analysis. Trans Am Ophthalmol Soc, 103, 173-184; discussion 184-176.

Drolet, D. W., Green, L. S., Gold, L., & Janjic, N. (2016). Fit for the Eye: Aptamers in Ocular Disorders. Nucleic Acid Ther, 26(3), 127-146. doi:10.1089/nat.2015.0573