News this Week

Science  12 Jan 2007:
Vol. 315, Issue 5809, pp. 170
1. STEM CELLS

Versatile Stem Cells Without the Ethical Baggage?

1. Constance Holden

Scientists this week reported that they have isolated a new type of cell from amniotic fluid that has many of the characteristics of embryonic stem (ES) cells without the ethical baggage. But other researchers, although enthusiastic about the work, are questioning just how new these so-called amniotic fluid-derived stem (AFS) cells are and are warning that they don't eliminate the need for ES cells.

The report, published online 7 January in Nature Biotechnology, seems likely to throw a new twist into this week's congressional debate over legislation to expand ES cell lines available to federally funded researchers. Congressional leaders were planning to make a splash by getting both houses to pass once again a measure that was vetoed last year by President George W. Bush. But if this much-touted paper persuades the public there's a ready alternative to ES cells, “the bill won't have the impact it would have had,” says bioethicist William Hurlbut of Stanford University in Palo Alto, California. The researchers themselves, led by Anthony Atala of Wake Forest University School of Medicine in Winston-Salem, North Carolina, say that AFS cells, obtained from amniocentesis samples, are no substitute for ES cells. But they see them as a unique type occupying an “intermediate” stage between embryonic and adult stem cells in terms of their versatility.

Several groups have already cultivated specialized tissue types from amniotic stem cells. But Atala insists that AFS cells are “absolutely totally different.” He says they are the only amniotic cells that are “fully undifferentiated” and pluripotent—by which he means capable of giving rise to representatives of all three embryonic germ layers. He concedes, however, that it is still unclear whether AFS cells can give rise to all cell types in the body, as can ES cells.

The team, which includes researchers from Children's Hospital and Harvard Medical School in Boston, has spent the past 7 years working up their evidence that AFS cells are capable of developing into fat, bone, muscle, nerves, liver, and the lining of blood vessels. They injected human AFS cells that had been coaxed to become neural precursor cells into the brains of newborn mice and found that they dispersed throughout the brains. And cells cultivated in a bone-growing medium not only produced mineralized calcium and other bone markers but also led to the growth of chunks of bonelike material when cultured on scaffolds and implanted into mice. AFS-derived liver cells secreted urea, a liver-specific function, in test tubes. Atala said at a press conference that the group has unpublished evidence that the AFS cells can also form blood cells. It has yet to produce pancreatic beta cells, needed to treat diabetes, but Atala says, “so far, we've been successful with every cell type we've attempted.”

Like ES cells, said Atala, the amniotic cells grow rapidly, doubling every 36 hours, and the cell lines are capable of extensive self-renewal without differentiation. Unlike ES cells, they can be readily obtained from amniocentesis without harm to the donor or fetus. And they multiply indefinitely without forming tumors—a big peril with ES cells.

Atala, whose university has applied for a patent on the cell type and the team's method for isolating them, said that amniotic cells may eventually be used as a repair kit for birth defects. He also predicted that banks of cell lines obtained from 100,000 pregnancies could offer reasonably good tissue matches to 99% of the population. Some scientists are deeply impressed. “I believe … that Dr. Atala's group has discovered a new stem cell,” says adult stem cell researcher Henry E. Young of Mercer University School of Medicine in Macon, Georgia.

Atala says AFS cells are the only type distinguished by C-Kit, a germ cell marker not reported in other papers about amniotic stem cells. Nonetheless, Dario Fauza of Children's Hospital, a pediatric surgeon unconnected with the Atala team who has pioneered in cultivating tissues from amniotic stem cells, says he doubts “whether they have indeed discovered a new stem cell. … I have the distinct impression we're just giving different names to the same cell.” Ming-Song Tsai, a stem cell researcher at Cathay General Hospital in Taipei, Taiwan, agrees. Atala's study is “excellent,” he says. But judging by surface markers and other characteristics, he believes “the cells described in this paper are the same cells” he and colleagues described last year in Biology of Reproduction. In that paper, the scientists reported cultivating “mesenchymal” stem cells from a single amniotic cell that could develop not only into multiple mesenchymal lineages but also into neuron-like cells. Tsai, who already has a U.S. patent on his method, adds that recently they revealed potential as liver cells.

Tsai predicts that amniotic stem cells may become a valuable tool given their “easy access [and] cultivation” and absence of ethical difficulties. But some researchers are taking a wait-and-see attitude. Harvard stem cell researcher Kevin Eggan is skeptical, especially because the field has been “burned” in recent years by hints of pluripotency in other cell types that haven't panned out.

2. ENERGY

Consortium Wins Big Drilling Technology Contract

1. Eli Kintisch

A consortium of big energy firms and universities has received $375 million, to be spread over 10 years, from the U.S. government for research on new ways to find and extract oil and gas. The money, awarded by the Department of Energy, comes from a controversial fund created by Congress in 2005 to encourage companies to pursue high-risk projects with potentially large payoffs. Opponents say the program is an unnecessary corporate subsidy in an era of rising energy prices. Last week's contract awarded to the Research Partnership to Secure Energy for America (RPSEA), a Sugarland, Texas, nonprofit whose members include energy giant Schlumberger and the Massachusetts Institute of Technology, will support development of new techniques to find fossil fuels from the deepest portion of the oceans and hard-to-obtain stores on land such as tar sands. It will also fund small energy companies that have traditionally eschewed research. RPSEA's Robert Siegfried says an “academic-industry powwow” will drive research priorities. RPSEA officials have yet to finalize the solicitation, but geophysicist Bob Hardage of the University of Texas, Austin, is hoping that the consortium will bolster his work on advanced seismic techniques for finding gas or oil in rock. Large amounts of natural gas are locked in icy cages called methane hydrates, he says, but oil firms have been leery of investing in what remains an unproven resource. “Hydrates may have great potential, or they may fall flat on their face. We need to get the answer,” says Hardage. A grant that ended in 2004 from RPSEA through a federal pot of money that has since dried up attracted corporate interest in his lab's algorithms for obtaining and interpreting seismic data. The new RPSEA funding won't focus on hydrates per se but could fund basic seismic work to unlock their potential. Critics question why the program, created by the 2005 energy bill, is run by a third party rather than by federal officials, who would be free of corporate ties. In 2005, President George W. Bush, a former oilman, said the government shouldn't pony up even a cent because the price of oil is sufficiently high for companies to afford risky research. Last summer, the U.S. House rejected a move to cancel the program by a vote of 161 to 255. “To call a federal R&D program a subsidy is like calling public education a social giveaway,” said one supporter, Representative Ralph Hall (R-TX), at the time of the vote. RPSEA officials point to rigorous reporting and oversight requirements designed to prevent conflicts of interest. The 2005 bill also provided$125 million to be administered by the National Energy Technology Laboratory, headquartered in Pittsburgh, Pennsylvania, to fund related research.

3. U.S. NATIONAL SECURITY

Head of Weapons Program Fired

1. Eli Kintisch

The U.S. government's top nuclear weapons official has been fired because of a series of security breaches at Los Alamos National Laboratory (LANL) in New Mexico. Although some legislators had previously called for the resignation of Linton Brooks, head of the National Nuclear Security Agency (NNSA), last week's announcement by Energy Secretary Samuel Bodman came as a surprise to many observers.

“I've never seen anything like it,” says Peter Stockton of the nonprofit Project on Government Oversight (POGO), which publicized an incident last October in which police found computer drives containing classified information from LANL during a neighborhood drug raid. “This is not a decision that I would have preferred, but it was made by a thoughtful and honorable man and is based on the principle of accountability,” says Brooks, a former arms-control negotiator.

