Primates' Unique Gene Regulation Mechanism: Little-Understood DNA Elements Serve Important Purpose

ScienceDaily (Feb. 9, 2011) — Scientists have discovered a new way genes are regulated that is unique to primates, including humans and monkeys. Though the human genome -- all the genes that an individual possesses -- was sequenced 10 years ago, greater understanding of how genes function and are regulated is needed to make advances in medicine, including changing the way we diagnose, treat and prevent a wide range of diseases.

Three baby orangutans from Tanjung Putting Orangutan Rehab Center in Borneo Indonesia. Scientists have discovered a new way genes are regulated that is unique to primates, including humans and monkeys.

"It's extremely valuable that we've sequenced a large bulk of the human genome, but sequence without function doesn't get us very far, which is why our finding is so important," said Lynne E. Maquat, Ph.D., lead author of the new study published February 9 in the journalNature.

When our genes go awry, many diseases, such as cancer, Alzheimer's and cystic fibrosis can result. The study introduces a unique regulatory mechanism that could prove to be a valuable treatment target as researchers seek to manipulate gene expression -- the conversion of genetic information into proteins that make up the body and perform most life functions -- to improve human health.

The newly identified mechanism involves Alu elements, repetitive DNA elements that spread throughout the genome as primates evolved. While scientists have known about the existence of Alu elements for many years, their function, if any, was largely unknown.

Maquat discovered that Alu elements team up with molecules called long noncoding RNAs (lncRNAs) to regulate protein production. They do this by ensuring messenger RNAs (mRNAs), which take genetic instructions from DNA and use it to create proteins, stay on track and create the right number of proteins. If left unchecked, protein production can spiral out of control, leading to the proliferation or multiplication of cells, which is characteristic of diseases such as cancer.

"Previously, no one knew what Alu elements and long noncoding RNAs did, whether they were junk or if they had any purpose. Now, we've shown that they actually have important roles in regulating protein production," said Maquat, the J. Lowell Orbison Chair, professor of Biochemistry and Biophysics and director of the Center for RNA Biology at the University of Rochester Medical Center.

The expression of genes that call for the development of proteins involves numerous steps, all of which are required to occur in a precise order to achieve the appropriate timing and amount of protein production. Each of these steps is regulated, and the pathway discovered is one of only a few pathways known to regulate mRNAs directly in the midst of the protein production process.

Regulating mRNAs is one of several ways cells control gene expression, and researchers from institutions and companies around the world are honing in on this regulatory landscape in search of new ways to manage and treat disease.

According to Maquat, "This new mechanism is really a surprise. We continue to be amazed by all the different ways mRNAs can be regulated."

Maquat and the study's first author, Chenguang Gong, a graduate student in the Department of Biochemistry and Biophysics at the Medical Center, found that long noncoding RNAs and Alu elements work together to trigger a process known as SMD (Staufen 1-mediated mRNA decay). SMD conditionally destroys mRNAs after they orchestrate the production of a certain amount of proteins, preventing the creation of excessive, unwanted proteins in the body that can disrupt normal processes and initiate disease.

Specifically, long noncoding RNAs and Alu elements recruit the protein Staufen-1 to bind to numerous mRNAs. Once an mRNA finishes directing a round of protein production, Staufen-1 works with another regulatory protein previously identified by Maquat, UPF1, to initiate the degradation or decay of the mRNA so that it cannot create any more proteins.

While the research fills in a piece of the puzzle as to how our genes operate, it also accentuates the overwhelming complexity of how our DNA shapes us and the many known and unknown players involved. Maquat and Gong plan on exploring the newly identified pathway in future research.

This research was supported by a grant from the General Medical Sciences Division of the National Institutes of Health and an Elon Huntington Hooker Graduate Student Fellowship.

Microsponges from Seaweed May Save Lives

ScienceDaily (Feb. 9, 2011) — Microsponges derived from seaweed may help diagnose heart disease, cancers, HIV and other diseases quickly and at far lower cost than current clinical methods. The microsponges are an essential component of Rice University's Programmable Bio-Nano-Chip (PBNC) and the focus of a new paper in the journalSmall.

