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Buck Institute research involves first genome-wide DNA methylation study in disease-free tissue

Our epigenome is a set of chemical switches that turn parts of our genome off and on at strategic times and locations. These switches help alter the way our cells act and are impacted by environmental factors including diet, exercise and stress. Research at the Buck Institute reveals that aging also effects the epigenome in human skeletal muscle. The study, appearing on line in Aging Cell, provides a method to study sarcopenia, the degenerative loss of muscle mass that begins in middle age.

The results came from the first genome-wide DNA methylation study in disease-free individuals. DNA methylation involves the addition of a methyl group to the DNA and is involved in a particular layer of epigenetic regulation and genome maintenance. In this study researchers compared DNA methylation in samples of skeletal muscle taken from healthy young (18 - 27 years of age) and older (68 - 89 years of age) males. Buck faculty and lead scientist Simon Melov, PhD, said researchers looked at more than 480,000 sites throughout the genome. "We identified a suite of epigenetic markers that completely separated the younger from the older individuals - there was a change in the epigenetic fingerprint," said Melov. "Our findings were statistically significant; the chances of that happening are infinitesimal."

In developing nations, rural areas, and even one's own home, limited access to expensive equipment and trained medical professionals can impede the diagnosis and treatment of disease. Many qualitative tests that provide a simple "yes" or "no" answer (like an at-home pregnancy test) have been optimized for use in these resource-limited settings. But few quantitative tests-those able to measure the precise concentration of biomolecules, not just their presence or absence-can be done outside of a laboratory or clinical setting. By leveraging their discovery of the robustness of "digital," or single-molecule quantitative assays, researchers at the California Institute of Technology (Caltech) have demonstrated a method for using a lab-on-a-chip device and a cell phone to determine a concentration of molecules, such as HIV RNA molecules, in a sample. This digital approach can consistently provide accurate quantitative information despite changes in timing, temperature, and lighting conditions, a capability not previously possible using traditional measurements.

In a study published on November 7 in the journal Analytical Chemistry, researchers in the laboratory of Rustem Ismagilov, Ethel Wilson Bowles and Robert Bowles Professor of Chemistry and Chemical Engineering, used HIV as the context for testing the robustness of digital assays. In order to assess the progression of HIV and recommend appropriate therapies, doctors must know the concentration of HIV RNA viruses in a patient's bloodstream, called a viral load. The problem is that the viral load tests used in the United States, such as those that rely on amplification of RNA via polymerase chain reaction (PCR), require bulky and expensive equipment, trained personnel, and access to infrastructure such as electricity, all of which are often not available in resource-limited settings. Furthermore, because it is difficult to control the environment in these settings, viral load tests must be "robust," or resilient to changes such as temperature and humidity fluctuations.

Many traditional approaches for measuring viral load involve converting a small quantity of RNA into DNA, which is then multiplied through DNA amplification-allowing researchers to see how much DNA is present in real time after each round of amplification, by monitoring the varying intensity of a fluorescent dye marking the DNA. These experiments-known as "kinetic" assays-result in a readout reflecting changes in intensity over time, called an amplification curve. To find the original concentration of the beginning bulk RNA sample, the amplification curve is then compared with standard curves representing known concentrations of RNA. Since assays, such as those for HIV, require many rounds of DNA amplification to collect a sufficiently bright fluorescent signal, small errors introduced by changes in environmental conditions can compound exponentially-meaning that these kinetic measurements are not robust enough to withstand changing conditions.

In this new study, the researchers hypothesized that they could use a digital amplification approach to create a robust quantitative technique. In digital amplification, a sample is split into enough small volumes such that each well contains either a single target molecule or no molecule at all. Ismagilov and his colleagues used a microfluidic device they previously invented, called SlipChip, to compartmentalize single molecules from a sample containing HIV RNA. SlipChip is made up of two credit card-sized plates stacked atop one another; the sample is first added to the interconnected channels of the SlipChip, and with a single "slip" of the top chip, the channels turn into individual wells.

In lieu of PCR, the researchers used a different amplification chemistry on this chip called digital reverse transcription-loop-mediated amplification (dRT-LAMP), which produces a bright fluorescent signal in the presence of a target molecule during the amplification process. The dRT-LAMP technique eliminates the need for continuous tracking of the intensity of fluorescence; instead, just one end-point readout measurement is used. The resulting patchwork of "positive" or "negative" wells on the device, in combination with statistical analysis, enables single molecules to be counted.

