New Therapy May Help People With Unexplained Symptoms of Pain, Weakness and Fatigue

ScienceDaily (July 28, 2011) A new type of therapy may help people with symptoms such as pain, weakness, or dizziness that can't be explained by an underlying disease, according to a study published in the July 27, 2011, online issue of Neurology, the medical journal of the American Academy of Neurology. These symptoms, which can also include fatigue, tingling and numbness, are also known as functional or psychogenic symptoms. "People with these symptoms make up one-third of all clinic visits, but the outcomes are poor," said study author Michael Sharpe, MD, of the University of Edinburgh in Scotland. Previous studies have shown that intense cognitive behavioral therapy can reduce the symptoms, distress and disability in people with these symptoms, but there are obstacles to providing this therapy. Many people do not feel psychological treatment is appropriate and resist referrals to mental health services, and therapists trained in cognitive behavioral therapy are not available in all communities. Cognitive behavioral therapy aims to improve people's physical symptoms, emotional state and functioning by helping them to understand, and where necessary change, how they think about and respond to their symptoms and life situation. For the study, the researchers developed a self-help workbook especially for patients with physical symptoms that was based on the therapy. A total of 62 people were given the workbook and over three months had up to four half-hour hour sessions guiding them in the use of the book with a nurse at their neurologist's office in addition to their usual medical care. They were compared to 63 people who received only their usual medical care. Most of the participants also had psychiatric diagnoses, such as panic disorder, anxiety disorder and depression. After three months, the people who received the extra therapy were approximately twice as likely to report improvements in their overall health as those who did not receive the extra therapy. A total of 13 percent more people who received the extra therapy reported that their health was "better" or "much better" than those who received only their usual care. After six months, there was no longer a significant difference in improvements in overall health between the two groups. However, those receiving the extra therapy continued to have greater improvement in their symptoms than those who received the usual care and also in their physical functioning. They were also more satisfied with their treatment. "This study suggests that cognitive behavioral therapy-based guided self-help may be a new and potentially useful first step in improving the management of these challenging symptoms,"

Sharpe said. "This approach needs further evaluation but can be a potentially effective and costeffective first step toward providing more help for these often neglected patients." The study was supported by the United Kingdom Medical Research Council.

A Cellular Protein Can Reduce the Growth and Spread of Cancer Cells
ScienceDaily (July 28, 2011) According to the Canadian Cancer Society, one in four Canadians will die of cancer. This year alone, the disease will kill an estimated 75,000 people. With incidence rates on the rise, more cancer patients are facing grave prognoses. Fortunately, Lawson Health Research Institute's Dr. John Lewis, Dr. Ann Chambers, and colleagues have found new hope for survival. Their new study released July 28 in Laboratory Investigation shows that maspin, a cellular protein, can reduce the growth and spread of cancer cells -- but only when it is in the nucleus. Maspin is believed to inhibit the formation, development, and spread of tumors in several aggressive cancers, including breast, ovarian, and head and neck cancers. Yet efforts to use this information to predict how cancer patients will fare have been challenging; the presence of maspin has been linked to both good and bad prognoses. Dr. Lewis, Dr. Chambers, and their team believed that this inconsistency was caused by the location of maspin in the cell, whether in the nucleus or in the cytoplasm, and sought to test this theory. To assess the effects of maspin on tumor growth and development, they tested two aggressive cancers: a highly invasive head and neck cancer, and a breast cancer known to spread to the lymph nodes and the lungs. The team introduced two forms of maspin into the cancer cells, one that went into the nucleus and one that was blocked from the nucleus. Then they injected the cells into both chicken embryo and mouse models of cancer and asked the simple question: which one slowed the cancer down? It turned out the answer was simple: when maspin was allowed to get into the nucleus of the cancer cells, the disease's ability to spread was significantly limited. In fact, the incidence of metastasis was lowered from 75% to 40%. When maspin was not established in the nucleus; however, this ability was reversed and cancer cells were far more likely to spread. These findings demonstrate that the location of maspin within the cell significantly influences cancer cells' behavior, determining how aggressive the disease will be and how positive patient outcomes will be. "The difference is night and day," Dr. Lewis says. "Metastasis is the cause of 90% of cancer deaths. We can now clearly see that maspin is working in the nucleus to dramatically reduce both the extent and the size of distant metastases." "This study resolves a mystery in which maspin was sometimes linked with poor patient prognosis and sometimes with good patient prognosis," Dr. Chambers explains. "Our new work

