# News this Week

Science  30 Nov 2001:
Vol. 294, Issue 5548, pp. 1802
1. BIOETHICS

# Cloning Announcement Sparks Debate and Scientific Skepticism

1. Eliot Marshall,
2. Gretchen Vogel

WASHINGTON, D.C., AND BERLIN—A small U.S. biotech firm made headlines around the world last week when it announced that it had cloned several human embryos for transplantation research. The breakthrough—as it was called by scientists who did the work at a privately held firm, Advanced Cell Technology (ACT) in Worcester, Massachusetts—prompted strong reactions. President George W. Bush denounced the research as unethical, European leaders discussed national controls on cloning, and the furor could spur efforts to pass a law in the United States that would ban research on human cloning. All this fuss over results whose scientific significance is questionable: Some scientists note that the six-cell clusters created by ACT barely qualify as embryos.

The experiments, according to ACT's chief executive Michael West, were designed to test a way of producing human embryos for use in transplantation therapy. The company is pursuing a vision known as “therapeutic cloning.” The goal is to transfer genes from a patient into an experimental embryo that can generate healthy new stem cells. The hope is that the new cells might then be reimplanted in the patient without causing an immune reaction, to treat common illnesses.

A six-member ACT team, including West, scientist Jose Cibelli, and vice president Robert Lanza, claims to have completed the first step of this process in October, cloning human embryos for the first time. They describe their “protocols for the generation of human embryos” in an online publication called e-biomed: The Journal of Regenerative Medicine, posted on 26 November. On the same day, they published a first-person narrative in Scientific American and were profiled in a glowing account in U.S. News & World Report.

ACT launched its human cloning project in early 2000, advertising for egg donors in Boston newspapers. It convened an advisory board under ethicist Ronald Green of Dartmouth College in Hanover, New Hampshire, to review and approve the procedures. The scientists obtained 71 eggs from seven paid volunteers: women between 24 and 32 years old who already had given birth to at least one child.

ACT attempted several kinds of procedures. First, the team removed the nucleus from an unfertilized egg and replaced it with a nucleus taken from a skin cell of another adult donor—a procedure called nuclear transfer. None of the 11 eggs treated in this way divided. The team had better luck when it transferred nuclei from cumulus cells, support cells that surround developing eggs in the ovary. Three of four eggs in this experiment began dividing, but only one developed to the six-cell stage.

The team also attempted to trigger embryonic development in unfertilized eggs, a process called parthenogenesis, by exposing the eggs to a mix of chemicals known to prompt cell division (see image). Twenty of these 22 chemically activated eggs divided at least once. Six formed what Cibelli and his colleagues call a “blastocoele cavity,” resembling the sphere of cells that a normal embryo forms a week after fertilization. However, they did not contain the crucial inner cell mass, a cluster of more than 100 cells that give rise to embryonic stem cells.

Many animal species can reproduce through parthenogenesis, but no mammals are known to do so. Because a parthenogenetically activated egg could never produce a full-term baby, some have speculated that stem cells derived from “parthenotes” might skirt the ethical questions that surround the use of human embryos. Bioethicists are skeptical. For example, Norman Fost of the University of Wisconsin, Madison, says he's not convinced there's a difference and notes that “the people who are against any use of human embryos in research are still going to be opposed.”

The fact that none of the experimental embryos developed to eight cells suggests that the inserted nucleus wasn't working properly, says developmental biologist John Eppig of the Jackson Laboratory in Bar Harbor, Maine. In normal human embryos, the nucleus begins to express its genes between the four- and eight-cell stage. The failure to survive to eight cells “strongly suggests that you're not getting gene activation” in the transferred nucleus, he says. “And if you're not getting that, what have you got? Nothing.” David Ayares of Edinburgh-based PPL Therapeutics, another company involved in animal cloning and attempts to generate human stem cell lines, agrees. “The fact that they only went to four cells implies incorrect gene activation. … Those embryos aren't going to go any further,” he says.

West agrees that the data so far are “admittedly scant.” Asked why he decided to go public at this time, he said: “We are asked all the time about where we are in our research; we made the decision that when we had enough reproducible data, we would publish.” He added: “Our next goal is to create blastocysts,” early-stage embryos that contain several hundred cells.

International reaction was mixed. The Vatican denounced ACT's research. Likewise, science ministers in Germany and France reaffirmed that such work was illegal in their countries. Earlier this month, France and Germany issued a joint statement calling for an international ban on human reproductive cloning, while their legislatures are debating whether to allow work on embryonic stem cells. In the U.K., which has the most tolerant rules in Europe on the use of human embryos in research, legislators approved research on nuclear transfer to derive stem cells early this year. The House of Commons is planning to debate a bill that would explicitly outlaw the implantation of such an embryo. Lawmakers say ACT's announcement adds urgency to the debate.

The U.S. Senate, which is divided on the subject, has not yet voted on human cloning, although the House passed a bill in July that would make it illegal. If the House bill were in effect today, ACT might be prosecuted. The main effect of ACT's announcement, stem cell researcher John Gearhart of Johns Hopkins University told Reuters, was to scuttle backstage talks among congressional staffers on how to reach a compromise on the use of embryos in research. ACT's announcement, one House aide says, “has made everyone a little queasy.”

2. MOLECULAR IMAGING

# Virus Infects Cell: Live and Uncut

1. Mary Beckman*
1. Mary Beckman is a writer based in southeast Idaho.

Reality TV has never been this good: After several brief kisses for its unwitting victim, a dazzling virus pushes inside the recumbent cell, while another radiant virus, unsuccessful in its flirtation, floats out of view. The camera pans to the cell interior, where glowing viruses glide along protein rails to the nucleus. There, some slip through nuclear pores, and others cruise through tunnels within the cell center.

Cut to laboratory: For the first time, researchers have viewed live scenes of viral infection. Their lens is an imaging technique that may open up gene therapy and antiviral research to highly detailed, blow-by-blow analysis.