The raid followed a breach earlier in 2006 of an unclassified federal computer system, in which a hacker obtained access to information on some 1500 government employees, many with top security clearances. Brooks, who had headed NNSA since 2003, didn't notify his boss for 9 months. “I do not believe that progress in correcting these [security] issues has been adequate,” Bodman said last week before naming former Brooks deputy Thomas D'Agostino as interim replacement.

Parts of the lab were shut down for as long as 7 months in 2004 after incidents involving missing disks and a laser accident. Prodded by Congress, the Department of Energy selected a new management team for the lab, which had been run by the University of California for more than 60 years (Science, 6 January 2006, p. 33). Some scientists fear that the new partnership, which includes the university and several major corporations, will result in more paperwork—much of it related to security and safety—and a smaller budget for the $2.2 billion facility. But the team passed its first test last fall when fewer researchers than projected chose to retire. 4. CHEMISTRY Platinum in Fuel Cells Gets a Helping Hand 1. Robert F. Service The behavior of nanoscopic bits of platinum may determine whether a hydrogen-powered car is in your future. The precious metal is the key ingredient in fuel cells that power electric cars with hydrogen, producing water as the only byproduct. Unfortunately, current models are expensive because they use so much platinum, and their performance degrades too quickly for practical use. But advances by two U.S.-led groups offer new hope for tackling these problems. The researchers targeted what is widely considered to be the biggest concern in fuel cells: improving the performance of the platinum on the positively charged electrode, or cathode—the part of the cell where chemicals react to split oxygen molecules in half. One group, led by materials scientists Vojislav Stamenkovic and Nenad Markovic at Argonne National Laboratory in Illinois, reports in a paper published online by Science this week (www.sciencemag.org/cgi/content/abstract/1135941) that it increased the catalytic activity of a platinum surface 90-fold over conventional cathode catalysts used today. Meanwhile, the other group, led by chemist Radoslav Adzic of Brookhaven National Laboratory in Upton, New York, reports on page 220 that adding tiny gold clusters to the outside of their cathode materials dramatically reduced the tendency of platinum to dissolve from the cathode over extended use. “Both of these results could be quite important if the concepts can be brought to fruition in a practical manner,” says Fred Wagner, a platinum catalyst expert at General Motors' fuel cell research center in Honeoye Falls, New York. Platinum is the key to fuel cells because of its unusually high catalytic properties. This ability comes into play first at the negative electrode, or anode, to split hydrogen molecules (H2) into two protons (2 H+) and two electrons (2e). The electrons then pass through a wire and power the car. At the end of their journey, they wind up at the cathode and pass to oxygen molecules, breaking them into negatively charged oxygen atoms (O22−). These oxygens then pair up with protons from the anode to create water molecules. Typically, catalyzing the reactions at each electrode are platinum nanoparticles that lightly coat a high-surface-area carbon skeleton. In practice, however, unwanted side reactions also occur around the cathode. Some charged oxygen atoms react with protons to create hydroxide molecules (OH) and likely other oxides as well. These oxides have an affinity for platinum atoms. They bind to the cathode surface, where they typically block access to as many as 45% of the platinum atoms, Markovic says. Even worse, the oxides tug on the platinum atoms and eventually pull many of them off the surface, drastically reducing the cathode's catalytic ability. Researchers have made some progress on both problems by alloying platinum with other metals. In previous work, Stamenkovic and colleagues studied polycrystalline platinum electrodes alloyed with other metals and found that some of the crystalline portions seemed to perform better than others. They suspected that the disparity reflected different ways platinum atoms can pack on a surface—such as a squarelike arrangement versus a hexagonal arrangement. To find out, for their current study Stamenkovic, Markovic, and colleagues created pure single crystals of platinum-nickel alloys with different atomic arrangements of their crystalline lattices. They compared the samples with single crystals of pure platinum as well as with conventional platinum-carbon fuel cell catalysts. They found that the most tightly packed arrangement of atoms, known in the materials lingo as a 111 surface, far outperformed all the others. The material wound up with a uniform layer of platinum atoms on top of a layer with 50% nickel atoms. All the layers under that had essentially a steady composition of three parts platinum to one part nickel (see diagram). Stamenkovic says the group's theoretical work shows that the 111 arrangement lowers the electronic interaction between platinum atoms on the surface and oxides seeking to bind to them. The upshot is that far fewer oxides bind to the platinum surface, leaving those sites open to carry out O2-splitting reactions. That setup boosts the PtNi alloy's activity 10-fold over a single-crystal platinum surface and 90-fold over the standard platinum-carbon combo. The reduced interaction also tugs less on the surface Pt atoms and therefore yanks fewer atoms off the surface. That increase in stability was echoed by the result from Adzic's team. Adzic and colleagues deposited tiny gold nanoclusters on the top of a conventional carbon-platinum fuel cell cathode. They found that the clusters produced a similar change in the electronic behavior of the surface of the cathode that prevented platinum atoms from dissolving into the electrolyte, while leaving the overall oxygen-splitting activity of the platinum unchanged. The key now, Wagner and others say, will be to create highly active, stable real-world catalysts. Markovic says his group is already working on creating octahedron-shaped platinum-nickel nanoparticles that theory shows should have all the desired 111 surfaces. If they work, hydrogen fuel cell-powered cars will take a major step toward widespread use. 5. HUMAN GENETICS In Asians and Whites, Gene Expression Varies by Race 1. Jennifer Couzin Genetic variation among races, long a political hot potato, has also been a scientific puzzle. Although researchers have cataloged different frequencies of inherited DNA among racial groups, and physicians have found that some groups are disproportionately susceptible to certain diseases, it's not clear how or even whether the two are linked. Do subtle differences in DNA between races really matter, medically speaking? Earlier this week, scientists described results from a new approach that may help answer that question: measuring gene expression levels among Caucasians and Asians. Because gene expression helps determine how a cell behaves, it can be more instructive than variations in inherited DNA. The researchers examined expression levels of more than 4000 genes in 142 banked cell lines drawn from individuals of European descent in Utah, and cohorts from Beijing and Tokyo. They found that 25% of the genes had expression patterns with statistically significant, although often small, differences depending on whether they came from a Caucasian or an Asian sample. Thirty-five genes had expression levels that differed, on average, as much as twofold. Still, “how that translates into traits of clinical interest is still a big question mark,” says Neil Risch, a human geneticist at the University of California, San Francisco. Although that critical bridge remains to be built, scientists say the expression patterns are intriguing. Indeed, geneticist Vivian Cheung of the University of Pennsylvania, who led the research team with her colleague Richard Spielman, was initially so taken aback by the number of genes whose expression varied that she suspected a technical glitch. “The 25% definitely shocked me,” says Cheung, who also works at the Children's Hospital of Philadelphia. But when she and her colleagues repeated the study on samples from 24 Chinese residents in Los Angeles, the results were virtually identical. All but one of the 35 genes with big variations in expression registered similar levels in the HapMap Asian samples and the Los Angeles cohort, they report online this week in Nature Genetics. “This lends support to the idea that there are genetically determined characteristics that tend to be clustered in