suggests that when maspin is located in the nucleus it blocks cancer growth and spread. This study may help doctors to understand how aggressive a patient's cancer will be, and may also lead to new targets for drug development." The study was funded through a Postdoctoral Fellowship Award from the Terry Fox Foundation, the Canadian Breast Cancer Research Alliance, the Canadian Cancer Society Research Institute, and the Canadian Institutes of Health Research.

A Heart-Rate-Reducing Medication Reduces the Risk of Heart Failure and Cardiac Fibrosis
ScienceDaily (July 28, 2011) The findings of a Montreal Heart Institute (MHI) study published in the scientific journal Cardiology suggest that ivabradine, a heart rate reduction medication, is also effective in reducing the risk of diastolic heart failure (left ventricular insufficiency) and cardiac fibrosis. The benefits of slower heart rate on mortality and morbidity associated with cardiovascular disease no longer need to be demonstrated. In this study, titled "Heart Rate Reduction by Ivabradine Reduces Diastolic Dysfunction and Cardiac Fibrosis," researchers sought to determine the effectiveness of ivabradine in treating diastolic dysfunction of the left ventricle, a condition affecting 40% of people with heart failure. The study was conducted on rabbits given a standard diet, a cholesterol-enriched diet or a cholesterol-enriched diet with ivabradine. It revealed that as well as improving the myocardial performance index, ivabradine greatly improved left ventricular diastolic dysfunction in animals receiving a cholesterol-enriched diet. Ivabradine also reduced fibrosis of the heart chambers. According to Dr. Jean-Claude Tardif, Director of the MHI Research Centre and professor of medicine at the Universit de Montral, the results are both interesting and encouraging. "The effectiveness of ivabradine in treating angina pectoris is now well known. However, few treatments are available to patients with diastolic heart failure. The beneficial effects of ivabradine demonstrated in laboratory suggest that this course of treatment should be further investigated." Clinical studies with subjects are expected to follow.

Increased Muscle Mass May Lower Risk of Pre-Diabetes: Study Shows Building Muscle Can Lower Person's Risk of Insulin Resistance

ScienceDaily (July 28, 2011) A recent study accepted for publication in The Endocrine Society's Journal of Clinical Endocrinology & Metabolism (JCEM) found that the greater an individual's total muscle mass, the lower the person's risk of having insulin resistance, the major precursor of type 2 diabetes. With recent dramatic increases in obesity worldwide, the prevalence of diabetes, a major source of cardiovascular morbidity, is expected to accelerate. Insulin resistance, which can raise blood glucose levels above the normal range, is a major factor that contributes to the development of diabetes. Previous studies have shown that very low muscle mass is a risk factor for insulin resistance, but until now, no study has examined whether increasing muscle mass to average and above average levels, independent of obesity levels, would lead to improved blood glucose regulation. "Our findings represent a departure from the usual focus of clinicians, and their patients, on just losing weight to improve metabolic health," said the study's senior author, Preethi Srikanthan, MD, of the University of California, Los Angeles (UCLA). "Instead, this research suggests a role for maintaining fitness and building muscle. This is a welcome message for many overweight patients who experience difficulty in achieving weight loss, as any effort to get moving and keep fit should be seen as laudable and contributing to metabolic change." In this study, researchers examined the association of skeletal muscle mass with insulin resistance and blood glucose metabolism disorders in a nationally representative sample of 13,644 individuals. Participants were older than 20 years, non-pregnant and weighed more than 35 kg. The study demonstrated that higher muscle mass (relative to body size) is associated with better insulin sensitivity and lower risk of pre- or overt diabetes. "Our research shows that beyond monitoring changes in waist circumference or BMI, we should also be monitoring muscle mass," Srikanthan concluded. "Further research is needed to determine the nature and duration of exercise interventions required to improve insulin sensitivity and glucose metabolism in at-risk individuals." Also working on the study was Arun Karlamangla, PhD, MD, of the David Geffen School of Medicine at UCLA.

