Research may improve performance of DNA detectors and aid diagnostics for HIV or cancer.

Kevin Plaxco (left) and Alexis Vallée-Bélisle, UC Santa Barbara

Over their 3.8 billion years of evolution, living organisms have developed countless strategies for monitoring their surroundings. Chemists at UC Santa Barbara and University of Rome Tor Vergata have adapted some of these strategies to improve the performance of DNA detectors. Their findings may aid efforts to build better medical diagnostics, such as improved HIV or cancer tests.

Their research is described in an article published this week in the Journal of the American Chemical Society.

Nature often serves as a source of inspiration for the development of new technologies. In the field of medical diagnostics, for example, scientists have long taken advantage of the high affinity and specificity of biomolecules such as antibodies and DNA to detect molecular markers in the blood. These molecular markers allow them to monitor health status and to guide treatments for diseases, including HIV, cancer and diabetes.

Kevin W. Plaxco, a professor of chemistry at UCSB, whose group carried out the research, notes that despite their great attributes, a main limitation of such biosensors is their precision, which is confined to a fixed, well-defined “dynamic range” of target concentrations. Specifically, the useful dynamic range of typical biomolecule binding events spans an 81-fold range of target concentrations.

“This fixed dynamic range complicates — or even precludes — the use of biosensors in many applications,” said Plaxco. “To monitor HIV progression and provide the appropriate medication, for example, physicians need to measure the levels of viruses over five orders of magnitude. Likewise, the two orders-of-magnitude range displayed by most biosensors is too broad to precisely monitor the concentrations of the highly toxic drugs used to treat many cancers. Our goal was, therefore, to create sensors with extended (for applications needing a broad dynamic range) or narrowed (for applications needing high measurement precision) dynamic ranges at will.”

The key breakthrough underlying their new approach came from the simple observation of nature. “All living organisms monitor their environments in an optimized way by using sensing molecules that respond to either wide or narrow change in target concentrations,” said Alexis Vallée-Bélisle, a postdoctoral fellow and the first author of the study. “Nature does so by combining in a very elegant way multiple receptors, each displaying a different affinity for their common target”.

Inspired by the optimized behaviors of these natural sensors, the UCSB research group teamed up with Francesco Ricci, professor at the University of Rome Tor Vergata to do their own mixing and matching of biomolecules to manipulate biosensors’ dynamic ranges. To validate their approach, they used a widely employed DNA-based biosensor used for detecting mutations in DNA called a “molecular beacon.”

By combining sets of molecular beacons all binding the same target molecule but with differing affinities, the international team was able to create sensors with rationally “tuned” dynamic ranges. In one case, they developed a sensor that monitors DNA concentrations over a six orders of magnitude range. In another example, they developed an ultrasensitive sensor that precisely detects small changes in target concentration over only a five-fold dynamic range. Finally, they also built sensors characterized by complex, “custom-made” dynamic ranges in which the sensor is insensitive within a window of desired concentrations (e.g., the clinically “normal” concentration range of a drug) and very sensitive above or below this “appropriate” concentration range. The researchers believe that these strategies can be in principle applied to a wide range of biosensors, which may significantly impact efforts to build better point-of-care biosensors for the detection of disease biomarkers.

This work was funded by the National Institute of Health, the Fond Québécois de la Recherche sur la Nature et les Technologies, the Italian Ministry of University and Research (MIUR) project “Futuro in Ricerca.”

Simple universal test spots biomarkers for group of disorders, may speed diagnosis.

Jeffrey Esko, UC San Diego

A team of scientists, led by researchers at the University of California, San Diego, School of Medicine and Zacharon Pharmaceuticals, have developed a simple, reliable test for identifying biomarkers for mucopolysaccharidoses (MPS), a group of inherited metabolic disorders that currently are diagnosed in patients only after symptoms have become serious and the damage possibly irreversible.

The findings will be published online Sunday in the journal Nature Chemical Biology.