"In each well, you are performing a qualitative experiment; the result is like a pregnancy test: either yes or no, positive or negative, for the presence of an HIV RNA molecule," says David Selck, a graduate student in Ismagilov's lab and a first author on the study. "But by doing a couple of thousand qualitative experiments, you end up getting a numerical, quantitative result: the concentration of HIV RNA molecules in the sample. By calculating the concentration from the number of wells that contain fluorescence-and therefore HIV-you're leveraging the robustness of many qualitative 'yes or no' experiments to fulfill the need for a quantitative, numerical result," he says.

UT Dallas researchers have demonstrated that treating tinnitus, or ringing in the ears, using vagus nerve stimulation-tone therapy is safe and brought significant improvement to some of the participants in a small clinical trial.

Drs. Sven Vanneste and Michael Kilgard of the School of Behavioral and Brain Sciences used a new method pairing vagus nerve stimulation (VNS) with auditory tones to alleviate the symptoms of chronic tinnitus. Their results were published on Nov. 20 in the journal Neuromodulation: Technology at the Neural Interface.

VNS is an FDA-approved method for treating various illnesses, including depression and epilepsy. It involves sending a mild electric pulse through the vagus nerve, which relays information about the state of the body to the brain.

"The primary goal of the study was to evaluate safety of VNS-tone therapy in tinnitus patients," Vanneste said. "VNS-tone therapy was expected to be safe because it requires less than 1 percent of the VNS approved by the FDA for the treatment of intractable epilepsy and depression. There were no significant adverse events in our study."

According to Vanneste, more than 12 million Americans have tinnitus severe enough to seek medical attention, of which 2 million are so disabled that they cannot function normally. He said there has been no consistently effective treatment.

The study, which took place in Antwerp, Belgium, involved implanting 10 tinnitus sufferers with a stimulation electrode directly on the vagus nerve. They received two and a half hours of daily treatment for 20 days. The participants had lived with tinnitus for at least a year prior to participating in the study, and showed no benefit from previous audiological, drug or neuromodulation treatments. Electrical pulses were generated from an external device for this study, but future work could involve using implanted generators eliminating the need for clinical visits.

The Cardiothoracic Surgical Trials Network (CTSN), whose Data and Clinical Coordinating Center is at Icahn School of Medicine at Mount Sinai, is reporting for the first time evidence on whether or not there is any significant difference between the two current surgical approaches to treat patients with severe ischemic mitral regurgitation -- mitral valve repair and mitral valve replacement.

The findings presented on November 18 at the American Heart Association's Scientific Sessions 2013 and published simultaneously by the New England Journal of Medicine, report similar clinical outcomes one-year following mitral valve surgery for both surgical approaches. Although patient clinical outcomes were similar, mitral valve replacement was shown to be more durable than repair.

"Ischemic mitral regurgitation is an important public health problem affecting as many as two million Americans. Our study is the first definitive clinical trial to address the relative merits of mitral valve repair versus mitral valve replacement surgery in patients suffering from severe ischemic mitral regurgitation," says Annetine C. Gelijns, PhD, Professor and Chair of the Department of Health Evidence and Policy at Icahn School of Medicine at Mount Sinai, who is the corresponding author for the NEJM study and principal investigator for the Data and Clinical Coordinating Center based at Mount Sinai for the NIH-sponsored Cardiothoracic Surgical Trials Network (CTSN) which conducted the clinical trial study.

Ischemic mitral regurgitation occurs when blood backflows into the left atrium from the left ventricle of the heart due to improper closure of the mitral valve. The condition often develops as a complication from a heart attack and subsequent enlargement of the left ventricle, the heart's main pumping chamber. If not surgically corrected, the condition has been reported to potentially have a one-year mortality risk as high as 40 percent.

Currently, practice guidelines recommend valve repair or replacement for severe ischemic mitral regurgitation. However, lack of evidence has led to uncertainty and variation in surgical practice, although surgeons' use of repair has increased over time.

Previous research has shown mitral valve repair has lower operative mortality and complications, while mitral valve replacement may provide better long-term correction of mitral regurgitation.