At the heart of Rice University's Programmable Bio-Nano-Chip is a grid that contains microsponges, tiny agarose beads programmed to capture biomarkers. The biomarkers help clinicians detect signs of disease in patients.

The paper by John McDevitt, the Brown-Wiess Professor in Bioengineering and Chemistry, and his colleagues at Rice's BioScience Research Collaborative views the inner workings of PBNCs, which McDevitt envisions as a mainstream medical diagnostic tool.

PBNCs to diagnose a variety of diseases are currently the focus of six human clinical trials. McDevitt will discuss their development at the annual meeting of the American Association for the Advancement of Science (AAAS) in Washington, D.C., Feb. 17-21.

PBNCs capture biomarkers -- molecules that offer information about a person's health -- found in blood, saliva and other bodily fluids. The biomarkers are sequestered in tiny sponges set into an array of inverted pyramid-shaped funnels in the microprocessor heart of the credit card-sized PBNC.

When a fluid sample is put into the disposable device, microfluidic channels direct it to the sponges, which are infused with antibodies that detect and capture specific biomarkers. Once captured, they can be analyzed within minutes with a sophisticated microscope and computer built into a portable, toaster-sized reader.

The biomarker capture process is the subject of the Smallpaper. The microsponges are 280-micrometer beads of agarose, a cheap, common, lab-friendly material derived from seaweed and often used as a matrix for growing live cells or capturing proteins.

The beauty of agarose is its ability to capture a wide range of targets from relatively huge protein biomarkers to tiny drug metabolites. In the lab, agarose starts as a powder, like Jell-O. When mixed with hot water, it can be formed into gels or solids of any size. The size of the pores and channels in agarose can be tuned down to the nanoscale.

The challenge, McDevitt said, was defining a new concept to quickly and efficiently capture and detect biomarkers within a microfluidic circuit. The solution developed at Rice is a network of microsponges with tailored pore sizes and nano-nets of agarose fibers. The sponge-like quality allows a lot of fluid to be processed quickly, while the nano-net provides a huge surface area that can be used to generate optical signals 1,000 times greater than conventional refrigerator-sized devices. The mini-sensor ensembles, he said, pack maximum punch.

The team found that agarose beads with a diameter of about 280 micrometers are ideal for real-world applications and can be mass-produced in a cost-effective way. These agarose beads retain their efficiency at capturing biomarkers, are easy to handle and don't require specialized optics to see.

McDevitt and his colleagues tested beads with pores up to 620 nanometers and down to 45 nanometers wide. (A sheet of paper is about 100,000 nanometers thick.) Pores near 140 nanometers proved best at letting proteins infuse the beads' internal nano-nets quickly, a characteristic that enables PBNCs to test for disease in less than 15 minutes.

The team reported on experiments using two biomarkers, carcinoembryonic antigens and Interleukin-1 beta proteins (and matching antibodies for both), purchased by the lab. After soaking the beads in the antibody solutions, the researchers tested their ability to recognize and capture their matching biomarkers. In the best cases, they showed near-total efficiency (99.5 percent) in the detection of bead-bound biomarkers.

McDevitt has expected for some time that a three-dimensional bead had greater potential to capture and hold biomarkers than the standard for such tests, the enzyme-linked immunosorbent assay (ELISA) technique. ELISA analyses fluids placed in an array of 6.5-millimeter wells that have a layer of biomarker capture material spread out at the bottom. Getting results through ELISA requires a lab full of equipment, he said.

"The amount of optical signal you get usually depends on the thickness of a sample," McDevitt said. "Water, for example, looks clear in a small glass, but is blue in an ocean or a lake. Most modern clinical devices read signals from samples in flat or curved surfaces, which is like trying to see the blue color of water in a glass. It's very difficult."

By comparison, PBNCs give the researchers an ocean of information. "We create an ultrahigh-surface-area microsponge that collects a large amount of material," he said. "The sponge is like a jellyfish with tentacles that capture the biomarkers."