On page 1929, a cadre of researchers led by physical chemist Christoph Bräuchle of Ludwig Maximilian University in Munich, Germany, reports having imaged—in real time—single adeno-associated virus (AAV) particles entering cells and moving into the nucleus. The closeup view was provided by a technique called single-molecule fluorescence spectroscopy, which until now had been used to view chemical reactions such as the enzymatic breakdown of adenosine triphosphate.

Although single-molecule imaging techniques have advanced significantly in the last few years (Science, 1 June, p. 1671), the technique had never before been used to watch a viral infection, says molecular virologist R. Jude Samulski, director of the University of North Carolina Gene Therapy Center in Chapel Hill. “This technique will be significant for helping us understand how the virus enters the cell,” he says. “We're usually taking a picture after the event happened, but this is real time, the live story.”

The documentary approach revealed new information about the small virus, which gene therapy researchers are hotly pursuing as a gene delivery vector. Among their findings: AAV poked through the cell membrane in about 64 milliseconds, much faster than expected, and reached the nucleus in about 15 minutes. That's about an eighth of the time in which conventional cell culture methods, which rely on viral gene expression, can detect infection. Also, the researchers were surprised to see that some particles, after floating toward the nucleus, hopped aboard microtubule-based “tracks” on the nuclear surface and began to move in a straight line. Other viruses followed along the same tracks. Bräuchle suggests that the tracks are tube-shaped invaginations of the nuclear membrane, recently discovered structures never before known to ferry viruses.

Bräuchle's team pieced together its imaging system from commercially available equipment and customized it to circumvent obstacles such as a cell's inconvenient habit of autofluorescing, which would outshine the virus's signal. Bräuchle says conventional methods of measuring and imaging viral entry average the properties of a population and may introduce artifacts that affect infection in unpredictable ways. For example, to see where viruses are concentrated, scientists have had to coat each particle with more than 300 fluorescent molecules, which may get in the way of the virus's activities.

To minimize such interference, the researchers tagged individual viruses with one or two fluorescent molecules, each of which is about 1/25 the size of the virus. After using light microscopy to get a good picture of the mammalian cells lying on a microscope slide, the researchers infected each cell with 10 to 1000 viral particles, which Bräuchle says is much closer than typical cell cultures to normal conditions in the body. The molecules' glow lasted 1 to 10 seconds before it bleached out. This gave the researchers ample time to capture the movement of individual viral particles with snapshots every 40 milliseconds. “Because they're carrying little flashlights around, we can see where the virus is,” says physical chemist Anne Myers Kelley of Kansas State University in Manhattan, an expert in single-molecule imaging who was not part of Bräuchle's team.

Bräuchle expects the technique to illuminate virus-host cell interactions for other types of viruses, as well as help screen antiviral drugs. “We can really see how drugs affect the uptake of virus into the living cell, at what stage of the infection pathway the drugs work, and to what extent [they interfere with infection],” Bräuchle says. The ability to view viral infection close up will ratchet up efforts to understand viral processes, says Samulski. “If we can understand processes at this level of detail, then we have to. When somebody breaks the mile record, it challenges everybody to run faster.”

3. RESEARCH COLLABORATIONS

# Asian Astronomers Build Closer Ties

1. Dennis Normile

TOKYO—The vastness of space is bringing Asian astronomers a little closer together. Meeting earlier this month in Taipei, astronomers from China, Japan, Korea, and Taiwan moved ahead with cooperative plans on both regional and international projects.

Since 1990, astronomers from the region have gotten together every 3 years or so to present recent results from their own instruments. But on 11 to 16 November, during the fifth East Asia Meeting of Astronomy (EAMA), participants broadened the scope of their discussion to include concrete ways to foster collaborations. “There is a sense of excitement about future prospects for more collaboration and exchanges and access to each other's facilities,” says K. Y. (Fred) Lo, director of Academia Sinica's Institute of Astronomy and Astrophysics in Taipei.

The first actual collaboration is likely to be an East Asian very long baseline interferometry network of radio antennas. Japan and Korea are near an agreement for joint observations beginning in 2005 on radio interferometry networks now under construction in each country (Science, 2 November, p. 977). Chinese astronomers are eager to add two more radio telescopes to the network, making a total of 12 antennas. Adding the two antennas, now used primarily for charting star positions, would allow Chinese astronomers to investigate star formation and other phenomena. “Radio astronomy is one area where collaboration among neighboring regions would be very natural,” says Se-Hyung Cho, vice president of the Korean Astronomy Observatory in Taejon.

