Time. Is it on My Side?

Time. Is it on My Side?

Time. There never seems to be enough of it. With our hectic lives, even the simplest of inconveniences, such as a car breaking down or a heart attack, can totally sour the afternoon and derail our well-laid plans. Wouldn’t it be nice to have advance warning for when we might expect to encounter an interruption to those plans? In part to gain us such a portent, several groups recently assessed a new technology for determining an individual’s cardiovascular disease risk, and the development of a warning test. Their combined efforts may indeed allow us to better plan our lives, or at least serve as a wake-up call that we need to change something in order to have more time to live.

The new technology that could play a crucial role in granting us more time is the SOMAscan® assay, which currently measures changes in over 1,130 proteins. To assess the practicality of SOMAscan in cardiovascular disease research, a research group led by Rob Gerszten at Beth Israel Deaconess Medical Center looked at “controlled heart attacks” to identify protein differences between pre- and post-heart attack in patients’ blood. Aside from identifying biomarkers that are well-established for heart injuries, the researchers also found several not previously seen. They also looked for biomarkers related to other traits that are known to elevate a person’s risk for cardiovascular disease (e.g. age, smoking, cholesterol levels, etc.). They noted the candidate biomarkers they discovered using SOMAscan may shed light into novel pathways that could in some way relate back to the development of cardiovascular disease. The researchers also noted that the SOMAscan assay was faster compared to mass spectrometry, an important consideration when assaying a large number of participants.

In addition to evaluating the SOMAscan assay for biomarker discovery, the research group also evaluated the accuracy of the individual SOMAmer® reagents in identifying their intended target proteins. Using mass spectrometry, the researchers found that all the SOMAmers tested did indeed hit the right targets.

In related work, a research group led by Peter Ganz at University of California San Francisco used the SOMAscan assay to identify a potential prognostic test for true cardiovascular risk in patients with stable coronary heart disease (CHD). The researchers initially analyzed plasma samples from CHD patients who took part in the Heart and Soul study (a study initially intended to assess how mental health affects heart disease patients) for biomarkers that could stratify risk, a measurement that has proved challenging when using traditional or even genetic methods. From the initial phase, the group identified nine proteins that passed the statistical rigors.

To further assess the accuracy of the nine-protein panel, Ganz and his group conducted another round of SOMAscan testing on samples from a completely different set of individuals (participants in the HUNT3 study whose medical data and samples were collected and could be used for further medical or social science research), they verified their findings from the Heart and Soul samples. The researches also evaluated paired samples from the Heart and Soul study participants to determine if the individuals’ risk change as a cardiovascular event approached. They found that indeed that the closer an individual came to a cardiovascular event, the greater the change for the nine-protein panel results when compared to baseline values. These changes were, in turn, shown to be a more reliable and accurate measure than the current clinical standards for assessing cardiovascular risk.

The related findings of these two research groups underline the ability of the SOMAscan assay to benefit cardiovascular disease research, and suggest that we are inching closer to being able to fine-tune our prediction of when a cardiovascular event may happen. Which in turn, may grant us the time we need to accomplish all our well-laid plans.

References:

Ngo, D., Sinha, S., Shen, D., Kuhn, E. W., Keyes, M. J., Shi, X., . . . Gerszten, R. E. (2016) Aptamer-Based Proteomic Profiling Reveals Novel Candidate Biomarkers and Pathways in Cardiovascular Disease. Circulation, 134(4), 270-285. doi:10.1161/CIRCULATIONAHA.116.021803

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

The Toll of Aging on the Quest for Gold

Although the summer Olympics are over for another four years, the world is again amazed at the amount of training these Herculean athletes endure in order to capture gold. A young body usually recovers quite easily after an intense training session; thus, permitting young athletes to continue their quest. However, age inevitably starts to take its toll on the body. Repairing damaged muscles becomes increasingly more difficult with advancing age, though “why” the ability to regenerate muscle is lost is largely unknown. To try to figure it out, a research group led by Jerome Feige and C. Florian Bentzinger at the Nestlé Institute of Health Sciences decided to look the effects of age on muscle stem cells, in the context of their normal contribution to muscle regeneration.