The agarose bead is engineered to become invisible in water. "That makes it an ideal environment to capture biomarkers, because the matrix doesn't get in the way of visualizing the contents. This is a nice use of novel biomaterials that are cheap as dirt, yet yield powerful performance," McDevitt said.

According to previous studies, only a fraction -- less than 10 percent -- of capture antibodies in the "gold standard" ELISA arrays are still active by the time a test begins. By comparison, nearly all of the antibodies in the agarose beads retain their ability to detect and capture biomarkers, McDevitt said.

Ultimately, he said, PBNCs will enable rapid, cost-effective diagnostic tests for patients who are ailing, whether they're in an emergency room, in an ambulance or even while being treated in their own homes. Even better, the chips may someday allow for quick and easy testing of the healthy to look for early warning signs of disease.

Co-authors of the paper included first author Jesse Jokerst, a National Institutes of Health postdoctoral fellow at Stanford University; postdoctoral students James Camp, Jorge Wong, Alexis Lennart, Amanda Pollard and Yanjie Zhou, all of the departments of Chemistry and Biochemistry at the University of Texas at Austin; Mehnaaz Ali, an assistant professor of chemistry at Xavier University; and from the McDevitt Lab at Rice, Pierre Floriano, director of microfluidics and image and data analysis; Nicolaos Christodoulides, director of assay development; research scientist Glennon Simmons and graduate student Jie Chou.

The National Institutes of Health, through the National Institute of Dental and Craniofacial Research, funded the research.

Fetal Surgery Takes a Huge Step Forward in Treating Children With Spina Bifida

ScienceDaily (Feb. 9, 2011) — Performing delicate surgery in the womb, months before birth, can substantially improve outcomes for children with a common, disabling birth defect of the spine. Experts at The Children's Hospital of Philadelphia (CHOP) co-led a new landmark study showing that fetal surgery for spina bifida greatly reduces the need to divert fluid from the brain, improves mobility and improves the chances that a child will be able to walk independently.

Part of the spinal cord and spinal nerves, usually encased in a sac, protrude through an opening in the back and is exposed to the amniotic fluid. The brainstem (hindbrain) descends, or herniates, into the spinal canal in the neck and blocks the flow of cerebrospinal fluid. This can cause a damaging buildup of fluid in the brain called hydrocephalus.

Spina bifida is the most common birth defect of the central nervous system, affecting about 1,500 babies born each year in the United States.

"This is the first time in history that we can offer real hope to parents who receive a prenatal diagnosis of spina bifida," said N. Scott Adzick, M.D., Surgeon-in-Chief at The Children's Hospital of Philadelphia, director of Children's Hospital's Center for Fetal Diagnosis and Treatment, and lead author of a federally sponsored study reporting results of a clinical trial of fetal surgery for myelomeningocele, the most severe form of spina bifida. Adzick, who led a team at CHOP that pioneered fetal surgeries for this condition and set the stage for this clinical trial, added, "This is not a cure, but this trial demonstrates scientifically that we can now offer fetal surgery as a standard of care for spina bifida."

Myelomeningocele is devastating, occurring when part of the spinal column does not close around the spinal cord, failing to protect it during stages of fetal development. Long-term survivors of the condition frequently suffer lifelong disabilities, including paralysis, bladder and bowel problems, hydrocephalus (excessive fluid pressure in the brain), and cognitive impairments.

Fetal surgery researchers have now reported long-awaited results from an unprecedented clinical trial that compared outcomes of prenatal, or fetal, surgery versus postnatal surgery, the conventional surgery for this disabling neurological condition. The study appears in an Online First article in theNew England Journal of Medicine.

Two and a half years after fetal surgery, children with spina bifida were better able to walk, when compared to children who received surgery shortly after birth. Patients who received fetal surgery also scored better on tests of motor function. Within a year after fetal surgery, they were less likely to need a shunt, a surgically implanted tube that drains fluid from the brain.