Japanese scientists are hoping to make an even bigger splash by bringing their regional neighbors into the fold on the $650 million Atacama Large Millimeter/Submillimeter Array (ALMA), a network of 64 dishes to be built and operated by European, Japanese, Canadian, and U.S. scientists at a site high in the Atacama Desert in northern Chile. Norio Kaifu, director general of Japan's National Astronomical Observatory in Mitaka, hopes to bring other Asian scientists into the project under Japan's umbrella. “That would make it a true world telescope,” he says. Lo says that talk of greater cooperation has been a staple at the EAMA meetings. But it wasn't until recently, he says, that each of the four neighbors achieved a critical mass of scientists, funding, and observational activities to make such joint efforts worthwhile. Yoshihisa Nemoto, senior specialist for space in the space policy division of Japan's Ministry of Education, Culture, Sports, Science, and Technology, calls the move “a natural and good thing” to do, adding that the ministry would be happy at some point to review any proposals for joint projects. The scientists who met at Taipei (some of whom are pictured at left) have set up a coordination committee to plan exchanges and the sharing of observation and computing facilities as well as additional conferences. Because of the tenuous political relations between Taiwan and mainland China, the group plans to establish smooth working relationships among scientists before approaching any government for support. 4. ULTRAFAST LASERS # Photoelectrons Show How Quick a Flash Is 1. Yudhijit Bhattacharjee* 1. Yudhijit Bhattacharjee is a science writer based in Columbus, Ohio. For more than a decade, scientists have captured the breaking of chemical bonds between atoms with the world's fastest strobe lights: flashes of laser light lasting a few femtoseconds. (A femtosecond is 1 millionth of a billionth of a second.) But bond breaking is a languid process compared with the lightning-fast activity of electrons inside atoms, which zip around the nucleus and hop between energy shells in less than a fifth of a femtosecond. To track such quicksilver movements, researchers have longed to generate and measure individual pulses of radiation as short as a few hundred attoseconds. (A femtosecond equals 1000 attoseconds.) Now they've got their wish. In this week's issue of Nature, researchers from the Vienna University of Technology in Austria, the National Research Council Canada, and Bielefeld University in Germany report that they have produced isolated x-ray pulses 650 attoseconds long. Using these pulses like flashbulbs, the researchers have traced the energy-level transitions of electrons in an atomic gas with a resolution of 150 attoseconds. “This is an important experiment,” says Anne L'Huillier, a physicist at the Lund Institute of Technology in Sweden. “It opens the door to the study of extremely fast electronic processes occurring inside atoms and molecules.” Generating the attosecond pulse was fairly straightforward, says Ferenc Krausz of the Vienna University of Technology, one of the authors of the paper. The researchers shined a strong beam of laser light at a volume of neon gas to produce x-ray photons of different frequencies. For the most part, the frequencies canceled each other out through destructive interference, leaving a spike where they added up. Because the laser flash lasted only a few wave cycles, Krausz and his colleagues were able to obtain a single spike instead of a train of spikes, something that a group of French and Dutch researchers achieved earlier this year (Science, 1 June, p. 1627). Measuring the duration of this pulse posed a bigger challenge. The researchers shot the pulse, along with the laser beam used to create it, into a chamber of krypton gas. The x-ray pulse ionized the krypton, causing electrons to fly out of their parent atoms with a certain kinetic energy. The electric field of the laser beam then subtly changed the kinetic energy of the electrons by amounts that depended on which point in its rising-and-falling cycle the field had reached at the instant the pulse knocked the electrons loose. Changing the birth moment of the photoelectrons—that is, the time the x-ray pulse struck the krypton atoms—determined how much the laser beam altered the electrons' kinetic energies. Shooting the x-ray pulse into the gas at various points in the laser cycle, Krausz's group observed that the change in kinetic energy rose or fell in a wavelike pattern, or modulation. The appearance of the modulation in the spectrum proved that the time of the krypton's ionization—and hence the duration of the x-ray pulse—fell within a window of time less than half the 1.2-femtosecond period of the laser cycle. “If the pulse were longer, it would not be possible to see the changing influence of the laser field as it goes through one cycle. The modulation would be smeared out,” Krausz explains. From the modulation, the researchers calculated that the x-ray pulse lasted a fleeting 650 attoseconds and that the krypton atoms released their electrons in less than 150 attoseconds. The modulation also gave information about the oscillating laser field. By combining it with the intensity of the laser beam, the researchers traced the beam's changing electric field over the course of one cycle—in effect, sketching the curve of a wave of light. It's only a matter of time, Krausz says, before the technique they have demonstrated is applied to meaningful attosecond experiments. One is the study of inner-shell relaxation—the filling up of vacancies in shells close to the nucleus by electrons jumping in from outer orbits. Attophysics is here, says Krausz; now for snapshots of the atomic interior. 5. CONSERVATION BIOLOGY # When Is a Coho Salmon Not a Coho Salmon? 1. Jocelyn Kaiser Should hand-reared fish be counted in efforts to save wild, imperiled salmon? A U.S. federal judge has said yes, and the U.S. government earlier this month decided not to challenge the ruling. As a result, up to two dozen West Coast salmon runs could be stripped from the endangered species list. These developments have outraged conservationists, who say counting hatchery fish is like tallying zoo animals when deciding whether their wild brethren are threatened with extinction. “It just doesn't make sense,” says Bill Bakke of the Native Fish Society in Portland, Oregon. For more than a century, the government has used hatcheries to bolster commercially valuable salmon runs, often to make up for spawning habitat lost to dams. The fish are dumped into rivers while they're still juveniles, then they swim out to sea and spend several years in the ocean maturing before returning to the hatchery. But although the pampered hatchery fish are the same species as their wild kin, they don't always act the same way: Behaviors and traits that help hatchery fish do well in captivity differ from those needed to survive in the wild. The National Marine Fisheries Service (NMFS), as a result, has ignored the hatchery fish when deciding whether a “distinct” salmon population—such as one that uses a specific river or coastal area—needs protection under the Endangered Species Act (ESA). In 1998, for instance, when NMFS listed the wild Oregon coast coho salmon as endangered, it did not take the hatchery fish into account because biologists said they weren't essential to the long-term survival of the struggling wild population. Two years ago, the Pacific Legal Foundation, a property rights group in Sacramento, California, that is critical of the ESA, challenged that omission in federal court. On 10 September a U.S. district judge agreed, ruling that NMFS had been “arbitrary” in distinguishing between “two genetically identical” salmon “in the same stream.” On 9 November, NMFS's parent agency—the National Oceanic and Atmospheric Administration—threw in the towel. It declined to appeal the ruling, signaling a change in policy. “It's time to stop fighting and start fixing” salmon runs, says Robert Lohn, NMFS Northwest Region administrator. But critics fear the decision will result in less protection for endangered salmon, because including hatchery fish will likely boost some populations beyond the threshold for protection. The agency must immediately delist the Oregon coast coho salmon, they note, and it must also consider removing from the list 23 other endangered salmon and steelhead populations that share waters with hatchery fish. The decision runs counter to salmon science, according to many biologists. “A whole sheaf of scientific studies” from the past 20 years suggests that hatcheries cause problems for wild fish, says Robin Waples of the NMFS Northwest Fisheries Science Center in Seattle. Hand-reared fish may be genetically similar to their wild cousins, for instance, but they often aren't as skilled at foraging or avoiding predators. As a result, interbreeding between hatchery and wild fish can produce less fit mongrels. Waples and other NMFS scientists hope to hash out the biological significance of these differences by next September, when the agency plans to release a new policy on the role that hatcheries should play in salmon restoration and then decide whether to delist some of the imperiled populations. NMFS will ponder whether hatcheries could help save wild populations, for instance, by rearing only eggs taken directly from wild fish. Waples says the agency will also consider whether seemingly plentiful runs that are composed largely of hatchery fish would survive on their own, as the ESA requires. 