The research group started with comparing gene expression in muscle stem cells from young and old mice. They found that the cells from the injured old mice showed lower expression of genes involved in the cell cycle regulation, higher expression of JAK-STAT and MAP kinase pathway-associated genes (major cell signaling pathways for a variety of functions), and multiple changes in the gene expression for proteins associated with the extracellular matrix (ECM) receptor pathway. The ECM findings were of particular interest, suggesting a role for ECM in muscle regeneration.

To confirm the ECM’s contribution to the muscle regeneration, the group used the SOMAscan® assay to directly measure and compare changes in protein levels in homogenized muscles from injured or uninjured, and young or old mice. They found that many ECM proteins showed higher levels in the old uninjured mice, an observation that is consistent with the usual increase in fibrosis seen in aging muscles. However, when looking at the injured mice, they saw that, in young mice, elevated levels of fibronectin occurred quickly after injury. However, in the old injured mice there was significantly less fibronectin present.

Upon further investigation, the overall importance of fibronectin in regenerating injured muscles solidified. Originating predominantly from lineage-positive cells (stem cells expressing markers seen in mature cells) near the sites of injury, fibronectin serves as an ideal substrate for muscle stem cells to adhere. The researchers found that loss of this ideal substrate led to alterations in several signaling cascades, in line with previous observations from other groups. These alterations may contribute to the aged muscle cells’ increased susceptibility to anoikis (cell-death induced by failure to anchor onto matrix).

Digging even deeper, the investigators looked into the role of a protein called focal adhesion kinase (FAK), which they noted is a known inhibitor of anoikis and dependent on fibronectin for activation. Like fibronectin, they observed the FAK levels to decrease with age in muscle stem cells, which may account for the increased susceptibility of the aged muscle stem cells to anoikis.

Is fibronectin the key to the fountain of youth for aged muscle stem cells? The researchers saw that aged muscle stem cells showed improved adhesion to matrices that included fibronectin, which reduced the cells’ susceptibility to anoikis and slightly improved their proliferation. The inclusion of fibronectin in the matrix also restored the FAK activity and subcellular localization in the aged muscle stem cells. When the researchers injected injured old mice with fibronectin, they saw more FAK in cells undergoing myogenesis, and the localization of the FAK within the cells was comparable to that of cells from young mice. In addition to the improved signaling, the researchers saw improved proliferation of the cells that would go on to become muscle cells. The resulting muscle fibers indeed showed fewer immature muscle fibers than seen in control mice, suggesting that the effects of age on muscle regeneration were mitigated by injection of fibronectin.

For the aging Olympian athletes, fibronectin injections would probably be seen as a new form of doping. For the rest of the aging populace, however, increasing fibronectin may be the winner as a way to maintain a more active lifestyle well into “old” age.

Link to paper: http://rdcu.be/i9ib (Lukjanenko et al., 2016)

Resources

Lukjanenko, L., Jung, M. J., Hegde, N., Perruisseau-Carrier, C., Migliavacca, E., Rozo, M., . . . Bentzinger, C. F. (2016) Loss of fibronectin from the aged stem cell niche affects the regenerative capacity of skeletal muscle in mice. Nat Med. doi:10.1038/nm.4126

Warding off the Theft of Independence and Memories by Alzheimer’s Disease

Alzheimer’s disease (AD) robs people of their memories and their independence. Determining those at greatest risk for the disease as early as possible may prove key for warding off AD for as long as possible. Although changes in the brain can show up between 4 and 17 years prior to manifestation of AD symptoms, these are only seen by using magnetic resonance imaging (MRI); not ideal for wide-spread population screening.1 What is needed is a non-invasive, readily accessible and inexpensive diagnostic test that would help clinicians manage patient health through early intervention.

As a first step towards such a diagnostic test, Steven Kiddle and his colleagues set out to identify biomarkers that associated with thought-based tests predictive of AD.2 Using relevant blood samples from the TwinsUK study, they acquired the subjects’ proteomic profiles using the SOMAscan® assay, and compared those findings with those obtained from traditional cognitive tests to see if they could identify potential AD biomarkers. The researchers also performed MRI scans to check for AD-related physical changes in the brain.