How the Fetal Surgery Trial was Conducted

Three fetal surgery centers participated in the Management of Myelomeningocele Study (MOMS) trial -- at The Children's Hospital of Philadelphia, Vanderbilt University, and the University of California San Francisco. The biostatistics center at George Washington University (GWU) served as the coordinating center and oversaw data collection and analysis, while the Eunice Kennedy Shriver National Institute of Child Health and Human Development sponsored the trial.

The MOMS study was a prospective, randomized clinical trial. One sign of its prominence is that all U.S. fetal surgery centers not participating in the trial agreed to perform no fetal surgery for spina bifida during the 7-year duration of the trial. The trial goal was to enroll 200 patients, but the NIH ended the trial in December 2010, after 183 surgeries had occurred, based on clear evidence of efficacy for the prenatal procedure.

Throughout the trial, women whose fetuses had been diagnosed with spina bifida contacted the trial's coordinating center at GWU if they chose to volunteer for the study. That center randomly assigned half of the eligible women to receive prenatal surgery, the other half to receive postnatal surgery.

Postnatal surgery entailed delivery by planned cesarean section at 37 weeks gestation, after which the surgical team repaired the opening in the newborn's spine, usually within 24 hours after birth. In prenatal surgery, done between 19 and 26 weeks' gestation, the surgical team made incisions in the mother and her uterus, then repaired the spina bifida lesion while the fetus was in the womb. Mothers in this group stayed near the center for ongoing monitoring, then underwent delivery by planned cesarean section at 37 weeks, or earlier, because many of the babies in the prenatal surgery group arrived prematurely.

The complex requirements of this fetal surgery require a highly sophisticated multidisciplinary team. The CHOP program includes specialists in fetal surgery, neurosurgery, obstetrics, maternal-fetal medicine, cardiology, anesthesiology and critical care, neonatology, and nursing.

In both study groups, surgeons used the same technique to cover the myelomeningocele with multiple layers of the fetus's own tissue. "This lesion leaves the spinal cord exposed, so it's essential to protect this tissue from neurological injury," said study co-author Leslie N. Sutton, M.D., Chief of Neurosurgery at The Children's Hospital of Philadelphia. Previous research had established that in myelomeningocele, amniotic fluid and other features of the intrauterine environment damage the exposed spinal cord.

Building on Decades of Research

Starting two decades ago, pioneering animal studies by Adzick and collaborators such as Martin Meuli, M.D. (now Surgeon-in-Chief at Zurich Children's Hospital in Switzerland) showed that the timing of the myelomeningocele repair was important, a finding borne out by clinical experience in fetal surgery done before the MOMS trial. "The damage to the spinal cord and nerves is progressive during pregnancy, so there's a rationale for performing the repair by the 26th week of gestation, rather than after birth," said Sutton.

The abnormal spinal development underlying myelomeningocele triggers a cascade of disabling consequences, including weakness or paralysis below the level of the defect on the spinal column. In addition, leakage of cerebrospinal fluid through the open spina bifida defect results in herniation of the brainstem down into the spinal canal in the neck -- a condition called hindbrain herniation. Hindbrain herniation obstructs the flow of cerebrospinal fluid within the brain, leading to hydrocephalus, a life-threatening buildup of fluid that can injure the developing brain. Surgeons must implant a shunt, a hollow tube that drains fluid from the brain into the child's abdominal cavity. However, shunts may become infected or blocked, often requiring a series of replacements over a patient's lifetime.

What the Study Found

The current study reports data on 158 patients who were followed at least one year after surgery. Clinicians who were independent of the surgical teams and blinded (not informed which of the two surgeries a given child received) evaluated the children from the study at one year of age and again at age 30 months.

--At one year of age, 40 percent of the children in the prenatal surgery group had received a shunt, compared to 83 percent of the children in the postnatal group. During pregnancy, all the fetuses in the trial had hindbrain herniation. However, at age 12 months, one-third (36 percent) of the infants in the prenatal surgery group no longer had any evidence of hindbrain herniation, compared to only 4 percent in the postnatal surgery group.