6. MARINE CONSERVATION # Reserves Found to Aid Fisheries 1. David Malakoff When California officials began holding public meetings last year on a controversial state plan to ban fishing in some coastal waters, some anglers raised a stink. Their anger, which included flinging dead fish at one session, stemmed in part from what they said was insufficient evidence that closing some fishing grounds would actually help boost catches in nearby areas. New findings presented on page 1920 could help clear the air. An international team of marine scientists reports marked increases in commercial catches and the number of trophy fish caught by sport anglers in and around small reserves in the Caribbean and off Florida. The authors say the results confirm the validity of their models and, more importantly, lend credence to global efforts to establish new reserves. The findings “will help remove a major logjam in the debate,” says lead author Callum Roberts, a biologist at the University of York, U.K. Some scientists, however, caution that closing off some areas won't be enough to restore healthy fisheries. And some influential fishing groups and politicians remain on the offensive, saying that reserves in U.S. waters threaten public access to the seas. Studies have shown that closing swaths of the sea to human activity can produce sizable ecological benefits within the reserve, from more diverse sea life to bigger schools of fish. But “whether reserves have spillover benefits is one of the most hotly debated and least studied issues in marine reserve research,” says Karen Garrison, a reserve advocate with the Natural Resources Defense Council in San Francisco, California. Such studies are difficult, she notes, because researchers must find areas where they can compare catches before and after a reserve was established, and monitor all the relevant variables, from how long fishers work to changing ocean conditions. In their study, Roberts and colleagues from the U.S. National Marine Fisheries Service and the University of the West Indies, Barbados, focused on a 5-year-old network of reserves off the Caribbean island of St. Lucia and an area off NASA's Cape Canaveral rocket launching site in Florida that had been closed for nearly 40 years. In St. Lucia, the researchers concluded that the reserves, which cover about one-third of a long-used coral reef fishing ground, increased catches in nearby areas by up to 90%, compared to prereserve numbers. Off Florida, they found that sport anglers fishing around a 40-square-kilometer area closed in 1962 for security reasons have landed a disproportionate number of world- and state-record fish from three species. Since 1985, for instance, most Florida record red and black drum came from the area. The study confirms that reserves can serve as sheltered nurseries for surrounding waters, say the researchers. And although the studied reserves were small, Roberts says the findings—when combined with sketchier data from bigger closures—“make a compelling argument that reserves will work across a wide spectrum of scales, in many geographical locations, and for many different fisheries.” One researcher, however, cautions that reserves are just one tool available to fisheries managers. “The question is, ‘What role should [reserves] play in the mix of regulatory options?’” says Ray Hilborn, a fisheries biologist at the University of Washington, Seattle. Hilborn argues that less severe moves—from closed seasons to size limits—could produce equally significant fisheries improvements. He also questions whether reserves can work wonders for some fisheries that are already highly regulated, such as those in the United States. Some sport and commercial fishing groups have challenged the California effort and asked the Bush Administration to reconsider a major reserve in the Hawaiian Islands (Science, 8 December 2000, p. 1873). In August, the American Sportfishing Association (ASfA) and other groups also convinced Senators John Breaux (D-LA) and Kay Bailey Hutchison (R-TX) to introduce the Freedom to Fish Act (S. 1314), which would require government planners to make reserves as small as possible. “Blanket closures and no-fishing zones should not be the first solution, but rather the last,” says an ASfA statement. Roberts agrees that reserves should be tailored to specific ecosystems and public goals. But “fishers have nothing to fear from reserves,” he says. The real danger “is a future without them.” 7. GENETICS # Fragile X's Missing Partners Identified 1. Emily Sohn* 1. Emily Sohn is a writer in Minneapolis, Minnesota. Three research teams have begun to decipher the molecular signals that lead to fragile X syndrome, one of the most common causes of mental retardation. People with this syndrome carry a mutated version of a gene on the X chromosome, and since 1991 researchers have known that this mutation blocks the production of a certain protein. But how that deficit causes the syndrome remains mysterious. Two new reports finger a set of messenger RNA (mRNA) molecules in the brain as crucial targets of the missing protein—findings that suggest these mRNAs play key roles in helping neurons communicate with each other. The third team reports that disabling a gene for one of these mRNAs eliminates symptoms of the mutation in a fruit fly model of fragile X, suggesting that eventually it may be possible to treat the syndrome. Together, the studies could help explain the role certain fragile X-related proteins play in the brain. “This is a major step toward understanding the biology of fragile X and understanding the biology of higher cognitive functions,” says geneticist Harry Orr of the University of Minnesota, Twin Cities. The genetic mutation in fragile X syndrome gives the chromosome a distinctive look: One arm appears to be hanging on by a thread. The mutation prevents the gene from producing the so-called fragile X mental retardation protein (FMRP). In addition to mental retardation, people with the disorder often have elongated faces, autism, and attention deficit disorder, among other symptoms. On the microscopic level, people with the syndrome have misshapen dendrites, neural projections that receive signals from other neurons. Scientists have struggled to find a link between the missing protein and these symptoms. In normal brain cells, FMRP binds to strands of mRNA, molecules that transmit blueprints for protein production. But with millions of differently shaped RNA molecules floating around in the cell, researchers have had trouble figuring out which are controlled by FMRP. To crack this problem, a group led by neuroscientist Robert Darnell at Rockefeller University in New York City manufactured trillions of different versions of mRNA. By seeing which stuck to FMRP and washing away the rest, the team narrowed its search down to a family of RNAs called G quartets, which are shaped like cubes on a stick—imagine square Popsicle flavored ices. The team then searched databases of the human genome to find sequences that produce similarly shaped mRNAs, suspecting that these mRNAs are targets of FMRP in the brain. Working independently, geneticist Stephen Warren of Emory University in Atlanta and his colleagues ground up mouse brains and mixed them with antibodies that specifically grab FMRP out of the mush. The team counted more than 400 mRNAs that came along with FMRP for the ride. When the two teams compared results, they identified a dozen mRNAs that have both a G quartet structure and cling to FMRP. Warren's group then looked in the brains of people with fragile X syndrome and found that eight of those mRNAs were either overexpressed or underexpressed. “These are the first eight mRNAs ever identified that are really different in fragile X patients,” Darnell says. Both teams report their results in the 16 November issue of Cell. Figuring out what these mRNAs do is the next step, but Darnell speculates that they build proteins that help brain cells communicate with each other. Knowing which mRNAs FMRP interacts with could potentially lead to