After collecting and processing data from the numerous tests, the research group analyzed the data to identify biomarker candidates. From the 1,129 proteins measured in samples from the TwinsUK group, the researchers found associations between the levels of three different proteins and brain volume changes as seen by the MRI scans. However, when the researchers compared these volume-associated proteins with measures of cognitive function, only one of them (called MAP2K4) showed an association, and only with the 10-year change in cognitive function testing. In looking at cognitive function scores only, the researchers also found an association between the 10-year change and MAPKAPK5. This protein, however, showed a nominal association with brain volume changes.

To confirm the findings, the researchers turned to a different set of samples that came from the AddNeuroMed study, again performing MRI scans and SOMAscan. From these data, the researchers did find an association between MAP2K4 plasma protein levels and brain volume. However, the smaller sample numbers requires additional studies to be done to potentially nail down a predictive biomarker for early intervention therapy in patients showing increased risk of developing AD. Though preliminary, the findings could eventually lead to making a huge difference in how long an at-risk person can live independently and still reminisce about the past.

References

1 Villemagne VL et al. (2013) Amyloid β deposition, neurodegeneration, and cognitive decline in sporadic Alzheimer’s disease: a prospective cohort study. Lancet Neurology 12 (pp. 357–67)

2 Kiddle SJ et al. (2015) Plasma protein biomarkers of Alzheimer’s disease endophenotypes in asymptomatic older twins: early cognitive decline and regional brain volumes. Translational Psychiatry 5(6): e584. doi: 10.1038/tp.2015.78.

SOMAmer Sandwich Assays: A New Diagnostic Flavor

The famous chef, Emeril Lagasse, delights his audience with his approach to cooking. As he cooks, he theatrically takes the dish to the “next level” by adding another ingredient to the flavor. Bam! The food is even better.

In many ways, laboratory work mirrors cooking. Researchers routinely look to bring out the best “flavors” in any particular experiment. Recently, a group of SomaLogic scientists figured out how to take the already impressive utility of SOMAmer® reagents (small pieces of synthetic DNA with modified nucleotides) up a notch. They recognized that the very properties that make SOMAmer reagents special could be harnessed for traditional antibody-based applications, such as diagnostic sandwich assays (where one binding reagent captures a protein and other one is used to detect the protein).1, 2  Because of ongoing problems with antibody consistency and availability, the use of SOMAmer reagents (easily made by synthetic means) in these assays could be significantly beneficial for both research and clinical applications.

The group devised a way to make a pair of SOMAmer reagents that bind to different places on the same protein. Using Clostridium difficile binary toxin (CdtA) as the target protein, Urs Ochsner and his colleagues modified the “recipe” for whipping up SOMAmer reagents to create tens of thousands of candidates.2, 3 The significant change to the process was including a new ingredient, a previously identified CdtA SOMAmer reagent 4758-6. For comparison, they performed the same procedure, but left out 4758-6.

Upon completion, Ochsner and colleagues looked at the results. In comparing the sequences generated by the method lacking 4758-6 to those from the method that included the older SOMAmer reagent, they saw clear differences in sequence patterns and sequence abundance, as well as some similar sequences. The differences observed suggest that addition of 4758-6 may have worked. Several sequences were chosen for testing in sandwich assays, which revealed that new sequence 5579–12 paired the best with 4758-6. The researchers also conducted additional experiments to confirm that these two SOMAmers did indeed bind to different sites on the protein: a SOMAmer reagent sandwich pair had been created.

Based on the success of their trial run, the investigators decided to make SOMAmer reagent pairs for eight more proteins. In the first phase, the researchers sifted through existing SOMAmer reagents for suitable pairings, finding existing candidates for three of the proteins. In the second phase, they performed the modified procedure to identify the best pairings for the five remaining proteins, successfully identifying SOMAmer reagent pairs for four of them (in line with the usual success rate in generating new SOMAmer reagents).