different ethnic groups,” says Phyllis August, a nephrologist at Weill Medical College of Cornell University in New York City, who has studied variation between blacks and whites in a gene involved in hypertension. “To deny that is really denying a lot of very obvious biological truths.” Researchers are careful to say that although mean expression between Asians and Caucasians differed in more than 1000 genes studied, the expression difference between individuals from each group was often not impressive. “These averages are not absolutes,” says Stephen Wooding, a population geneticist at the University of Texas Southwestern Medical Center in Dallas. He compares the variation in gene expression to height in men and women; although men on average are taller, plenty of individual women are taller than individual men. To analyze expression levels, Cheung and her colleagues began with samples collected for the International HapMap Project, which aimed to catalog genetic variation to help identify disease genes. They used microarray technology to measure gene expression in several thousand genes at once and found measurable expression in 4197 genes. Then, they compared mean expression levels in the three different sets of samples. At first, the researchers separated the Chinese and Japanese samples but then lumped them together after finding that only 27 genes registered different mean expression levels between the two. The different expression levels seemed to correspond to patterns of inherited variation in single-nucleotide polymorphisms (SNPs)—for example, if one DNA stretch with a particular SNP was rare in a higher percentage of Asians than Caucasians, average gene expression in the first group might be lower. It's still not clear whether the SNPs themselves might be regulating gene expression, or whether they travel together with other DNA that's the regulator. The question now is whether and how these expression differences affect health. One gene, called UGT2B17, is deleted more often in Asians than Caucasians and had a mean expression level that was 22 times greater in Caucasians than Asians, the most dramatic variation seen. “That one really stuck out,” says Wooding, who notes that this gene is involved in steroid metabolism and, possibly, drug metabolism as well. Spielman agrees that genes such as UGT2B17 and others that showed up in the list of 35 should be looked at individually to determine what the expression differences might mean. Next up for his group: examining gene expression in other ethnicities, including Africans, to see what patterns materialize. 6. NUCLEAR WASTE With Plutonium, Even Ceramics May Slump 1. Valerie Brown* 1. Valerie Brown is a writer in Portland, Oregon. A Promising idea for immobilizing nuclear waste may not be so solid after all. Researchers have pointed to crystalline ceramics such as zircon as a strong medium for holding Plutonium, a fission product in sent commercial fuel and a security risk with a half-life of 24,000 years. But a new study by mineral physicist Ian Farnan of the University of Cambridge, U.K., and colleagues reveals that al ha radiation could break down this ceramic's structure more rapidly than assumed. A zircon mix containing 10% plutonium-239 (239Pu), for exam le, could become amorphous in just 1400 years—far short of the U.S. containment target of 210,000 years. This experimental finding, experts say, points to a need for more research on alternative forms of waste storage. Zircon (ZrSiO4) is frequently studied in modeling waste storage because it can contain natural inclusions of long-lived radioactive elements such as uranium and thorium. Some such samples are as old as Earth. The Farnan study, published in the 11 January issue of Nature, used nuclear magnetic resonance (NMR) to directly measure the number of silicon atoms displaced by each emitted alpha particle, first in natural zircon containing 238U and 232Th, and then in zircon doped with 239Pu. Previous estimates of such displacement were in the range of 1000 to 2000 atoms; Farnan observed a much larger displacement of about 5000 atoms, indicating that the structure would fail sooner. Bruce Begg of the Australian Nuclear Science and Technology Organisation calls the Farnan team's work “very significant” but says it does not address the “key question”: whether the alpha-induced transformation of ceramic to an amorphous state “has any detrimental impact on the ability of the waste form to lock up plutonium.” Many researchers believe it does. Linn Hobbs of the Massachusetts Institute of Technology Department of Nuclear Science and Engineering says that a form that becomes amorphous can change “the way that various elements are surrounding other elements.” This could allow significant “dimensional changes” in the structure, according to Hobbs, which “may or may not have larger leach rates” into the surrounding environment. The U.S. storage plan for a significant portion of its weapons waste relies on a completely amorphous medium: glass. The U.S. Department of Energy (DOE) is melting radioactive material together with borosilicate glass in a program to immobilize millions of liters of mixed liquid waste at the Hanford Nuclear Reservation near Richland, Washington, and the Savannah River Site near Aiken, South Carolina. DOE chose this “vitrification” option because tank waste is so complex that no single crystal structure could accommodate all its components. However, most of the plutonium and uranium has been removed, so “there's essentially no probability of a criticality event” in vitrified tank waste, says J. Russell Dyer, chief scientist with DOE's Office of Civilian Radioactive Waste Management. The U.K. and France also vitrify reprocessed power-plant fuel, but only after removing the plutonium. The biggest reservoir of plutonium-bearing waste is in spent but unreprocessed commercial nuclear power fuel, most of it stored onsite at utility companies, expected to reach 62,000 metric tons by 2010. The federal weapons complex owns about 7000 metric tons of reprocessed weapons waste and spent fuel, also containing plutonium. Experts say that research is needed to narrow down the candidates for optimal plutonium storage. Vitrification is a “completely unstable” method of storing wastes, says Kurt Sickafus of the Materials Science and Technology Division at Los Alamos National Laboratory in New Mexico. He argues that ceramic forms can be made “highly stable,” but not the silicate-based forms such as zircon. He suggests fluorite crystal structures instead because their amorphousness lies somewhere between that of glass and the rigid silicates. This makes them able to tolerate radiation-induced defects without severe disruption of the crystal lattice, he says. Other researchers look to pyrochlores and zirconolites, outgrowths of the work on the titanium-based SYNROC (“synthetic rock”) by A. E. Ringwood in the 1970s. U.S. funding for research on ceramic waste forms has been stagnant or declining for years, says Sickafus. Despite the obstacles, Farnan says the problem is “tractable.” However, “if you take a material and ask what its behavior is going to be in 10,000 years, the uncertainties become very large.” Even so, there is good news in these findings, Sickafus notes: This “very sensitive and elegant” NMR technique can help whittle down uncertainty about the robustness of alternative materials relatively quickly. 7. OCEAN POLICY Panel Urges Environmental Controls on Offshore Aquaculture 1. Erik Stokstad A blue-ribbon panel is calling for tight environmental standards on farmed fish in U.S. ocean waters. Although few commercial aquaculture operations currently exist outside shallow coastal zones, the panel predicts a boom in offshore enterprises and says now is the time to craft regulations to prevent future ecological damage. Many of the recommendations in the 142-page report, released this week, would also help make existing aquaculture operations more benign and sustainable, the panel says. “I think the report makes very helpful, practical recommendations, anchored in good science,” says Jane Lubchenco of Oregon State University in Corvallis, who wasn't involved. Farmed fish and other fruits of marine aquaculture—worth$200 million in the United States last year—are currently grown within 5 kilometers of shore, a swath of water regulated by states. But heightened demand and new technologies, such as storm-resistant pens, are promising to carry f ish farming into open waters under the jurisdiction of the federal government. In 2005, the Pew Charitable Trust and Lenfest Foundation asked the Woods Hole Oceanographic Institution (WHOI) in Massachusetts to convene a task force of stakeholders to examine the risks and benefits of offshore aquaculture and how it could be regulated.