Scientist Converts Human Skin Cells Into Functional Brain Cells

ScienceDaily (July 28, 2011) A scientist at the Gladstone Institutes has discovered a novel way to convert human skin cells into brain cells, advancing medicine and human health by offering new hope for regenerative medicine and personalized drug discovery and development.

Rendering of the brain. A scientist at the Gladstone Institutes has discovered a novel way to convert human skin cells into brain cells, advancing medicine and human health by offering new hope for regenerative medicine and personalized drug discovery and development. (Credit: ktsdesign / Fotolia) in a paper being published online July 28 in the scientific journal Cell Stem Cell, Sheng Ding, PhD, reveals efficient and robust methods for transforming adult skin cells into neurons that are capable of transmitting brain signals, marking one of the first documented experiments for transforming an adult human's skin cells into functioning brain cells. "This work could have important ramifications for patients and families who suffer at the hands of neurodegenerative diseases such Alzheimer's, Parkinson's and Huntington's disease," said Lennart Mucke, MD, who directs neurological research at Gladstone. "Dr. Ding's latest research offers new hope for the process of developing medications for these diseases, as well as for the possibility of cell-replacement therapy to reduce the trauma of millions of people affected by these devastating and irreversible conditions." The work was done in collaboration with Stuart Lipton, M.D., Ph.D., who directs the Del E. Webb Neuroscience, Aging and Stem Cell Research Center at Sanford-Burnham Medical Research Institute. Dr. Ding, one of the world's leading chemical biologists in stem-cell science, earlier this year joined Gladstone and the faculty at the University of California San Francisco (UCSF), as a professor of pharmaceutical chemistry. Gladstone, which is affiliated with UCSF, is a leading and independent biomedical-research organization that is using stem-cell research to advance its work in its three major areas of focus: cardiovascular disease, neurodegenerative disease and viral infections. Dr. Ding's work builds on the cell-reprogramming work of another Gladstone scientist, Senior Investigator Shinya Yamanaka, MD, PhD. Dr. Yamanaka's 2006 discovery of a way to turn adult skin cells into cells that act like embryonic stem cells has radically advanced the fields of cell biology and stem-cell research.