Compared to antibodies (the traditional protein-binding reagents used in most sandwich assays), SOMAmer reagents are chemically synthesized resulting in better consistency between batches. They also have tremendous multiplexing capabilities, with the possibility of combining thousands of SOMAmer reagents into same experiment. The use of diagnostic sandwich assays incorporating SOMAmer reagent sandwich pairs rather than antibodies may indeed take disease research and diagnostic proteomics up a notch. Bam!

References

Davies DR et al. (2012) Unique motifs and hydrophobic interactions shape the binding of modified DNA ligands to protein targets. Proc. Natl. Acad. Sci. 109: 19971–19976.

Ochsner UA et al. (2014) Systematic selection of modified aptamer pairs for diagnostic sandwich assays. BioTechniques 56(3): 125–133.

Gold LD et al. (2010) Aptamer-based multiplexed proteomic technology for biomarker discovery. PLoS ONE 5: e15004.

Attention to Details

During the 2016 Olympics, an oversight occurred. At first, it might have seemed minor, but it really ruffled some feathers and caused major embarrassment for the Olympic committee. So, what happened? The Olympic committee approved and used incorrect Chinese flags with the smaller yellow stars pointing in the wrong direction.1 The mistake was caught and deep apologies were made. If greater attention had been paid to even these seemingly tiny details, then this situation would not have happened.

In science, minute changes that may seem subtle or inconsequential can also have a huge impact. For example, a single-nucleotide polymorphism (SNP) is a change of one nucleotide that can occur anywhere in a genome. Many times, the single change is no big deal. Other times, the change can happen in a spot that affects how a resulting protein is made, perhaps leading to disease, changing a patient’s response to medication, or making a person more vulnerable to toxins.2 Negative outcomes from these seemingly innocuous scenarios are not inconsequential.

Detecting SNPs at the DNA level is the conventional way for finding them. However, SNPs can also be detected at the protein level by SomaLogic’s SOMAscan assay. SomaLogic researchers have documented that certain SOMAmer reagents (a modified single-stranded DNA that is seminal to the SOMAscan assay) can distinguish between proteins resulting from SNPs and normal proteins in human plasma.3 For example, a SNP causes an amino acid change (histidine to arginine) at position 167 in the low affinity immunoglobulin gamma Fc region receptor II-a protein (FCGR2A for short) in about 44% of the population. The protein version of FCGR2A used to create the SOMAmer reagent carried the SNP. When the SOMAscan assay was performed using samples from healthy individuals, a “bimodal distribution” was observed in both plasma and serum, suggesting that some of the individuals did not possess the SNP version of the protein. In other words, the SOMAmer reagent in the assay could not bind to the “non-SNP” version of the protein.

To confirm this hypothesis, the scientists performed a series of binding experiments. Using purified normal FCGR2A and mutant FCGR2A (i.e., the one that contains the SNP), they measured the affinity of the SOMAmer reagent to these two proteins. While they observed very good affinity to the mutant FCGR2A, the binding to the normal protein was significantly weaker. An additional experiment using a closely related protein to dissect the binding pattern of the SOMAmer reagent ultimately supported the hypothesis.

This work demonstrates how the subtlest change in a protein can affect experimental results. Paying attention to the bimodal data of the SOMAscan assay revealed marvelous insight into the SOMAmer reagent’s specificity for that particular protein. This should not be the cause for any embarrassment, and no apologies are needed.

References

Brocchetto, M. (2016). Rio 2016 officials apologize to China for using wrong flag. Retrieved from http://www.cnn.com/2016/08/08/americas/rio-2016-china-wrong-flag-trnd/

What are single nucleotide polymorphisms (SNPs)? Genetics Home Reference Website. https://ghr.nlm.nih.gov/primer/genomicresearch/snp Published August 9, 2016.

Wilcox S. (2015). SOMAscan Assay: A Proteomic Platform that Can Also Detect SNPs. Poster presentation shown at Human Proteome Organziation (HUPO).

Putting the Pain on Ice: IL-6 SOMAmer Reagent Inhibits Rheumatoid Arthritis

The seasons are changing. The days are getting shorter. The air is getting cool and crisp. Soon, the outdoors will be covered by a lovely blanket of fresh snow. As serene as this image may be, the fact is that the changing seasons maybe painful for those who suffer from rheumatoid arthritis (RA) (Savage et al., 2015).