MPS is caused by the absence or malfunctioning of a lysosomal enzyme required to break down and recycle complex sugar molecules called glycosaminoglycans, which are used to build bone, tendons, skin and other tissues. If not degraded and removed, glycosaminoglycans can accumulate in cells and tissues, resulting in progressive, permanent damage affecting appearance, physical abilities, organ function and often mental development in young children. The effects range from mild to severe.

There are 11 known forms of MPS, each involving a different lysosomal enzyme. A number of treatments exist, including enzyme replacement therapy and hematopoietic stem cell transplantation, but efficacy depends upon diagnosing the disease and its specific form as early as possible. That has been problematic, said Jeffrey D. Esko, Ph.D., professor in the Department of Cellular and Molecular Medicine and co-director of the Glycobiology Research and Training Center at UC San Diego.

“The typical time from seeing first symptoms to diagnosis of MPS is about three years. Since the early signs of disease are common childhood issues like ear infections and learning disorders, the disease is usually not immediately recognized,” Esko said.

“A child often has multiple visits with their pediatrician. Eventually they are referred to a metabolic disease specialist, where rare diseases are considered. It takes an expert to identify MPS and its most likely form in each patient. Every subclass of MPS has its own specific diagnostic test, so developing better diagnostics is an essential part of effective treatment. ”

In their paper, the scientists describe an innovative method to detect tell-tale carbohydrate structures specific to glycosaminoglycans in the cells, blood and urine of MPS patients. The biomarker assay identifies all known forms of the disease.

Esko is collaborating with Zacharon Pharmaceuticals, a San Diego-based biotechnology company, to develop a commercial diagnostic assay for differentiating forms of MPS from urine and blood samples, a screening test for newborns and a tool for measuring the biochemical response of MPS patients to existing and novel therapies.

“Since the severity of the disease is highly variable among patients, this could provide a tool that a doctor can use to optimize dosing or treatment,” said Brett Crawford, vice president for research at Zacharon. “Currently, all patients are treated with the same dose of drug.”

The biomarker test may also be used to discover new forms of MPS and better characterize existing ones.

Disclosure: Esko co-founded Zacharon Pharmaceuticals in 2004 with Brett E. Crawford and Charles Glass. He is a scientific advisor to the company.

Co-authors include Roger Lawrence and William C. Lamanna, UC San Diego Department of Cellular and Molecular Medicine, Glycobiology Research and Training Center; Jillian R. Brown, James R. Beitel and Brett E. Crawford, Zacharon Pharmaceuticals; Geert-Jan Boones and Kanar Al-Mafraji, University of Georgia, Athens.

Funding for this research came, in part, from the National Institutes of Health, a Kirschstein National Research Service Award and the National MPS Society.

Clinical trial will test a new diagnostic approach for the disorder, which affects Venus Williams and millions of others.

David Wong, UCLA

In August, tennis star Venus Williams withdrew from the U.S. Open, saying she was suffering from fatigue and other symptoms related to Sjögren’s syndrome, an autoimmune disorder that results in the loss of the ability to produce saliva and tears. Her announcement focused public attention on this malady, which affects nearly 4 million Americans.

While women are nine times more likely than men to develop Sjögren’s, the disorder affects virtually every racial and ethnic group. Most patients develop symptoms after age 40, including dry eyes, dry mouth and often joint pain and chronic fatigue. And because of their paucity of saliva and the antibacterial chemicals it contains, patients may also develop tooth decay and cavities.

While much is known about the symptoms of Sjögren’s, the disease is complex and poorly understood, and in some cases, it can take more than six years to be diagnosed.

The UCLA School of Dentistry has now received a $2.8 million grant from the National Institute of Dental and Craniofacial Research, part of the National Institutes of Health, to support a multi-center clinical trial of a diagnostic test that uses patients’ saliva to determine whether they have Sjögren’s syndrome. This simple, non-invasive test will permit a diagnosis within minutes, rather than the weeks currently required when using blood or other tissue samples.