--At age 30 months, children in the prenatal group had significantly better scores in measurements of motor function. While the ability to walk depends on the level of the spina bifida lesion, the study found a twofold increase in the proportion of children able to walk without crutches or other assistive devices -- 42 percent in the prenatal group compared to 21 percent in the postnatal group.

--As with any surgery, fetal surgery carries risks. Fetal surgery in this study raised the risk of premature birth and scarring in the mother's uterus. "Additionally, the surgical site in the uterus used for fetal surgery requires that the mother will have to undergo a cesarean section for any subsequent births," said study co-author Mark P. Johnson, M.D., obstetrics director at the Center for Fetal Diagnosis and Treatment (CFDT).

"The mothers, children and families who participated in this MOMS trial, and who are continuing to be available for follow-up studies, have made an important contribution to our knowledge and treatment of spina bifida," said Lori J. Howell, R.N., M.S., Executive Director of the CFDT, and a study co-author. "Because of their involvement, we are better able to accurately counsel other families about what it will mean to have a child with spina bifida -- and to offer a rigorously tested, innovative prenatal surgical treatment."

Next Steps in Fetal Surgery Although the trial results mark a milestone in spina bifida treatment, not every woman carrying a fetus with spina bifida may be a suitable candidate for fetal surgery. For example, severely obese women were not included in the current study because they have a higher risk of surgical complications. Adzick noted that further research will continue to refine surgical techniques and improve methods to reduce the risks to mothers and fetuses.

In the meantime, concluded Adzick, "Both the experimental outcomes of animal studies and the results of the MOMS trial suggest that prenatal surgery for myelomeningocele stops the exposure of the developing spinal cord to amniotic fluid and thereby averts further neurological damage in utero. In addition, by stopping the leak of cerebrospinal fluid from the myelomeningocele defect, prenatal surgery reverses hindbrain herniation in utero. We believe this in turn mitigates the development of hydrocephalus and the need for shunting after birth."

Adzick added that this demonstrated success for fetal surgery may broaden its application to other birth defects, many of which are rarer but more uniformly lethal than spina bifida. Children's Hospital's comprehensive center already offers fetal surgery for selected life-threatening fetal conditions.

The Children's Hospital of Philadelphia began performing fetal surgery for spina bifida in 1998, three years after Adzick launched the Center for Fetal Diagnosis and Treatment. The Center's reports of neurological improvements in spina bifida, based on 58 fetal surgeries through 2003, helped lay the groundwork for the MOMS trial. For Adzick, who has been working to advance fetal surgery since performing preclinical studies in the early 1980s, "It's very gratifying to take this idea forward over 30 years, starting with a concept and now offering hope -- to families, mothers and the children themselves."

This trial was sponsored by the Eunice Kennedy Shriver National Institute of Child Health and Human Development. Additional funding for spina bifida research at the CFDT at The Children's Hospital of Philadelphia was provided by Katherine and Michael Mulligan, the March of Dimes Foundation and the Spina Bifida Association.

Entering a Wild Frontier: Testing Vaccines in Apes for Apes

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Credit: New Iberia Research Center

Tomorrow, chimpanzees will take part in a vaccine experiment that, for the first time, aims to help chimpanzees. Researchers at the New Iberia Research Center, a branch of the University of Louisiana, Lafayette, plan to inoculate six chimpanzees with a vaccine against Ebola, which is decimating wild ape populations. The experiment will not test whether the vaccine works, which would require injecting the animals with a "challenge" dose of the deadly Ebola virus. Rather, it will simply assess the safety of the vaccine and its ability to trigger an immune response.