There are several treatments for RA available today (briefly reviewed in Hirota et al., 2016), including non-steroidal anti-inflammatory drugs, small molecule disease-modifying anti-rheumatic drugs, and a growing number of biologics that specifically target inflammatory pathway components. None of these therapeutics can cure the disease, but they can make life a bit more comfortable for RA sufferers (though not without some significant side-effects).

One protein target of particular interest for new RA treatments is interleukin-6 (IL-6) (briefly reviewed in Hirota et al., 2016). As a cytokine (a protein involved in the communication between cells), IL-6 is involved in the immune response, inflammation, hematopoiesis (making of new blood cells) and bone metabolism. During an RA response, IL-6 and the IL-6 receptor-α together bind to signal transducing protein gp130 (CD130), which activates the cell signaling pathway known as “JAK-STAT3” and phosphorylation (the addition of phosphates) of STAT3.

A research team led by Dr. Masao Hirota recently developed a new approach to inhibit the IL-6 signaling pathway (Hirota et al., 2016). They developed SL1026, a SOMAmer reagent that has a strong binding affinity for both human and monkey IL-6.

Dr. Hirota and his team determined that SL1026 inhibits IL-6-induced STAT3 phosphorylation in human peripheral blood lymphocytes (a type of white blood cell). First, they incubated whole blood with IL-6 alone, or with IL-6 in combination with either SL1026 or tocilizumab (a current drug that specifically inhibits IL-6 signaling in RA). The researchers then isolated and analyzed the cells and, as expected, IL-6 treatment increased STAT3 phosphorylation. Treatment of the cells with either SL1026 or tocilizumab inhibited STAT3 phosphorylation.

The researchers tested whether or not SL1026 could delay the actual progression of RA in a primate collagen-induced arthritis model (an established model system for evaluating the therapeutic and preventative effects of existing RA drugs). Similar to human RA, the monkeys experienced an autoimmune-mediated polyarthritis, joint inflammation and erosion of bone and cartilage. While one group of monkeys went untreated, a subset of the monkeys received intravenous doses of SL1026. The animals were monitored and scored for behavior and movement. The researchers also scored the degree of swelling and rigidity in numerous joints.

SL1026 not only reduced arthritis symptoms in the monkey model, but also delayed arthritis onset in treated monkeys for 20 days compared to 13 days for the untreated monkeys. The monkeys treated with higher doses of SL1026 also had a reduced arthritis score at day 34 that was significantly different than the untreated monkeys’ score.

One concern with any potential new therapeutic is an allergic reaction on the part of the recipient. To see if this might happen, the team measured levels of anti-SL1026 antibodies. None were identified in the plasma of treated monkeys, indicating that the SL1026 did not trigger an immune/allergic response. In addition, the researchers noted that the SL1026 was well tolerated by the monkeys, and no adverse events occurred.

The efforts put forth by Dr. Hirota and his team established that SOMAmer reagents can be effective therapeutics as a result of their exquisite binding affinities for their target proteins. This particular work demonstrates that the SL1026 is a potent antagonist of the IL-6 signaling pathway. SL1026 may be a promising drug candidate for RA and potentially in other IL-6 mediated diseases. If it makes it to market, SL1026 may prove to be a better treatment option for RA suffers during these cooler and darker days.

References

Hirota, M., Murakami, I., Ishikawa, Y., Suzuki, T., Sumida, S., Ibaragi, S., . . . Schneider, D. J. (2016). Chemically Modified Interleukin-6 Aptamer Inhibits Development of Collagen-Induced Arthritis in Cynomolgus Monkeys. Nucleic Acid Ther, 26(1), 10-19. doi:10.1089/nat.2015.0567

Savage, E. M., McCormick, D., McDonald, S., Moore, O., Stevenson, M., & Cairns, A. P. (2015). Does rheumatoid arthritis disease activity correlate with weather conditions? Rheumatol Int, 35(5), 887-890. doi:10.1007/s00296-014-3161-5