treatments for the disease, suggests a third study. Drosophila have a gene similar to the FMRP-producing gene, and scientists can disable it to induce a version of fragile X syndrome. Neurogeneticist Kendal Broadie of the University of Utah in Salt Lake City and colleagues made fragile X fly mutants and noticed an increase in concentrations of the fly version of map1b—a protein built by one of the mRNAs Warren's team found to be overexpressed in people with fragile X syndrome. When the team increased the levels of map1b in normal flies, fragile X symptoms appeared—a sign that map1b is behind the fly syndrome. But the clincher came when the team simultaneously disabled the fragile X gene and, to compensate, hobbled the gene that makes map1b. The researchers ended up with normal flies, they report in the 30 November issue of Cell. “This was completely startling,” Broadie says. “There were no defects left over whatsoever.” That suggests, he says, that a single mRNA target might be the key to explaining what causes fragile X syndrome, at least in Drosophila. If the same proves true in humans, “that takes us to the point where we might be able to treat and cure” fragile X syndrome. Other experts are more circumspect about treatment possibilities. “At this time,” says Orr, “it is a bit simplistic to think that the whole story of fragile X syndrome is controlled by the ability of this protein to regulate a single mRNA,” especially considering the hundreds of potential targets found by Warren's group. Still, Orr agrees with other experts that the recent convergence of fragile X research is a major step toward understanding what causes the disorder. 8. ANTHRAX # Taking Anthrax's Genetic Fingerprints 1. Martin Enserink Armed with a vast collection of strains and a refined DNA fingerprinting system, a research team in Arizona may help solve who's behind the anthrax attacks—and nail other bioterrorists in the future FLAGSTAFF, ARIZONA, AND BATON ROUGE, LOUISIANA—Paul Keim loves talking about his work. But ask him whether he's been enlisted by the Federal Bureau of Investigation (FBI) to help identify the source of the anthrax spores that have so far killed five people in the United States, and there's a good chance he'll recite a line he knows by heart: “I can't deny or confirm that, but we would be pleased to work with any federal officials if they so request.” But it's hardly a secret that Keim, a geneticist at Northern Arizona University (NAU) in Flagstaff, is one of the key scientists in the current anthrax inquiry—and one of the very few who are not employed by a government lab. His role: to produce a genetic fingerprint of the anthrax spores used in every one of the mail attacks. Colleagues say his lab will soon receive two new samples, if it doesn't have them already: one from a letter to Senator Patrick Leahy (D-VT), discovered on 16 November, and one from the 94-year-old Connecticut woman who mysteriously died of anthrax last week. Keim's work should provide the most detailed description of the anthrax spores possible, and, when compared to the fingerprint of bacilli stored in labs around the country, it may help investigators home in on the perpetrator. The assaults have turned life in Keim's lab on its head. Graduate students, logging long hours and working weekend shifts, find themselves in the thick of a suspense novel come to life—and having to keep quiet about it. Keim, walking around with two pagers, says he's getting calls from headhunters and has become a local hero in this small university town. The lab was inundated with media calls last month—which Keim says he has answered haphazardly—and TV vans lined up in front of the building. Meanwhile, security has been tightened: Armed police officers guarded the lab around the clock in the first days after the discovery of the first anthrax letter, while new iron doors and locks were hastily added. But for Keim, a tall and cheerful Idaho native who joined NAU 13 years ago, the attacks were also a scientific challenge that came at just the right time. Keim's primary interest is how genomes evolve, and he has studied genetic variation in a wide variety of species, from microbes to endangered birds. But 5 years ago, with funding from the Department of Energy, he embarked on a program to develop fingerprinting techniques for a series of potential bioweapons. Bacillus anthracis—which heads almost every list of biothreats—was one of the first organisms he took on. In fact, his identification system for anthrax—using short pieces of repetitive DNA sequences that vary from one strain to the other—has worked so well that Keim was already shifting his focus to other potential bioweapons, such as Brucella, Burkholderia, and Francisella tularensis. Other researchers in the field say Keim's strain-typing system for anthrax is the most advanced yet—and the way to go for several other organisms as well. “We, the anthrax community, have been very excited by what they have been able to achieve,” says Peter Turnbull, a researcher formerly at the Centre for Applied Microbiology and Research in Porton Down, United Kingdom. To develop a fingerprinting system for anthrax—or any other organism—state-of-the-art molecular tools are not enough. Another prerequisite is a large collection of strains that researchers can search for distinctive genetic differences. That's why Keim is quick to credit another lab for his current role as a disease detective: that of veterinary epidemiologist Martin Hugh-Jones at Louisiana State University, Baton Rouge. In the mid-1990s Hugh-Jones started building a huge anthrax collection that now comprises more than 1200 isolates. Hugh-Jones, a transplanted Brit nearing retirement, chairs a World Health Organization (WHO) working group on anthrax and moderates animal disease reports for ProMED, a popular electronic mailing list about emerging infectious diseases. He's fascinated by the ecology and epidemiology of anthrax as a disease of domesticated and wild animals: where it arose, how it survives in the soil, and how human travel and trade have helped spread it around the globe. Hugh-Jones was a member of a team of U.S. scientists who traveled to Russia in 1992 to investigate a 1979 outbreak of inhalational anthrax in the city of Sverdlovsk (now called Yekaterinburg). Two years later, one of the Russian scientists involved in the epidemic came to the U.S. with tissue samples from 42 of the patients. Hugh-Jones arranged for 11 of them to be tested for genetic traces of B. anthracis by Paul Jackson's group at Los Alamos National Laboratory in New Mexico. Keim, visiting Jackson's lab as part of a sabbatical, was specializing in strain typing and did part of the work. The study, published in 1998, showed that the Sverdlovsk patients had been infected with four different strains of B. anthracis. This further bolstered the earlier group's conclusion, published in Science in 1994 (18 November 1994, p. 1202), that the outbreak had been caused by an accidental release of anthrax spores from a nearby military research facility—and not by contaminated meat, as Russian authorities had long insisted. Keim and Hugh-Jones have worked closely together ever since. Collaborating with a well-known veteran in the field helped Keim, a relative newcomer, “break into the smoke-filled backrooms of anthrax,” he says. It also gave him access to Hugh-Jones's collection. Through “a combination of persuasion, financial help, and blackmail,” Hugh-Jones says he constantly pressures researchers and diagnosticians from around the world to send him anthrax collections and samples they have gathered. Over the years, 60 samples arrived from Turkey, 55 from Italy, 225 from China, and so on. The entire collection—believed to be the biggest and most diverse in the world—still fits in a freezer the size of a household refrigerator. (Keim also keeps a copy of every one of Hugh-Jones's isolates, so that the collection is now stored in both Baton Rouge and Flagstaff.) Over the past 5 years, Keim's group has been refining a technique that Hugh-Jones needs for his epidemiologic studies—and the FBI needs for its criminal investigation: a way to tell different anthrax strains apart. (Researchers refer to an isolate as something they find in the field; a strain is thought to be a set of microbes that are genetically close, although what that means is not well defined.) Anthrax is probably the most genetically homogenous species known, says Keim: Until recently, every isolate ever found seemed to have exactly the same DNA as every other isolate. It was as if 1000 potential murderers all shared the same fingerprints, height, race, and eye color. Historically, researchers have given only three isolates a specific name: Ames, isolated from a cow in Iowa; Vollum, a strain originally isolated in Britain; and