First off, says panel chair Richard Pittenger, a former vice-president of WHOI, Congress should put the National Oceanic and Atmospheric Administration (NOAA) in charge and mandate it to evaluate the risks of offshore aquaculture before granting any permits. In the panel's view, major hazards include pollution from excess waste and feed. Although the open ocean is better than coastal ecosystems at dispersing these pollutants, the panel says that water-quality standards are needed. Another worry is that escaped fish could harm wild populations. That's why the panel says non-native fish, regardless of whether they're in coastal or open waters, should not be allowed, unless they have been shown to pose no risk.

In addition to strict regulations, the panel also called for market-based incentives that would encourage industry to invest in sustainable aquaculture operations. “I think we've arrived at a reasonable balance that would improve the environmental performance of the U.S. industry,” says task force member Bill Dewey of the Taylor Shellfish Company in Shelton, Washington. Also important for the long-term health of aquaculture is reducing and replacing fishmeal. On average, it takes 6.6 kg of wild-caught fish to grow 1 kg of farmed fish, the panel notes, and the supply fisheries are fully or overexploited.

NOAA wants to add offshore aquaculture to its bailiwick. In the previous Congress, the agency proposed a bill—the National Offshore Aquaculture Act of 2005—that would give it authority in federal ocean waters. Opponents criticized the bill because it didn't mandate environmental standards (Science, 8 September 2006, p. 1363), and it never made it out of a Senate committee. Two key senators are expected to reintroduce the bill, which NOAA is revising to include recommendations from the panel.

8. EPIDEMIOLOGY

Agent Orange's Bitter Harvest

1. Richard Stone

New findings paint a more sinister picture of the Vietnam War herbicide; scientists are trying to revive an epic study of its effects on U.S. veterans and clarify its legacy in Vietnam

HANOI—Several children and young adults sit at a table, fiddling with plastic blocks and colored rings with the self-absorption of toddlers. “We teach them small skills. How to wash hands. How to play with toys, distinguish colors,” explains Nguyen Thi Oanh, a teacher at Friendship Village, a rehabilitation center in Van Canh, west of Hanoi. The students, 9 to 24 years old but with limited mental development, will spend a few years here and then return to their home villages. During rehab, Oanh says, “some kids get a little bit better.” Others do not.

This scene may resonate among health workers around the world who have run similar rehab sessions. But in Vietnam, it resonates with the trauma of war. The 120 children and young adults from 34 provinces at Friendship Village share one thing in common: Their parents or grandparents claim to have been in areas where the U.S. military 4 decades ago used herbicides—the most notorious being Agent Orange—to destroy crops and strip forest canopy to flush out the enemy.

Vietnam claims that the children's disabilities were caused by parental exposures to Agent Orange. Western scientists have long been at odds with their Vietnamese counterparts over the strength of evidence correlating exposure to dioxin—a toxic contaminant of the herbicide—and illnesses in individuals, particularly birth defects. “The Vietnamese government is using malformed babies as a symbol of Agent Orange damage,” says Arnold Schecter, a toxicologist at the University of Texas School of Public Health in Dallas, who remains cautious about making associations after studying Agent Orange for more than 20 years.

In Vietnam, there is far less ambiguity. “The number of child victims could be in the 100,000s,” says Dang Vu Dung, director of Friendship Village, run on donations from overseas veterans. Countrywide, roughly 3 million people are Agent Orange victims, asserts Nguyen Trong Nhan, vice president of the Vietnam Association for Victims of Agent Orange/Dioxin (VAVA), a nongovernmental organization in Hanoi.

The long-term effects of Agent Orange may never be known, now that an ambitious attempt to analyze them has ended. Late last year, the U.S. Department of Defense pulled the plug on a 20-year-long health study of U.S. veterans involved in Operation Ranch Hand, which sprayed 95% of the Agent Orange and other herbicides used in Vietnam. The $140 million research effort was “the most detailed study of human exposures ever done,” says epidemiologist Joel Michalek of the University of Texas Health Science Center in San Antonio, who until 2005 was a principal investigator of the Air Force study. The firmest link it uncovered was between Agent Orange and an elevated risk of diabetes. Otherwise, Michalek says, “there has been little or nothing to say—until now.” A cancer signal is just beginning to emerge from the data, he claims, as are subtle physiological changes such as suppressed testosterone levels and prostate growth. The decision to halt Ranch Hand stunned many researchers. “It will be a tremendous loss to science if it is not continued,” says Linda Birnbaum, chief of the U.S. Environmental Protection Agency's (EPA's) experimental toxicology division in Research Triangle Park, North Carolina. A proposal to resurrect it is circulating on Capitol Hill. By law, the Air Force must transfer custody of existing Ranch Hand data and specimens to the U.S. National Academies, which hopes to make them available for further research. Another day of reckoning is on the horizon—this one for the Vietnamese who claim to have been injured by Agent Orange. This spring, in a U.S. appeals court, oral arguments are expected to begin in a class-action suit brought by Vietnamese citizens against Agent Orange manufacturers. (The claims had been dismissed by a lower court in 2005.) The claimants demand compensation like that given to U.S. veterans who handled Agent Orange and contracted certain illnesses. “It is time for the U.S. government and chemical companies involved in the war to take responsibility for the damage caused by their actions and products,” says epidemiologist Tuan Nguyen of the Garvan Institute of Medical Research in Sydney, Australia. Bitter feelings threaten the blossoming relationship between the United States and Vietnam. “Agent Orange is a very sensitive, very delicate, very political issue—and very controversial,” Schecter says. In a small gesture, the U.S. government has pledged to assist Vietnam in cleaning up several hot spots where soil dioxin levels are sky-high. Researchers from both countries hope this will kindle fresh interest in a joint probe. “We are really ready for cooperation with the United States—as long as it is based on mutual benefits and mutual respect,” says toxicologist Le Ke Son, director general of Vietnam's “national steering committee for the overcoming of the consequences of toxic chemicals used by USA in the war in Vietnam,” or simply “Committee 33.” But U.S. experts have found Committee 33 rigid and opaque and therefore hard to work with. Says Michalek, “Studies in Vietnam are going to be difficult.” True colors The U.S. and South Vietnamese air forces, mostly using military transport planes, began spraying herbicides in the fall of 1962. Over the next decade, they unloaded some 77 million liters of herbicides on 2.6 million hectares of south and central Vietnam. For the first few years, the main herbicide was Agent Purple, a mix of 2,4-dichlorophenoxyacetic acid (2,4-D) and two forms of 2,4,5-trichlorophenoxyacetic acid (2,4,5-T). Then in 1965, the military deployed Agent Orange, a faster-acting defoliant consisting of 2,4-D and a single form (n-butyl ester) of 2,4,5-T. In a painstaking reanalysis of herbicide use during the Vietnam War, Columbia University chemist Jeanne Mager Stellman and her colleagues estimated that over 6 years, 45 million liters of Agent Orange were sprayed (Nature, 17 April 2003, p. 681). These agents were laced with a long-lived contaminant, 2,3,7,8-tetrachlorodibenzoparadioxin (TCDD). It's unclear precisely how much dioxin rained down on Vietnam. Stellman's group adopted a “conservative” value of 3 parts per million of TCDD in Agent Orange, although levels “could be fourfold or more higher,” they assert. About 10% of Vietnam took a direct hit. By the late 1960s, Western researchers had evidence that 2,4-D and 2,4,5-T cause birth defects in mice; they were alarmed as well by anecdotal reports of birth defects in Vietnam attributed to the herbicides. In a resolution at its annual meeting in 1969, AAAS (publisher of Science) urged the Defense Department to “immediately cease all use of 2,4-D and 2,4,5-T in Vietnam.” As criticism of the war intensif ied, the U.S. military banned the herbicides in April 1970, although Ranch Hand operations didn-t cease until late in 1971, and South Vietnamese forces continued to dip into herbicide stockpiles until the war ended in 1975. But whereas 2,4-D and 2,4,5-T “are not innocuous compounds,” Birnbaum notes, evidence soon pointed to a darker villain: dioxin. A toxic trail In the past 3 decades, studies have revealed that dioxin causes many harmful effects in animals—birth defects, cancers, and endocrine disorders—sometimes at vanishingly low concentrations. In a rogue's gallery of 75 known forms of dioxin, TCDD is the nastiest. “From fish through primates, it's the most toxic,” Birnbaum says, perturbing “lots of different systems in the body.” Significantly, it binds to the aryl hydrocarbon receptor, a key regulatory protein. As a result of this unholy coupling, dioxin throws a wrench into processes as diverse as normal homeostasis and aging. (Ukraine's president, Victor Yushchenko, was deliberately poisoned with TCDD in 2004.) It has, however, been difficult to probe for links between dioxin and human illness. “Thank goodness, very few people in the world are ever exposed to high levels,” Birnbaum says. But those with high exposures—in rare occupational accidents and industrial disasters—have suffered chloracne, a severe skin disorder, and transient symptoms of poisoning. Studies have also indicated that dioxin might trigger or abet cancer development and possibly heart disease years after exposure. Exposures in Vietnam are hard to quantify. Stellman's team estimates that more than 3000 villages with at least 2.1 million people were “sprayed directly” with herbicides, although the number potentially exposed could be as high as 4.8 million. “There are no good records as to who lived in a certain village at a certain time,” says Michalek. In more than 30 trips to Vietnam since 1983 to document TCDD in humans, wildlife, food, and soil, Schecter and John Constable of Harvard University have found elevated dioxin levels in many of the roughly 4000 people they have tested. Schecter says that a handful of individuals living near a wartime herbicide storage area, Bien Hoa, had TCDD blood levels exceeding 400 parts per trillion. (The U.S. population averages 1 or 2 ppt.) In the United States, in response to pressure from veterans' groups, the Air Force in the late 1970s began planning a study to track the health of some 1200 Ranch Hand veterans and a control group: veterans not exposed to Agent Orange. The research also examined both cohorts' roughly 8500 children. “We launched the study knowing next to nothing about the exposure profiles”—how much dioxin each vet absorbed, says Michalek, who started on the project in the late 1970s when he was with the Air Force Research Laboratory at Brooks Air Force Base in Texas. With veterans blaming Agent Orange for an array of ills, the Air Force scientists opted for a broad approach to data collection—and took some heat for that. “The study was seen as seriously flawed,” asserts Stellman, who states that it began as “too much of a fishing expedition, measuring everything and anything with too few scientific hypotheses.” In 1987, Ranch Hand researchers began to measure dioxin levels in veterans' blood samples. It was revelatory. “Many people who thought they were highly exposed actually were not,” says Birnbaum. “There were very few people with high levels.” Michalek and his colleagues sorted veterans into low-, medium-, or high-exposure categories. In 1995, that rough cut at estimating exposure turned up a clear hit: Diabetes risk increased with exposure. Over the next decade, however, other findings were frustratingly indistinct. Michalek has since reanalyzed the data, zeroing in on veterans who were in Vietnam during or prior to 1968 and were involved in at least 90 days of herbicide spraying. He also excluded vets who spent more than 2 years in Southeast Asia. (Veterans in the control group with such extended deployments are at higher risk of cancer—possibly from exposure to DDT during a World Health Organization campaign in the 1960s to eliminate malaria in the region, Michalek speculates.) The new analysis uncovered “a stronger and clearer trend” of a dose-dependent risk for diabetes and cancer, says Michalek, who intends to submit his findings to a peer-reviewed journal later this month. He expects heavy flak: “Critics will accuse me of slicing and dicing the data,” he says. He and others say it would be a mistake to walk away now. “Certain chronic effects can take years and years to develop,” says Birnbaum. And although some experts assailed the study's design, a panel of the National Academies' Institute of Medicine (IOM) concluded last year that “the data appear to be of high quality and the specimens well preserved.” The Air Force will transfer Ranch Hand data and specimens to the academies by the end of September. “If we subsequently receive funding to manage the assets and permission from the research subjects, we intend to make the materials available for further analysis,” says David Butler, an IOM senior program officer. And IOM next month will convene a panel to advise the Department of Veterans Affairs (VA) on how to apply the Stellman group's exposure model to studies of U.S. veterans. Michalek's university, meanwhile, sent a proposal late last year to several members of Congress and key committees seeking support for a$2-million-per-year Ranch Hand extension.