The project will be led by Dr. David Wong, associate dean for research and the Felix and Mildred Yip Endowed Professor in Dentistry at the UCLA School of Dentistry. For Dr. Wong and his colleagues, who have been conducting research on using saliva as a diagnostic tool for biomarkers of oral cancer, early-stage pancreatic cancer and other maladies for several years, this is an important step in moving from the research realm to actual clinical trials and, eventually, to use by medical and dental practitioners.

“This clinical trial will make it possible to validate the effectiveness of salivary diagnosis and move us a step closer to eventual FDA approval and clinical product development,” Wong said. “The establishment of scientifically credible biomarkers for this chronic autoimmune disease that are not invasive, painful or embarrassing is our goal.”

Clinical trials will be conducted at three major rheumatology centers, at University Medical Center Groningen in the Netherlands, the University of Minnesota and the Oklahoma Medical Research Foundation. Centers will enroll patients exhibiting sicca symptoms of dry eye and dry mouth and will perform the saliva biomarker assay based on a panel of highly discriminatory salivary biomarkers developed at UCLA. Researchers will benchmark the outcome with the current clinical practice of six clinical tests, including serology and a lip biopsy, to diagnose Sjögren’s syndrome (AECC 2002 criteria).

“The UCLA School of Dentistry is very proud to be at the forefront of this international effort to advance the field of saliva diagnostics from the research laboratory to clinical trials,” said No-Hee Park, dean of the UCLA School of Dentistry. “The prospect of early detection of Sjögren’s syndrome, and possibly other serious illnesses, in the future through this methodology is truly exciting.”

The UCLA School of Dentistry is dedicated to improving the oral health of the people of California, the nation and the world through its teaching, research, patient care and public service initiatives. The school provides education and training programs that develop leaders in dental education, research, the profession and the community; conducts research programs that generate new knowledge, promote oral health and investigate the cause, prevention, diagnosis and treatment of oral disease in an individualized disease-prevention and management model; and delivers patient-centered oral health care to the community and the state.

Berkeley Lab-developed technique could help pinpoint Alzheimer’s in its early stages.

Alzheimer’s disease is the most common form of dementia, currently affecting more than 35 million people worldwide. Although many genetic and hereditary factors are thought to contribute to the telltale deterioration of memory and cognitive functions resulting from Alzheimer’s, a central aspect to this disease is an accumulation of misfolded proteins in the brain.

Now, scientists at Berkeley Lab have engineered a universal, highly sensitive technique for detecting misfolded proteins in biological fluids. This groundbreaking nanoscience capability could help pinpoint Alzheimer’s in its early stages and enable researchers to discover new therapies for this devastating disease.

When a protein doesn’t fold into its normal shape, it also doesn’t perform its normal functions. This disruption in behavior could lead to proteins that aggregate into plaques or deposits and become toxic to cells. In Alzheimer’s disease, aggregates of a protein called beta-amyloid form in the central nervous system, causing damage to cells in the brain and triggering dementia.

An analytical capability for measuring tiny clusters of these proteins — before irreversible damage occurs — would be a powerful tool in the early detection of Alzheimer’s and other misfolded protein diseases. However, despite significant research efforts, there are currently no diagnostic tools available to selectively detect small-scale aggregates of misfolded proteins in biological fluids, such as blood or spinal fluid.

“This collaboration illustrates how a biomedical problem can also be a nanoscience problem, in which a chemical reagent is needed to recognize partially aggregated proteins,” said Ron Zuckermann, director of the Biological Nanostructures Facility at the Molecular Foundry, a nanoscience user facility at Berkeley Lab. “We were faced with the challenge of synthesizing a material that’s capable of specifically detecting this aggregated protein and not any of the other proteins in the blood.”

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New technologies for the diagnosis of cancer are rapidly changing the clinical practice of oncology. As scientists learn more about the molecular basis of cancer, the development of new tools capable of multiple, inexpensive biomarker measurements on small samples of clinical tissue will become essential to the success of genetically informed and personalized cancer therapies.