Primatologist Peter Walsh, the driving force behind the experiment, ultimately wants to vaccinate wild chimpanzees and gorillas against Ebola, and he hopes this test will help him clear a few remaining hurdles. "The objective is to show the conservation community that the vaccine won't kill chimpanzees or gorillas," says Walsh, an ecologist who worked until recently at the Max Planck Institute for Evolutionary Anthropology in Leipzig, Germany. A study led by Walsh and published in the 8 December 2006 issue ofScience documented that Ebola had killed about 5000 gorillas in the area his group studied in Gabon and the Republic of Congo. "Ebola has killed one-third of the gorillas in the world," estimates Walsh.

Walsh has pushed for vaccinating wild apes for several years and says he has met some steep resistance. "Vaccination is scary both in international conservation circles and to many people in Africa," says Walsh. But Max Planck primatologist and great ape conservationist Christophe Boesch, who has worked with Walsh in the past, supports his efforts. "Vaccination of wild apes is certainly a new and somehow contentious notion," says Boesch. "However, pristine nature is becoming rarer and rarer in Africa, and if we want apes to survive in Ebola-infected areas, vaccination is one of the few realistic solutions. Peter has the stamina to bring this very challenging project to completion, and I am happy to see the concrete steps starting soon."

The vaccine the chimpanzees will receive presents little risk. Developed by Integrated BioTherapeutics Inc. in Gaithersburg, Maryland, which hopes to develop a product to protect humans against biowarfare, the vaccine contains Ebola virus proteins in what's known as a viruslike particle that cannot copy itself or cause disease. In a monkey experiment published in the 15 November 2007 Journal of Infectious Diseases, investigators from the company and the U.S. Army showed that the vaccine completely protected five monkeys against a lethal challenge dose of Ebola virus. But monkeys are not great apes, and captive chimpanzees were the next logical rung in the testing process, especially given Walsh's desire to conduct vaccination of their relatives in the wild.

New Iberia plans to immunize the chimpanzees tomorrow and then 28 days later. The researchers will collect blood samples from the animals, which will require anesthetizing them. "There's a minimal risk there," acknowledges Thomas Rowell, who heads the New Iberia facility. But he stresses that the potential benefit to chimpanzees and other great apes offsets the risks. The tests will also look for Ebola antibodies triggered by the vaccine in chimp stools, which will mirror what Walsh hopes to do in the wild.

Walsh, who has consulted many experts about how best to immunize wild apes, for safety reasons does not want to anesthetize wild apes. Instead, he plans to use darts that contain the vaccine; similar darting has been used to treat wild gorillas with antibiotics. Collection of subsequent fecal samples should allow Walsh and his team to hunt for vaccine antibodies and to pluck out DNA to identify individuals.

The New Iberia experiment is taking place against a backdrop of increasing opposition to the use of chimpanzees in biomedical research. Researchers for many decades have turned to our closest relatives to test vaccines against a wide range of diseases, including polio, hepatitis B and C, respiratory syncytial virus, and AIDS. But this "animal model" largely has fallen out of favor, primarily because the ethical landscape has changed and costs have escalated. Several countries have banned all invasive biomedical research experiments with this endangered species, and the United States and Gabon remain the only two that house captive chimps for research and allow it.

The debate about chimpanzees in research went to a full boil in the United States last year. First, Congress began considering a bill, the Great Ape Protection Act, that would altogether ban this type of biomedical research. Proponents of the bill—including the Humane Society of the United States and primatologist Jane Goodall—teamed with then-New Mexico Governor Bill Richardson and launched a high-powered campaign over the fate of a colony of 186 "research" chimpanzees that live in Alamogordo, New Mexico, and are owned by the U.S. National Institutes of Health (NIH). Scientists have not conducted studies with these chimpanzees for several years, and when NIH announced that it intended to move them to a facility in Texas that again may use them in research, a furor broke out. At the behest of three U.S. senators, the National Academy of Sciences agreed in December to analyze the current and future need for chimpanzees in biomedical research, and NIH put plans for the Alamogordo chimps on hold. New Iberia's Rowell says the analysis will likely look at the impact such a ban would have on chimpanzee health by slowing development of products like an Ebola vaccine that could help both humans and apes.

If all goes well, Walsh expects to begin tests of the Ebola vaccine in Gabon later this year.