Congress has intervened before: It passed the Agent Orange Act in 1991, mandating care for veterans known to have been exposed to Agent Orange. The act also called for a definition of illnesses attributable to Agent Orange, as a basis for compensating sick veterans. Toward this end, the VA enlisted IOM to review the health effects of exposure to herbicides used in Vietnam. IOM's landmark report, Veterans and Agent Orange, came out in 1994; by law it must be updated every 2 years until 2014. The latest update, published in 2004, concludes that there is “sufficient evidence of an association” between herbicide exposure and five ailments: chronic lymphocytic leukemia, soft-tissue sarcoma, non-Hodgkin's lymphoma, Hodgkin's disease, and chloracne (see table).

Of all categories of illness blamed on Agent Orange, the most divisive, perhaps, is birth defects. This “remains one of the most contentious issues in science,” says Nguyen of the Garvan Institute. According to VAVA's Nhan, the rate of severe congenital malformations in herbicide-exposed Vietnamese populations is 2.95%, compared to 0.74% in nonexposed populations. Grandchildren are afflicted at a similar disproportionate rate, Nhan notes. Government publications about the herbicides are filled with heartrending pictures of deformed children. Reports of families with multiple malformed children abound.

In contrast, the IOM panel has noted “limited or suggestive” evidence linking herbicide exposure and one congenital defect: spina bifida, a malformation of the spinal cord. For all other birth defects, the panel concluded that evidence for an association was “inadequate or insufficient.”

This long-running debate has been reignited. A team led by Nguyen for the first time pooled published data with unpublished data from Vietnamese studies of veterans and sprayed civilians. Their meta-analysis of 22 studies, half of which were unpublished, found a “substantially greater” association between Agent Orange exposure and birth defects in Vietnamese populations than in U.S. veterans. Overall, people who believe they were exposed to Agent Orange were almost twice as likely to have a child with birth defects as were unexposed people, Nguyen's group reported last October in the International Journal of Epidemiology.

The study has received mixed reviews. “I don't think using unpublished data is a good way to do a meta-analysis,” says Schecter, who believes that poor nutrition, infections, and genetic flaws are responsible for most malformations seen in Vietnamese children. Michalek, on the other hand, says Nguyen and colleagues “did the best they could with available data.” Nguyen notes that the Vietnamese researchers have had a “hard time” submitting their findings to international journals. “I certainly hope that they will publish their work,” he says.

Whether the health effects can be brought into sharper focus is unknown. A few years ago, prospects were looking good. In March 2002, the U.S. and Vietnamese governments signed a research framework to probe Agent Orange effects. “Agreeing to do the research is the easy part,” Anne Sassaman, then an official with the U.S. National Institute of Environmental Health Sciences (NIEHS), said at the time. “The more difficult task will be to develop research studies that are definitive and address the underlying causes of disease in Vietnam.”