Researchers at UCLA have now developed a microfluidic image cytometry (MIC) platform that can measure cell-signaling pathways in brain tumor samples at the single-cell level. The new technology combines the advantages of microfluidics and microscopy-based cell imaging.

The ability to make these in vitro molecular measurements, or “fingerprints,” marks a new advance in molecular diagnostics that could ultimately help physicians predict patient prognosis and guide personalized treatment.

“The MIC is essentially a cancer diagnostic chip that can generate single-cell ‘molecular fingerprints’ for a small quantity of pathology samples, including brain tumor tissues,” said Dr. Hsian-Rong Tseng, a UCLA associate professor of molecular and medical pharmacology and one of the leaders of the research. “We are exploring the use of the MIC for generating informative molecular fingerprints from rare populations of oncology samples — for example, tumor stem cells.”

The research, which appears in the Aug. 1 issue of the journal Cancer Research, represents the teamwork of 35 co-authors from UCLA’s Jonsson Comprehensive Cancer Center with expertise in surgery, pathology, cancer biology, bioinformatics and diagnostic devices.

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Paul Hansma’s face lights up when he talks about what his latest research might mean for people who suffer after breaking their hips or other bones that become more and more brittle as they age. Statistics show that a woman is more likely to die in the next year after a hip fracture than if she’s had a heart attack.

Hansma, a professor of physics at UC Santa Barbara who has spent much of the past 20 years developing Atomic Force Microscopes, has focused on biophysical research and the study of human bones. His renowned bone tissue research has led him to what he believes will be a significant step in the study of biomaterials: development of a new medical diagnostic tool — the Reference Point Indentation (RPI) instrument.

Hansma is a co-author of a new study published in the Journal of Bone and Mineral Research. In the study, “Microindentation for in vivo Measurement of Bone Tissue Mechanical Properties in Humans,” Hansma and his co-authors say they have validated the RPI as a new tool for measuring the strength and quality of bones by using live human test subjects.

“This is a revolutionary breakthrough,” Hansma said of the RPI. “People get excited when they hear about this.”

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A two-day wait for blood test results turned into a hospital stay for Rodrigo Martinez-Duarte’s wife, Anne-Carole. A routine infection spread to her kidneys, making her very sick, while confirmation of her diagnosis was pending.

“If the results hadn’t taken so long, doctors could have begun treatment sooner and my wife might have recovered quicker,” says Martinez-Duarte, a UC Irvine doctoral candidate in mechanical & aerospace engineering.

He hopes a device he’s building will make blood and other cell-sorting tests faster – and cheaper. The technology someday could help diagnose and treat diseases ranging from Alzheimer’s and diabetes to leukemia and HIV.

To support his research efforts, Martinez-Duarte – who was born in Mexico and studied electrical engineering at Tecnologico de Monterrey before coming to UCI in 2005 – has been awarded a $10,000 Public Impact Fellowship from UCI’s Graduate Division.

Many diagnostic tests and therapies are based on sorting and isolating cells. Current methods are costly and time-consuming because of their complexity. Sorter machines can sell for thousands of dollars and weigh hundreds of pounds. Antibodies needed to identify cells are also expensive and require special handling by skilled personnel.

The device Martinez-Duarte is developing sorts cells using electric fields instead of antibodies. Cells have properties that respond to different frequencies. Turn a particular electric field on, and targeted cells can be manipulated at will. The technique, called dielectrophoresis, dates to the 1970s but hasn’t gained popularity in clinical settings because the equipment involved is often bulky and complicated.

Martinez-Duarte’s innovation was to adapt DEP to a medium the size and shape of a compact disc. Cell-containing fluid on the disc can be controlled by changing the disc’s rotation speed and other parameters. The goal is an automated process in which a technician could load a sample, choose a sorting program and obtain results in just a few minutes.

Martinez-Duarte and his colleagues in Chancellor’s Professor Marc Madou’s lab hope the device will be in clinical use within the next few years.