Embryonic stem cells -- "pluripotent" cells that can develop into any type of cell in the human body -- hold tremendous promise for regenerative medicine, in which damaged organs and tissues can be replaced or repaired. Many in the science community consider the use of stem cells to be key to the future treatment and eradication of a number of diseases, including heart disease and diabetes. But the use of embryonic stem cells is controversial -- which is one reason why Dr. Yamanaka's discovery of an alternate way to obtain human stem cells, without the use of embryos, is so important. Dr. Ding's work extends Dr. Yamanaka's by offering still another method for avoiding the use of embryonic stem cells and creating an entirely new platform for fundamental studies of human disease. Rather than using models made in yeast, flies or mice for disease research, all cellreprogramming technology allows human brain, heart and other cells to be created from the skin cells of patients with a specific disease. The new cells created from the skin cells contain a complete set of the genes that resulted in that disease -- representing the potential of a farsuperior human model for studying illnesses, drugs and other treatments. In the future, such reprogrammed skin cells could be used to test both drug safety and efficacy for an individual patient with, for example, Alzheimer's disease. "This technology should allow us to very rapidly model neurodegenerative diseases in a dish by making nerve cells from individual patients in just a matter of days -- rather than the months required previously," said Dr. Lipton. In the experiments being reported July 28, Dr. Ding used two genes and a microRNA to convert a skin sample from a 55-year-old woman directly into brain cells. (MicroRNAs are tiny strands of genetic material that regulate almost every process in every cell of the body.) The cells created by Dr. Ding's experiments exchanged the electrical impulses necessary for brain cells to communicate things such as thoughts and emotions. Using microRNA to reprogram cells is a safer and more efficient way than using the more common gene-modification approach. In ensuing experiments, Dr. Ding hopes to rely only on microRNAs and pharmaceutical compounds to convert skin cells to brain cells, which should lead to more efficient generation of cells for testing and regenerative purposes. "This will help us avoid any genome modifications," said Dr. Ding. "These cells are not ready yet for transplantation. But this work removes some of the major technical hurdles to using reprogrammed cells to create transplant-ready cells for a host of diseases." Dr. Ding is a senior investigator at the Gladstone Institute of Cardiovascular Disease and a UCSF professor of pharmaceutical chemistry. Dr. Ding, who performed the work described in this paper at The Scripps Research Institute, has pioneered the development and application of innovative chemical approaches to stem-cell biology and regeneration.

Researchers Target, Switch Off SerotoninProducing Neurons in Mice; New Insights May Be Relevant to Sudden Infant Death Syndrome
ScienceDaily (July 28, 2011) Researchers have developed a toolkit that enables them to turn off targeted cell populations while leaving others unaffected. Led by Susan Dymecki, a professor of genetics at Harvard Medical School, the group focused on serotonin-producing neurons, observing how mice behave in a normal environment when suddenly their serotonin neurons are turned down. While their findings affirm earlier studies, the researchers used a technique that is non-invasive and does not require anesthesia, surgeries, or knocking out a gene -- each of which can cause problems when interpreting results. "By selectively and abruptly switching off the serotonin-producing cells, we can get a definite idea of what bodily functions the serotonin cells specifically control," said Dymecki. "These findings and the new tools in neuroscience that it brings to the table will help us understand the role of serotonergic neurons in many human disorders." One such disorder particularly relevant to these findings is Sudden Infant Death Syndrome, or SIDS. These findings will appear in the July 29 edition of the journal Science. The mammalian brain contains multiple chemical messengers, called neurotransmitters, which transfer information between nerve cells in order to regulate basic behaviors and functions like walking, eating, and sleeping. Serotonin is a major brain neurotransmitter produced solely by cells in the lower brain, or brainstem. Cells that make serotonin can convey information to large numbers of neurons distributed throughout the brain and can affect behavior as complex as mood. In order to better understand how these serotonin-producing cells in the brain relate to basic physiology, Russell Ray and Rachael Brust, a postdoctoral researcher and a graduate student in Dymecki's lab, along with Jun Chul Kim, a prior postdoctoral fellow in Dymecki's lab who is now at the University of Toronto, and Andrea Corcoran, a postdoctoral researcher in the lab of Eugene Nattie at Dartmouth Medical School along with George Richerson, a professor of neurology at the University of Iowa, developed and characterized a method for selectively silencing neurons that produce serotonin. The group began with a molecule genetically engineered by Bryan Roth and his colleagues at the University of North Carolina School of Medicine. Using a method that Dymecki's group had developed and optimized over the years called "intersectional genetics," they incorporated this