NIEHS thought it had a viable project in sight. In 2003, the agency committed $3.5 million to a study led by David Carpenter of the University at Albany in New York, to probe the possible relation between Agent Orange and birth defects. But talks over a U.S.-Vietnam cooperation agreement foundered. “Without it, the research was impossible to implement,” says Committee 33's Son. U.S. officials, including the ambassador to Vietnam and the health attaché, “worked very hard with the Vietnamese but ran into constant roadblocks,” says one U.S. scientist. With talks stalemated, NIEHS shelved the Albany study in February 2005. Seeking closure In a common room of a dormitory at Friendship Village, Tran Van Tham, a retired lieutenant in the Vietnam People's Army, and several other veterans are lounging under a portrait of Ho Chi Minh, the leader with the white-streaked Fu Manchu mustache and goatee who orchestrated the North's victory 30 years ago. Whereas disabled children stay for rehabilitation for up to 3 years, veterans cycle through for a month at a time for health checks. “We reminisce, but mostly are here to enjoy life. We feel better, spiritually,” says Tham. Years ago, Tham's two babies succumbed to hydrocephaly and other defects, he says. He blames wartime Agent Orange exposure. Nevertheless, Tham says, eyes glistening, “we can forgive American veterans.” But Agent Orange victims are a burden on Vietnam, he says. “We support our government's policy to close the past and look to the future with the United States,” adds Nhan. “But we cannot ignore Agent Orange victims.” In 2000, Vietnam introduced a program to compensate people who claim disability from Agent Orange exposure. But Nguyen says that each person gets only a few U.S. dollars per month. He estimates that Vietnam needs hundreds of millions of dollars to care for all victims. In 2004, VAVA, exasperated after years of pleas for U.S. aid went unanswered, filed a class-action suit in U.S. District Court against 37 companies that supplied herbicide chemicals to the U.S. military during the Vietnam War. “We had hoped the United States would respond with goodwill and regarded the lawsuit as a last resort,” says Nhan. The claims were dismissed in March 2005. In a 233-page decision, Senior District Judge Jack B. Weinstein ruled that the companies could not be sued as government contractors. Nor was he persuaded by the scientific case. “No study or technique presented to the court has demonstrated how it is now possible to connect the herbicides supplied by any defendant to exposure by any plaintiff to dioxin from that defendant's herbicide,” he wrote. The decision “was a great surprise,” says Nhan. The plaintiffs appealed to the 2nd Circuit Court of Appeals in New York City, and oral arguments could be heard as early as April. The plaintiffs' first challenge is to convince the appeals court that the companies can be sued. If they succeed, they would then have to refute Weinstein's conclusions about the science. “The fact that diseases were experienced by some people after spraying does not suffice to prove general or specific causation,” the judge wrote. “Proof of causal connection depends primarily upon substantial epidemiological and other scientific data.” That's a tough argument to overcome, given the paucity of solid epidemiological data. To carry out a high-quality study of human health effects in Vietnam would require “a huge amount of money,” says Birnbaum. The “real hurdle,” adds Sassaman, who recently retired from NIEHS, “is to get the appropriate scientists and scientif ic expertise engaged in truly collaborative research.” With that in mind, she says, NIEHS has just launched a program to fund junior researchers from Vietnam and other developing countries to work up to 2 years in labs of NIEHS-funded scientists. Others are taking direct action to eliminate dioxin hot spots in Vietnam. International experts, working with Vietnamese counterparts, have identif ied nearly 100,000 square meters of heavily contaminated soil in several places where herbicides were stored during the war, says Son. Near Da Nang Airport, he says, TCDD levels in soil reach 35 parts per billion—35 times the permissible level. “Hundreds of thousands of tons” of soil will have to be dug up and stored or treated to remove dioxins, Son says. Last month, the Ford Foundation awarded$460,000 to Hatfield Consultants, an environmental firm in West Vancouver, Canada, to assist at Da Nang.

The U.N. Development Programme, with support from EPA and the Ford Foundation, is setting up a \$60 million trust fund for cleanup efforts and to improve the economy of villages near the hot spots. Vietnam's Ministry of Defense has already commenced cleanup at Bien Hoa. “We should get rid of these hot spots,” says Birnbaum. “We know that dioxin is bad stuff.”

There may be no consensus on exactly how potent dioxin is as a cause of disease and disfigurement. But people do seem to agree that purging the land of the last vestiges of the Vietnam War—particularly the chemical residues of Agent Orange—is something worth fighting for.

9. NEUROBIOLOGY

Fruit Fly Fight Club

1. Greg Miller

Fruit flies brawl over mates and territory. Now some scientists are betting that these battles can help them unravel the genetic basis of aggression

BOSTON, MASSACHUSETTS—After a furious combination of blows, the pugilist has his opponent backed up to the edge of the ring. Punches fall like rain as the opponent teeters on the brink. But just when it looks like he can take no more, the fighter employs a surprising tactic. Planting one wing on the ground, he regains his balance and drives back his adversary with four wildly swinging legs. The combatants here are fruit flies, the ring is a thimble-sized cup of agar, and the fighting venue is a laboratory here at Harvard Medical School.

Sibu Mundiyanapurath, a visiting medical student from Germany, is reviewing videos of a recent series of bouts with a genetically modified strain of Drosophila melanogaster. Even unaltered fruit flies fight, Mundiyanapurath says, but this strain is unusually combative. “These guys just keep on going after each other,” he says.

Who knew fruit flies were such pugnacious little beasts? Very few people until recently, says Harvard neurobiologist Ed Kravitz, Mundiyanapurath's research adviser. In a research paper published in 1915, the noted geneticist Alfred Sturtevant mentioned tussles between male flies competing for mates, but only a smattering of papers on fly aggression appeared subsequently in the scientific literature. That seems to be changing now.

Since 2002, Kravitz and colleagues have described a surprisingly diverse repertoire of aggressive behaviors in these tiny insects. They've recently found that flies remember previous opponents and that vanquished flies seem to develop a “loser's mentality” that virtually ensures defeat in subsequent bouts. The biologists also discovered that male and female flies have distinct fighting styles, and they have taken advantage of the powerful genetic techniques available to Drosophila researchers to investigate the basis of such differences.

Other scientists have turned into fruit fly fight promoters too. Last year, researchers in California and North Carolina independently reported on changes in gene expression in fly strains bred for aggression. Understanding the genetic basis of aggression in flies may eventually lead to a better understanding of aggression in other animals, including humans, Kravitz and others suggest. “Drosophila are a great model system for looking at the genetic basis and evolution of aggressive behavior,” says Ary Hoffmann, a geneticist at the University of Melbourne in Australia who published a series of papers on fly aggression in the 1980s. Hoffmann had shelved his work on aggression, but he says the new research has rekindled his interest, and his lab now plans to look for genetic variations that account for individual differences in fly aggression.

Lobster versus fly

Kravitz first heard of fighting flies about 10 years ago when he gave a lecture on his studies of aggression in lobsters. That research, begun in the late 1970s, had established that levels of neurotransmitters such as serotonin fluctuate when the crustaceans fight to establish social status. Afterward, someone from the audience told him about fighting flies. “I don't think I was too impressed,” Kravitz recalls. But the researcher sent him some papers, and that got Kravitz thinking about the advantages of working with the insects.

One of the most fundamental questions he'd been trying to address was how complex patterns of behavior get wired into nervous systems. “If you want to ask a question like that, you have to be able to manipulate genes,” Kravitz says. “And there was no easy way to do that with lobsters.”