Sterne, an avirulent strain widely used as a vaccine. But apart from the obvious fact that the Sterne strain causes no disease, researchers had no way of telling these three apart—nor any other member of their anthrax collections—some of which presumably are duplicates. Researchers suspect this uniformity arises from anthrax's unique life cycle. After killing an animal, the bacteria sporulate and ooze with blood from the carcass into the soil, where they can remain dormant for years, decades, or perhaps centuries. Only when another animal comes into contact with the spores—it's unclear exactly what conditions favor this process—does the cycle start anew. So, although anthrax may have caused vicious outbreaks for more than 10,000 years, its evolution has moved in slow motion. In 1996, Kenneth Wilson's team at the Department of Veterans Affairs Medical Center in Durham, North Carolina, detected a single spot where anthrax strains varied genetically from each other. In that region, a short stretch of DNA was repeated two times in some bacteria and up to six times in others. Finally, researchers had a way to classify anthrax strains into five different groups, based on the number of repeats in this area. Keim and his colleagues spent the next 3 years painstakingly searching for more of these so-called variable-number tandem repeats (VNTRs), or markers. By 1998, they had discovered seven new ones; using all of them and the VNTR discovered by Wilson, the team fingerprinted 426 isolates from Hugh-Jones's worldwide collection. The results, published last year, revealed that many of the strains were identical: The 426 isolates had only 89 unique genomes. Then the genomic revolution came to anthrax. In 1999, Timothy Read of The Institute for Genomic Research in Rockville, Maryland, posted the bulk of the sequence of the B. anthracis genome online. Keim's colleague Jim Schupp vividly recalls searching the data for the first time: “I was enthralled,” he says. “I could not sleep, I sat here until 4 in the morning staring at my screen.” What Schupp saw—represented graphically as a series of boxes and lines—were hundreds of potential new VNTRs that could help discern infinitely more differences among anthrax strains. Some of these newfound VNTRs have changed slowly over time, making them suitable for comparing strains that are far apart genetically; others have evolved rapidly, making them perfect for studying groups of closely related strains that were indistinguishable in previous analyses. Keim's group is now working with an arsenal of 50 markers and adding new ones all the time. “If we had known the stakes would be this high 6 months ago, we might have had 100 or 150 markers ready to go,” says Keim. Using that growing arsenal should make it possible to distinguish almost any isolate from any other, says Keim. Even within a conservative bug like B. anthracis, changes occur from generation to generation, he says, and some of the new markers could pick those changes up. The number of mutations may even provide an estimate of the number of generations between the original sample and one used in an attack. That is exactly the kind of resolution needed to help solve a bioterrorist crime. Keim won't say whether his analysis has helped investigators much, except to repeat the scant information issued so far by Homeland Security director Tom Ridge: that the isolates from New York, Washington, and Florida are the same, and that they all belong to the so-called Ames strain. But other researchers speculate that the FBI may learn far more from Keim's lab than they are letting on. Over the past 2 decades, the U.S. Army Medical Research Institute of Infectious Diseases in Fort Detrick, Maryland, sent the Ames strain to several research labs. And as it was passed around and grown in different labs, it may well have accumulated minute new changes. “The Ames strain can be many different things,” says Hugh-Jones. “A very detailed fingerprint could reveal very minor variations.” That's why comparing the strain used in the anthrax attacks to those stored in freezers around the United States could well pinpoint the lab that the spores came from, says Keim. “So far, I haven't heard that any cultures have been subpoenaed,” he says. “But that would be a logical next step.” A genetic fingerprint may also form part of the evidence if researchers ever apprehend suspects in the case—which both Keim and Hugh-Jones are convinced is just a matter of time—and anthrax spores are found in their home or possessions (see sidebar on p. 1811). Meanwhile, anthrax researchers are grappling with the unsettling possibility that the microbes used in the attacks could have come from their labs. “I've given that a lot of thought recently,” says Keim, who thinks the prospect is highly unlikely. Hugh-Jones, too, says he could think of no one fitting the bill when federal agents questioned him recently. But he did post the FBI's psychological profile of the perpetrator on ProMED, believing that some subscribers might have more information. “It's a small world,” says Hugh-Jones. “I'm sure I know somebody who knows him.” 9. ANTHRAX # Can Lab Sleuths Clinch the Case? 1. Martin Enserink It would make a terrific episode of Law and Order: The police have finally nabbed a lonesome lab technician from Trenton, New Jersey, and charged him with sending anthrax-laced letters to members of the media and the Senate. But the evidence is circumstantial, and the prosecution's case hinges on the similarity between the DNA of anthrax spores found in his apartment and those found in the letters. Would the evidence hold up in court? At the moment, few researchers would bet on it, because microbial genetic analysis is not as standardized as human DNA analysis is. “It's very scary; none of us is fully prepared for that,” says Paul Keim of Northern Arizona University in Flagstaff. “I'd hate to be across the witness stand from somebody like Barry Scheck,” a high-profile lawyer with expert knowledge about human DNA fingerprinting. Over the past decade, human DNA from blood stains, semen, or hair has become an accepted forensic tool, and debates about its reliability have subsided as the technology has advanced. But the rare cases when nonhuman DNA went to court—like one in which seed pods were traced back to a single palo verde tree in Arizona (Science, 14 May 1993, p. 894)—have been highly controversial. Similarly, “I would expect a fairly extensive hearing” about the admissibility of evidence linking a disease outbreak to a suspected bioterrorist, says David Kaye, a lawyer and DNA evidence expert at Arizona State University in Tempe. Even if the judge allowed such evidence, he says, lawyers would certainly try to sow doubts in the jury's mind by having other scientists highlight its weaknesses. Whether DNA evidence would be allowed, says Kaye, would depend on how well accepted the underlying science was—in this case, the use of repetitive DNA sequences to identify pathogens. DNA evidence would be less likely to be accepted, he says, if it involved a technique that was still being developed—such as the anthrax typing system used by Keim—or if there was debate about the likelihood that two DNA samples would match. At the moment, such debates seem highly likely if anthrax or other agents of bioterrorism are involved, says Keim. Human DNA is shuffled during sexual reproduction, giving each individual a set of genes that's unique in the world. But microbes reproduce asexually, and every new generation is an almost exact copy of its ancestor. With anthrax, especially, the genetic differences among strains—even if they're found in far-flung parts of the world—are extremely small, so an apparent match could easily be a coincidence. On the other hand, adds Keim, if the perpetrators used a very rare strain, DNA evidence might clinch the case. To study the validity and credibility of microbial DNA tests, Keim plans to assemble a panel—he's hoping for support from the National Academies of Science or the National Science Foundation—to study the issue, much like past committees that hammered out consensus about human DNA testing. “We need to get some of these things figured out as a scientific community,” he says. “If we're fighting like cats and dogs among ourselves, how are we going to convince a judge?” 10. ANTHRAX # A Second Anthrax Genome Project 1. Martin Enserink The bioterrorist assaults in the United States have spawned a scientific novelty: the first genome project ever triggered by a crime. Last month, the National Science Foundation (NSF) announced that it would give The Institute for Genomic Research (TIGR) in Rockville, Maryland, almost$200,000 to sequence the entire genome of the Bacillus anthracis strain used in the attack on American Media, a publishing company in Boca Raton, Florida.