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As a result of improved imaging technology, pancreatic cysts are increasingly diagnosed in asymptomatic individuals who undergo scans for other reasons. And while most of these cysts follow a benign course, a small but significant number are either malignant at the time of diagnosis or have the potential to develop into pancreatic cancer during a patient’s lifetime.

The dilemma for both patient and clinician is determining which cysts to leave alone and which to surgically remove. Existing treatment guidelines don’t clearly address many treatment options beyond the removal of part of the pancreas — a major undertaking for an asymptomatic lesion.

Now, a UCLA-Veterans Affairs research team has developed an evaluation tool to help guide asymptomatic pancreatic cyst treatment. Published in the February issue of the journal Gastroenterology, the tool takes into account overall health, age, cyst size, surgical risk and patients’ views about quality of life.

“Surgery may not be the best initial approach for all patients diagnosed with a specific pancreatic cyst. The new tool may help with decision-making and mapping out a treatment plan,” said study author Dr. Brennan Spiegel, director of the UCLA-VA Center for Outcomes Research and Education at the David Geffen School of Medicine at UCLA and the VA Greater Los Angeles Healthcare System.

The diagnosis of asymptomatic cysts has increased fivefold over the past decade, due partly to an aging population and to improved diagnostics. Current imaging techniques — including computed tomography (CT), magnetic resonance imaging (MRI) and endoscopic ultrasound, in which a small camera is inserted down the throat and into the stomach and small bowel to image the pancreas — combined with pancreatic cyst fluid analysis, offer an 80 percent accuracy in cyst diagnosis.

“Pancreatic cysts are most often diagnosed in an older population, and although many are benign, these must be carefully tracked, since a small percentage can develop into pancreatic cancer,” said study author Dr. James J. Farrell, associate professor of digestive diseases at the Geffen School of Medicine and director of UCLA’s Pancreatic Diseases Program.

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UC Davis Health System’s clinical laboratory now offers a test for respiratory viruses that its laboratory directors believe is more rapid, sensitive and comprehensive than any other test methods currently available in the Sacramento region.Known as the Respiratory Viral Panel (RVP) by RT-PCR, the test simultaneously detects 10 common respiratory viruses from patients with cold and flu-like symptoms, providing definitive results within 24 to 36 hours. In contrast, the tests used by most clinical laboratories detect fewer viruses and often take longer to generate results, potentially leading to delayed or inappropriate medical care.

The Respiratory Viral Panel by RT-PCR is particularly beneficial for severely ill patients when a fast and accurate diagnosis is needed to determine the optimal course of treatment. This was particularly true during the recent 2009 H1N1 influenza pandemic, when the RT-PCR test allowed rapid determination of whether a virus was the novel 2009 H1N1 strain or seasonal flu.

Physicians in the Sacramento area may request the RVP by RT-PCR test by ordering it through the UC Davis Health System Medical Diagnostic service.

“Rather than limiting this test to UC Davis patients, we would like to make the RVP by RT-PCR available to the entire Sacramento community, with the hope of improving diagnosis and care for as many patients as possible,” said Ralph Green, medical director of the health system’s Medical Diagnostics.

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Doctors may soon be able to quickly and accurately diagnose the cause of pneumonialike symptoms by examining the chemicals found in a patient’s urine, suggests a new study led by UC Davis biochemist Carolyn Slupsky.

Pneumonia is a lung infection that annually sickens millions of people in the United States, resulting in approximately 500,000 hospitalizations and thousands of deaths. A rapid, accurate diagnostic test for pneumonia could save lives by enabling doctors to begin appropriate treatment earlier.

Using technology known as nuclear magnetic resonance spectroscopy, the researchers were able to identify a chemical “fingerprint” for the type of pneumonia caused by the bacterium Streptococcus pneumoniae, and compare this to the chemical fingerprints for other types of pneumonia and noninfectious lung diseases.

Findings from the study, conducted by Slupsky and colleagues in Canada and Australia, are discussed in a research profile in the December issue of the Journal of Proteome Research. A patent is pending on the diagnostic procedure.

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