Before he started tinkering with fly genes, Kravitz wanted a better understanding of the insect's fighting behavior. Many of the early experiments put a bunch of flies, males as well as females, in a small space. It was basically a free-for-all, with courting, fighting, and mating going on simultaneously. Kravitz simplified the situation by pitting just one fly against another. It took some trial and error to get the setup right, but the arena now consists of a small cup of agar enclosed by Plexiglas. A dab of yeast paste—a delicacy for Drosophila—in the middle of the cup gives the flies something to fight over. For male flies, the researchers up the ante by sticking a headless female in the center of the ring. (The males seem to find decapitated females just as attractive as intact ones, and the headless ones can't fly away.) After poring over more than 2000 videotaped interactions between male flies, Kravitz and colleagues identified nine distinct acts of aggression in a 2002 paper in the Proceedings of the National Academy of Sciences (PNAS). These moves included “wing threats” in which one fly faces another and suddenly raises both wings, “fencing” in which one fly pokes a leg at another fly, “lunges” in which one fly stands up on two hind legs and slams down on his opponent, and “boxing,” which looks about like it does in humans, if you add two limbs and subtract the gloves.

Whichever fly started the fight was most likely to win, especially if his first move was a strong one, the researchers also found. For example, an instigator that used a slow “approach” move, in which he lowered his body and walked toward his opponent, had 3-to-1 odds of ultimately making his opponent retreat. But flies that started with a more intense move, such as fencing or wing threat, improved their odds to 16 to 1.

“The videos were just absolutely stunning,” says Robert Huber, an animal behaviorist at Bowling Green State University in Ohio who helped Kravitz with some of the behavioral analysis. What struck Huber most about the fly fights were the intricacy of the different moves and the fact that the insects used certain combinations far more often than others. “It all seemed to be going on according to very strict rules,” he says. Huber speculates that a consistent pattern of fight escalation gives the insects an efficient way to establish dominance hierarchies: Fights between mismatched flies get resolved quickly with visual displays and other low-intensity maneuvers, whereas only closely matched flies have to go through their entire aggressive repertoire to determine who's the champ.

Recent work by Kravitz's team sheds further light on how flies form and maintain hierarchical relationships. When flies that had lost their first fight reentered the ring after a 30-minute time-out, they almost never won. First-time losers had a 0-5-5 (win-loss-draw) record in rematches with their first opponent and a similarly feeble 0-6-6 record against naïve opponents who'd never fought another fly, Kravitz and colleagues reported in the 16 November 2006 PNAS. First-time losers lunged less and retreated more in their second fights, and they rarely made the first move; they only managed wins against other losers.

The researchers also found that flies appear to remember not just the outcome of their first fight but also the opponent. In second fights, familiar opponents had fewer aggressive encounters than did unfamiliar opponents. First-time losers tried out a few more lunges early on in fights against unfamiliar winners than in fights with the fly they'd lost to previously.

Now Kravitz and colleagues are hunting for changes in gene expression that may underlie the memory of past battles. Many researchers have investigated learning and memory in flies in relatively simple classical conditioning experiments, Kravitz points out. “But the learning we see happens during a social experience, and we want to know if the same genes are involved and whether we can see differences in gene expression that accompany becoming a winner or becoming a loser.”

Girl fights

Like males, female fruit flies don't shy away from conflict. They may not be as easily provoked as males, but given a dab of delicious yeast to fight over, a pair of females will do their worst. (“They might be interested in headless males,” Kravitz says. “We haven't looked.”) Although males and females employ some common moves, female fights never escalate to “boxing” and “tussling” (a barroom-brawl mix of holding, punching, and rolling around on the ground) as do the most intense fights between males, Kravitz and colleagues reported in PNAS in 2004. Instead, females frequently head butt and shove—tactics rarely used by males. Females also showed no evidence of dominance hierarchies. Unlike fights between males, in which a clear victor typically emerges, fights among females seesaw indefinitely.

More recently, Kravitz's team has begun to investigate the genetics behind these gender differences. The group's initial experiments have focused on a gene called fruitless (fru) that has long been studied for its role in determining sex-specific courtship behavior. The fru gene is spliced differently in males and females, creating distinct messenger RNA transcripts. The male transcript can be used to make protein, but the female transcript apparently cannot. In 2005, Barry Dickson of the Research Institute of Molecular Pathology in Vienna, Austria, and colleagues reported in Cell that female flies genetically altered to make the male version of fru performed courtship behaviors usually seen in males and courted other females (Science, 3 June 2005, p. 1392). Male flies given female fru barely courted at all.

The fru gene has a similar effect on fighting styles, Kravitz, Dickson, and colleagues reported in the December 2006 issue of Nature Neuroscience. Males with the female version of fru were more likely to fight females than to court them. The altered males also fought like females, using head butts and shoves; they never boxed. In addition, males with female fru did not appear to form dominance relationships with other males. Conversely, female flies with the male version of fru tended to fight like males. Overall, the findings suggest that fru establishes the neural circuitry for aggressive behavior, just as it does for courtship behavior.

Links between fighting and courting aren't unique to flies, Kravitz says. One of the most basic decisions any animal has to make is how to respond to another of its kind, he says. “Is this someone I want to court or someone I want to fight?” Kravitz's lab now hopes to identify the neural circuitry and chemical signals underlying such decisions by expressing female fru in specific subsets of neurons in male flies.

Bred for battle

Other labs have taken a different approach to studying aggression in Drosophila. Last September, two research teams reported breeding flies to be hyperaggressive. In one study, geneticists Ralph Greenspan and Herman Dierick of the Neurosciences Institute in San Diego, California, selected aggressive flies by introducing 120 males and 60 virgin females into an enclosure with 11 small cups filled with fly food. Males' first priority was mating, but after that they settled down on the food cups and started defending their territories.

In most encounters between males, one fly was clearly dominant from the beginning and would chase any intruders on his cup, Dierick says. But a few would stand their ground and fight back. These males are the most interesting, in Dierick's view. “The real question to me is what happens when a male decides to reciprocate?”

To get at that question, he extracted these dauntless flies from the fight cage and mated them with random females from the same generation. Then he started the process all over. After 21 generations, he'd created a superaggressive line of flies that were quicker to fight and fought longer and more intensely than a line of flies created by selecting random males from the fight cages. Next, Dierick used DNA microarrays to look for changes in gene expression in the aggressive flies. In this strain, 42 genes had increased or decreased their activity by 25% or more, Dierick and Greenspan reported in the September 2006 issue of Nature Genetics. These genes, they noted, have diverse roles, including muscle contraction, energy metabolism, and cuticle formation.

One gene in particular, Cyp6a20, has stood out so far as having a potentially significant influence on aggressive behavior. Cyp6a20 was less active than normal in the aggressive line of flies, and deactivating it in a normal strain made the flies more aggressive. The gene encodes an enzyme that plays a role in many physiological processes, including pheromone signaling, and Dierick suspects that an underactive Cyp6a20 gene makes flies more aggressive by making them hypersensitive to pheromones.

In the September 2006 issue of PLoS Genetics, a team led by Trudy Mackay of North Carolina State University in Raleigh reported the results of an attempt to pinpoint genes related to aggression in their own line of hyperaggressive flies. Mackay's group identified a much larger set of candidate genes—nearly 1500—and has so far found 15 that alter aggressive behavior when mutated. As in Greenspan and Dierick's study, the candidate genes covered a wide range of physiological functions.

One puzzle is that neither set of experiments turned up genes related to serotonin, the neurotransmitter with the longest legacy in the literature on aggression. One explanation, Dierick suggests, is that the breeding experiments didn't enhance (or repress) serotonin-related genes because there was little variation in these genes in the starting populations. Going forward, he says, establishing whether serotonin plays a role in fly aggression will be important for evaluating how applicable fly studies are for understanding aggression in other animals.

The broader implications of this work on fighting flies remains an open question. “It's far too early to speculate on what these studies might tell us about vertebrate aggression,” cautions Hoffmann. Kravitz is more optimistic. Genes shape complex behaviors such as aggression in all animals, he notes. “If we understand how that happens in flies, it will give us some real information about how it might happen in other animals.”