molecule, a receptor, into the serotonin-producing brain cells in mice. As a result, the mice naturally generated this "unnatural" receptor on the surface of their serotoninergic neurons. Receptors are key players in cellular communications, the initial recipients of chemical signals sent by other cells. Here, the researchers injected the mice with clozapine-N-oxide, a chemical compound designed to bind to and trigger the engineered receptor. Within minutes, the chemical and the foreign receptor acted together as a kind of dimmer switch, dampening the action of serotonin networks in the brains of these mice. "This gave us the ability to selectively shut down serotonergic neuron function in the mouse brain," said Ray. "The mice remained awake, thus we could study their behavior in a normal environment." When serotonergic neuron activity was diminished, the mice lost their capacity to maintain body temperature, and their temperatures plummeted to that of their surrounding environment. Also, the ability of the mice to physiologically respond to elevations in carbon dioxide levels -typically in the form of heavy, rapid breathing to rid the body of excessive carbon dioxide buildup before it might reach dangerous levels -- was roughly half that of normal mice when serotonergic neuron activity was low. "What is particularly powerful to note is that we were able to study the mice both before and after we switched off the serotonergic neurons," said Ray. "We were able to demonstrate that prior to activating this foreign receptor switch -- that is prior to silencing the serotonin neurons -the mice responded normally to temperature and carbon dioxide challenges." The researchers believe this work may help us better understand the mechanisms underlying SIDS in humans. Recent findings from the lab of Hannah Kinney at Children's Hospital Boston suggest that SIDS babies may have a deficiency of serotonin in circuits in the brainstem. Such deficiencies may lead directly to abnormal responses to elevated levels of carbon dioxide, such as when a baby rebreathes exhaled stale gases with high carbon dioxide levels while lying facedown. "These infants may be vulnerable to sudden death due to impaired serotonin function in brainstem circuits important for protective responses to life threatening challenges, such as increased levels of carbon dioxide," said Dymecki. "What's more, a SIDS-vulnerable infant may be less equipped to maintain a normal body temperature." Dymecki, along with her lab members and her colleagues at Dartmouth and University of Iowa, are now investigating how serotonergic neurons influence vital functions in young mice that are in the comparable age range to human infants at peak risk for SIDS. They also plan to use this genetic platform to selectively turn off subsets of serotonergic neurons, to better understand their specific functioning in health, and in other serotonin-linked disorders.

Traumatic Brain Injury Linked With Tenfold Increase in Stroke Risk

ScienceDaily (July 29, 2011) If you suffer traumatic brain injury, your risk of having a stroke within three months may increase tenfold, according to a new study reported in Stroke: Journal of the American Heart Association. "It's reasonable to assume that cerebrovascular damage in the head caused by a traumatic brain injury can trigger either a hemorrhagic stroke [when a blood vessel bursts inside the brain] or an ischemic stroke [when an artery in the brain is blocked]," said Herng-Ching Lin, Ph.D., senior study author and professor at the School of Health Care Administration, College of Medicine, Taipei Medical University in Taiwan. "However, until now, no research had been done showing a correlation between traumatic brain injury and stroke." It is the first study that pinpoints traumatic brain injury as a potential risk factor for subsequent stroke. Traumatic brain injury occurs when an external force such as a bump, blow or jolt to the head disrupts the normal function of the brain. Causes include falls, vehicle accidents, and violence. In the United States alone, approximately 1 in 53 individuals sustain a traumatic brain injury each year, according to 2004 statistics from the Centers for Disease Control and Prevention. Worldwide, traumatic brain injuries are a major cause of physical impairment, social disruption and death. Using records from a nationwide Taiwanese database, researchers investigated the risk of stroke in traumatic brain injury patients during a five-year period. The records included 23,199 adult traumatic brain injury patients who received ambulatory or hospital care between 2001 and 2003. The comparison group comprised 69,597 non-traumatic brain injury patients. The average age of all patients was 42 and 54 percent were male. During the three months after injury, 2.91 percent of traumatic brain injury patients suffered a stroke compared with only 0.30 percent of those with non-traumatic brain injury -- a tenfold difference. Stroke risk in patients with traumatic brain injury decreased gradually over time, researchers said:

After one year, the risk was about 4.6 times greater for patients who suffered a traumatic brain injury than for those who had not. After five years, the risk was 2.3 times greater for traumatic brain injury patients.