TIGR had already sequenced more than 95% of the genome of the so-called Ames strain of B. anthracis, a common variety used in research labs. (The remaining hard-to-sequence part is expected to be in the bag within a few months.) The bioterrorists also used Ames—but their version may have subtle differences that set it apart from other Ames cultures. According to NSF, having a second anthrax genome will give researchers a better understanding of anthrax's diversity; and the project may just help officials investigating the case, says Maryanna Henkart, NSF's division director for molecular and cellular biosciences.

The genome project should reveal whether the perpetrators genetically altered the anthrax, for instance, to make it more virulent, says Arthur Friedlander of the U.S. Army Medical Research Institute of Infectious Diseases in Fort Detrick, Maryland. So far, he says, there's nothing to suggest that the microbes have been tinkered with—but that doesn't mean it didn't happen.

But the project is less likely to be of help in tracing the bioterrorists' identities, says Paul Keim of Northern Arizona University in Flagstaff. To do that, researchers cannot just compare the new strain with that already sequenced by TIGR; they'd have to sequence yet another strain—say, spores found in a suspect's lab or car—and compare the two new genomes. But the error rate of current sequencing technology—at least 1 in every 100,000 bases—would swamp the rare single-base pair differences between two closely related strains, says Keim.

Still, most researchers applaud the project, if only because it will for the first time give them the full sequence of the two plasmids—circular minichromosomes that harbor anthrax's virulence genes—of the Ames strain. The first TIGR project had skipped those because plasmids from different strains had already been sequenced by other groups.

11. ANTHRAX

# A 'Sure Killer' Yields to Medicine

1. Kathryn Brown

The clinical course of inhalational anthrax hasn't changed in centuries, but speedy diagnosis and new drugs can alter its outcome

It was Tuesday morning when a patient dubbed T.T. began complaining that his head and back ached. He left work at lunchtime. That afternoon, his doctor diagnosed the flu, giving him aspirin and codeine. It seemed to do the trick—for a day. But then something went terribly wrong. By Friday, T.T. was dead.

Now, we know the correct diagnosis: inhalational anthrax. But T.T. was not a victim of bioterrorism, and he didn't die last month. In fact, he died 44 years ago, in the fall of 1957. Three more deaths followed, in the biggest 20th century outbreak of inhalational anthrax reported in the United States. The victims all worked in a Manchester, New Hampshire, goat hair mill, where they likely inhaled anthrax spores from the hair of infected animals. These cases would have remained a historical footnote, except that history is repeating itself.