10. AGBIOTECH

GM Technology Develops in the Developing World

1. Gunjan Sinha*
1. Gunjan Sinha is a writer in Berlin, Germany.

The first genetically modified crop developed entirely in Africa is gearing up for field trials. Its success would be a milestone

About 100 km north of Durban, South Africa, in a greenhouse chamber no larger than a walk-in closet, Frederik Kloppers clips a slender vial to a baby maize plant's new leaf. Inside the tube sits an insect with a potentially deadly bite, at least deadly to corn. This African leafhopper (Cicadulina mbila) carries maize streak virus, a scourge endemic to sub-Saharan Africa that devastates fields. Kloppers, a plant pathologist and technical manager at Pannar Seeds in Greytown, South Africa, gathers a dozen more tubes from the insect house and clips them to additional plants. Tomorrow, after the bugs have eaten their fill, he'll remove the tubes and then wait.

The fruit of more than a dozen years of effort, these maize plants have been genetically altered to resist infection by the virus. In greenhouse studies so far, the plant is highly resistant. If it proves equally hardy in field trials scheduled to begin in late 2007, it would be a milestone: the first-ever genetically modified (GM) crop developed by Africans for Africa.

But Kloppers and the plant's inventors, microbiologist Jennifer Thomson, virologist Edward Rybicki, and collaborators at the University of Cape Town (UCT), have much larger goals in mind. In a region where chronic hunger is the norm, GM maize could help alleviate grain shortages and potentially even boost economic development, says Thomson. And because plans call for selling the seed to small-scale and subsistence farmers for minimal profit, the inventors also hope it will help burnish the dim reputation of GM technology.

None of that is assured, Thomson and Rybicki concede. The plant could still fail in the field, as other African GM crop varieties such as sweet potato and cassava have done. The failures not only have disappointed the technology's advocates, but they've also fanned the flames of anti-GM sentiment. Although South Africa is one of the few African countries to permit farmers to plant GM crops within its borders, naysayers there, who still have substantial clout, have condemned the technology as a mere moneymaking tool for Western companies. Moreover, they remain unconvinced that homegrown efforts such as UCT's maize will succeed. Another failure would give anti-GM groups even more ammunition. The stakes are high, and the UCT scientists are treading carefully.

The problem

Maize is not native to Africa. It likely sailed across the Atlantic from the New World as cargo during the early 1500s, according to historian James McCann of Boston University. Maize flourished and displaced other native crops during the 20th century because it grows in only a few months and requires relatively little labor—one pass of the plow instead of the three or four necessary for crops such as sorghum and millet. In sub-Saharan Africa, maize has become the staple food; it makes up more than 50% of calories in local diets. In Malawi alone, maize occupies 90% of cultivated land and accounts for 54% of Malawians' caloric intake.

Maize streak virus is likely homegrown, say scientists. It lives in native grasses. At some point, the virus adapted itself to maize and is now able to jump between grasses and corn through the bite of an infected leafhopper, which itself isn't sickened by the virus.

Like any other infection, the wrath of maize streak waxes and wanes with different environmental conditions. Some years, crop losses are minimal. But in bad years, such as 2006, it can wipe out from 5% to 100% of a farmer's maize crop.

For the past 25 years, African crop scientists have been trying to breed resistant maize by crossing plants that carry some degree of natural resistance. But the task has not been wholly successful. The trait is conferred by several genes on different chromosomes and isn't consistently transmitted to the next generation. “It's not quite clear how resistance genes are inherited,” says Kloppers of Pannar Seeds. Moreover, traditionally bred varieties do not completely resist the virus, Kloppers explains. Many tolerate an infection but still produce stunted or deformed cobs.

A solution

In 1988, when Thomson took over as head of microbiology at UCT, GM technology seemed a perfect solution. Rybicki's plant virology group there was already intensively studying the virus. Perhaps they could engineer a way to stop it in its tracks?

The design seemed simple enough: The team studied the proteins necessary for the virus to replicate. If they inserted a mutated viral gene into the plant, which in turn expressed a mutated protein necessary for the virus to replicate at very high levels, it could beat out the virus's normal protein and immobilize the virus, they reasoned.

But getting the genes in proved tough, Thomson says. The UCT team first tried infecting maize with a widely used vector, Agrobacterium tumefaciens, carrying the genes, but to no avail. Ultimately, they successfully shot DNA into the plant using a gene gun. The GM maize plant carries a mutated form of a gene from the maize streak virus and two additional regulatory genes, one derived from maize itself and another from Agrobacterium.

Into the field

That was 6 years ago. Since then, the UCT scientists have been working closely with Kloppers at Pannar Seeds to test the plant's hardiness against infection. Kloppers has bred a previous version of the plant that carried an antibiotic-resistance gene through four generations. So far, it resists infection consistently. Moreover, the trait appears to be inherited in a dominant fashion.

Kloppers is repeating the experiment with a new group of plants that, because of environmental safety concerns, no longer carry an antibiotic-resistance gene. He expects to carry on crossing and checking inheritance and resistance through the next few months. Provided there are no major setbacks, he expects to apply for field trials during the latter part of this year.

Field trials are crucial to assess environmental and health risks, says Dionne Shepherd, a UCT postdoc who has been working on the project for the past 10 years. The scientists plan to examine whether the crop affects soil microorganisms and also whether it affects insects that feed on it. Other studies will also ensure that the added protein is indeed digestible and not an allergen.

If all goes well, the resistant maize will be the first GM crop to be field-tested in South Africa; to date, all GM crops planted in the country have been developed and tested elsewhere. The government is now developing its own expertise to evaluate environmental and human safety, says Shepherd, and because “UCT's maize is the most advanced locally produced GM product, they want to use our plant as a guinea pig,” she adds.

To avoid the pitfalls that have beset other African GM crop varieties, the UCT scientists and Pannar have been working with regulators all along. At stake, they say, is not only their crop's fate, but also the technology's reputation.

A few years ago, Kenyan scientist Florence Wambugu, who was trained and supported by Monsanto, developed a sweet potato plant resistant to the feathery mottle virus. But when scientists field-tested the crop, traditionally bred resistant varieties outperformed it. Other efforts have also stumbled during field tests. Just a few months ago, scientists at the nonprofit Donald Danforth Plant Science Center in St. Louis, Missouri, announced that cassava plants genetically modified to resist cassava mosaic disease lost the trait after a few generations.

Both setbacks have fueled ongoing skepticism about GM technology. “All this talk about the technology's benefit for Africa is just a lot of PR hype to garner funding,” says Mariam Mayet of the African Centre for Biosafety, an anti-GM lobby group in Richmond, South Africa. Most of the GM crops in the world are grown for animal feed or go toward food aid, Mayet says. “The benefit mainly goes to industrial agriculture, not to small-scale farmers.”

Because UCT's maize is homegrown and was supported with very little corporate money—Pannar was the project's only corporate contributor—Thomson and Rybicki hope it can dodge some of these criticisms. Private foundations that typically give money with no strings attached and the South African government funded the project's bulk. To recoup its share of investment, Pannar expects the seed to cost no more than 15% higher than non-GM seed, says Kloppers. Small-scale or subsistence farmers would likely be charged much less, he adds.

If UCT's plant succeeds, it would be the first GM crop developed by a developing country. But Africans might not be the only beneficiaries. It might also become the poster child of what many argue is a useful and important technology—and for better or worse, one that desperately needs a public relations makeover.