Stroke risk among traumatic brain injury patients with skull bone fractures was more pronounced than in traumatic brain injury patients without fractures, researchers said. During the first three months, those with skull bone fractures were 20 times more likely to have a stroke than patients without skull bone fractures. The risk decreased over time. Furthermore, the risk of subarachnoid hemorrhage (bleeding in the area between the brain and the thin tissues that cover the brain) and intracerebral hemorrhage (bleeding in the brain caused by the rupture of a blood vessel) increased significantly in patients with traumatic brain injury versus non-traumatic brain injury patients. After considering age and gender, patients with traumatic brain injury were more likely to have hypertension, diabetes, coronary heart disease, atrial fibrillation and heart failure than nontraumatic brain injury patients. Early neuroimaging examinations -- such as MRI -- and intensive medical monitoring, support and intervention should be required following a traumatic brain injury, especially during the first few months and years, Lin said. Moreover, better health education initiatives could increase public awareness about the factors that cause strokes and the signs and symptoms of stroke in patients with traumatic brain injuries. "Stroke is the most serious and disabling neurological disorder worldwide," said Lin. "Our study leads the way in identifying stroke as an additional neurological problem that may arise following traumatic brain injury." Co-authors are: Yi-Hua-Chen, Ph.D, lead author and Jiunn-Horng Kang, M.D.

Rate of Stroke Increasing Among Women During, Soon After Pregnancy

ScienceDaily (July 29, 2011) The stroke rate for pregnant women and those who recently gave birth increased alarmingly over the past dozen years, according to research reported in Stroke: Journal of the American Heart Association. Researchers gathered data from a large national database of 5 to 8 million discharges from 1,000 hospitals and compared the rates of strokes from 1994-95 to 2006-07 in women who were pregnant, delivering a baby and who had recently had a baby. Pregnancy-related stroke hospitalizations increased 54 percent, from 4,085 in 1994-95 to 6,293 in 2006-07. "I am surprised at the magnitude of the increase, which is substantial," said Elena V. Kuklina, M.D., Ph.D., lead author of the study and senior service fellow and epidemiologist at the Centers

for Disease Control and Prevention's Division for Heart Disease and Stroke Prevention in Atlanta, Ga. "Our results indicate an urgent need to take a closer look. Stroke is such a debilitating condition. We need to put more effort into prevention. "When you're relatively healthy, your stroke risk is not that high," Kuklina said. "Now more and more women entering pregnancy already have some type of risk factor for stroke, such as obesity, chronic hypertension, diabetes or congenital heart disease. Since pregnancy by itself is a risk factor, if you have one of these other stroke risk factors, it doubles the risk." For expectant mothers, the rate of stroke hospitalizations rose 47 percent. In pregnant women and in women who had a baby in the last 12 weeks (considered the postpartum period), the stroke rate rose 83 percent. However, the rate remained the same for stroke hospitalizations that occurred during the time immediately surrounding childbirth. Pregnant and post partum women ages 25 to 34 were hospitalized for stroke more often than those who were younger or older. Furthermore, high blood pressure was more prevalent in pregnant women who were hospitalized because of stroke. In 1994-95, among pregnant women with stroke, researchers found high blood pressure in:

11.3 percent of the pregnant women prior to birth; 23.4 percent of those at or near delivery; and 27.8 percent of those within 12 weeks of delivery.

In 2006-07, they discovered high blood pressure among stroke patients in:

17 percent of those pregnant; 28.5 percent of those at or near delivery; and 40.9 percent of women in the postpartum period.

It's best for women to enter pregnancy with ideal cardiovascular health -- without additional risk factors, Kuklina said. Next, she suggests developing a comprehensive, multidisciplinary plan that gives doctors and patients guidelines for appropriate monitoring and care before, during and after childbirth. A major problem is that pregnant women typically aren't included in clinical trials because most drugs pose potential harm to the fetus. Therefore, doctors don't have enough guidance on which medications are best for pregnant women who have an increased risk for stroke. "We need to do more research on pregnant women specifically," said Kuklina, who found only 11 cases of pregnancy-related stroke in her review of previously published literature.

Co-authors are: Xin Tong, M.P.H.; Pooja Bansil, M.P.H.; Mary G. George, M.D., M.S.P.H.; and William M. Callaghan, M.D., M.P.H. Author disclosures are on the manuscript.

ncreasing Potency of HIV-Battling Proteins

ScienceDaily (July 28, 2011) If one is good, two can sometimes be better. Researchers at the California Institute of Technology (Caltech) have certainly found this to be the case when it comes to a small HIV-fighting protein. The protein, called cyanovirin-N (CV-N), is produced by a type of blue-green algae and has gained attention for its ability to ward off several diseases caused by viruses, including HIV and influenza. Now Caltech researchers have found that a relatively simple engineering technique can boost the protein's battling prowess. "By linking two cyanovirins, we were able to make significantly more potent HIV-fighting molecules," says Jennifer Keeffe, a staff scientist at Caltech and first author of a new paper describing the study in the Proceedings of the National Academy of Sciences (PNAS). "One of our linked molecules was 18 times more effective at preventing infection than the naturally occurring, single protein." The team's linked pairs, or dimers, were able to neutralize all 33 subtypes of HIV that they were tested against. The researchers also found the most successful dimer to be similar or more potent than seven well-studied anti-HIV antibodies that are known to be broadly neutralizing. CV-N binds well to certain carbohydrates, such as the kind found in high quantities connected to the proteins on the envelope that surrounds the HIV virus. Once attached, CV-N prevents a virus from infecting cells, although the mechanism by which it accomplishes this is not well understood. What is known is that each CV-N protein has two binding sites where it can bind to a carbohydrate and that both sites are needed to neutralize HIV. Once the Caltech researchers had linked two CV-Ns together, they wanted to know if the enhanced ability of their engineered dimers to ward off HIV was related to the availability of additional binding sites. So they engineered another version of the dimers -- this time with one or more of the binding sites knocked out -- and tested their ability to neutralize HIV. It turns out that the dimers' infection-fighting potency increased with each additional binding site -- three sites are better than two, and four are better than three. The advantages seemed to stop at four sites, however; the researchers did not see additional improvements when they linked three or four CV-N molecules together to create molecules with six to eight binding sites. Although CV-N has a naturally occurring dimeric form, it isn't stable at physiological temperatures, and thus mainly exists in single-copy form. To create dimers that would be stable

under such conditions, the researchers covalently bound together two CV-N molecules in a headto-tail fashion, using flexible polypeptide linkers of varying lengths. Interestingly, by stabilizing the dimers and locking them into a particular configuration, it seems that the group created proteins with distances between binding sites that are very similar to those between the carbohydrate binding sites in a broadly neutralizing anti-HIV antibody. "It is possible that we have created a dimer that has its carbohydrate binding sites optimally positioned to block infection," says Stephen Mayo, Bren Professor of Biology and Chemistry, chair of the Division of Biology, and corresponding author of the new paper. Because it is active against multiple disease-causing viruses, including multiple strains of HIV, CV-N holds unique promise for development as a drug therapy. Other research groups have already started investigating its potential application in prophylactic gels and suppositories. "Our hope is that those who are working to make prophylactic treatments using cyanovirin will see our results and will use CVN2L0 instead of naturally occurring cyanovirin," Keeffe says. "It has higher potency and may be more protective." The work was funded by the National Security Science and Engineering Faculty Fellowship program, the Defense Advanced Research Projects Agency Protein Design Processes program, and the Bill and Melinda Gates Foundation through the Grand Challenges in Global Health Initiative.

The experimentally determined structure of one of the engineered dimers (CVN2L0). One CV-N repeat is shown in green, while the other appears in blue. The polypeptide linker is not shown. (Credit: Caltech/Jennifer Keeffe)