Since early October, 11 patients with inhalational anthrax have arrived at the doors of various doctors. At press time, five had died, two of whom were sent home by doctors who mistook the disease for a stomach virus or bad cold. Like the mill workers half a century ago, the recent patients had early aches that were hard to pin down. Some, too, inhaled Bacillus anthracis spores at work: a thin dust of bioterror coating mail.

The surge of new cases has given physicians a fresh look at an old disease in unprecedented clinical detail. Researchers familiar with anthrax say the cases look remarkably familiar, except perhaps for one aspect: Physicians have long thought that death from inhalational anthrax is almost inevitable once a patient shows advanced symptoms. Yet quick-thinking doctors armed with a trove of drugs and techniques have saved six of the 11 patients this fall—an impressive success rate. “There's nothing like seeing a patient leave the hospital with a disease that was supposed to have killed him,” remarks Thomas Mayer, head of emergency medicine at Inova Fairfax Hospital in Falls Church, Virginia, where two of the survivors were treated.

But unresolved questions linger. Will the survivors face lung problems in the years to come? Did others get mild cases of anthrax that went undiagnosed? And, perhaps most pressing, could those exposed to anthrax weeks ago still fall ill? “We're learning as we go,” remarks John Jernigan, an epidemiologist with the Centers for Disease Control and Prevention (CDC) and Emory University in Atlanta.

Anthrax gets its name, anthracis, from the Greek word for coal, a reference to the legendary coal-black scabs of cutaneous, or skin, anthrax. According to the 1999 book Anthrax: The Investigation of a Deadly Outbreak by Boston College sociologist Jeanne Guillemin, anthrax may have been the sixth plague in the Book of Exodus, mentioned in Homer's Iliad, and lamented by Virgil in ancient Rome. Early microbiologists clearly knew the bacteria well. In 1876, Robert Koch first showed that B. anthracis causes anthrax in animals—and in the process, established Koch's postulates. Five years later, Louis Pasteur demonstrated a method of vaccinating sheep and cattle against the disease.

Even then, anthrax struck terror as it wiped out livestock. Grazing animals are most at risk for the disease, consuming anthrax endospores that are hidden in soil. During the 19th century, mill workers who handled infected animal hairs, wools, or hides came down with anthrax so often that the condition became known as woolsorters' or ragpickers' disease. Even in the early 1900s, about 130 people in the United States contracted some form of anthrax every year. The disease became a modern bioweapon in World Wars I and II, as countries including Germany, Japan, and the United Kingdom reportedly tested it.

Anthrax comes in three forms, of varying lethality. Skin anthrax—caused when B. anthracis spores seep into a cut—is the most common and treatable form of the disease. During the current outbreak, at least seven people have gotten skin anthrax. In the 1957 episode, four people did; all recovered. Less common but more dangerous is gastrointestinal anthrax, from eating tainted meat. Finally, there is inhalational anthrax, the most feared variety. A 1994 paper in Clinical Infectious Diseases sums up the traditional thinking: “Inhalation anthrax is virtually always fatal.”

At the outset, however, the disease seems deceptively benign: Patients may feel achy, feverish, nauseated, or fatigued. This first phase typically lasts several days, during which time macrophage cells are gobbling up anthrax spores and carrying them to the lymph nodes near the lungs. There, the spores germinate into full-fledged bacteria, break free from the phages, and multiply.

Then the real trouble starts. During the second stage of infection, anthrax bacteria flood the bloodstream, releasing toxins that make many tissues swell and bleed. Within hours, a patient's blood pressure can plummet, oxygen levels dip, and organs fail. “This is not a diagnosis you want to sit around and ponder,” remarks H. Clifford Lane, clinical director of the National Institute of Allergy and Infectious Diseases in Bethesda, Maryland. “The key is to act quickly.”

The problem is, rare outbreaks of inhalational anthrax catch doctors off guard. “The cases in 1957 came as a real surprise,” recalls infectious disease specialist Philip Brachman of Emory University, who was testing a vaccine on mill workers at the time. “We'd only seen sporadic cases of anthrax in the U.S., and these workers just came in with flulike symptoms. By the time doctors realized they were dealing with anthrax, it was usually too late.” Today, anthrax still falls under the radar of most physicians. “I've never seen a case of anthrax, and I've never seen anybody who's seen a case of anthrax,” comments Bill Roper, dean of the school of public health at the University of North Carolina, Chapel Hill.

Researchers did have one recent glimpse of anthrax bioterror in action. In the spring of 1979, at least 66 people in the industrial city of Sverdlovsk (now Yekaterinburg), Russia, died. At first, authorities blamed tainted meat. But death certificates cited influenza, sepsis, and pneumonia, among other conditions. Finally, in 1992, a team of Russian and U.S. scientists concluded that a local military facility had accidentally released a cloud of anthrax bacteria, killing scores of people and animals who inhaled it.

The outbreak might have provided a wealth of anthrax data, but the KGB confiscated most relevant medical records, leaving only autopsy notes and tissue samples behind. “What we glimpsed was the clinical endpoint: the end of life with anthrax,” says David Walker, a pathologist at the University of Texas Medical Branch in Galveston who studied the Sverdlovsk cases.

## Dusty data

One puzzling question is who succumbs to the bacteria and why. The Sverdlovsk and New Hampshire cases provide some clues.

They show just how mercurial inhalational anthrax is: sickening some but not others; causing illness within days—or weeks—after exposure; prompting heavy sweating or just fatigue. In Sverdlovsk, for instance, people fell ill anywhere from 2 to 43 days after inhaling bacterial spores. Some who died were farther from the point of exposure than were 11 reported survivors. Of 77 known patients, 55 were men, with a mean age of 42. For some reason, no children were reported ill. Although the demographics are sketchy, researchers say these numbers raise questions about differences in individual susceptibility to anthrax.

The same questions are being asked today. Why did these 11 patients get sick with inhalational anthrax—and others not? Was their exposure level higher, or did something else make them particularly vulnerable? One possibility, speculates Jernigan, is that some additional people did contract the disease, but in a mild form that went undetected.

Other lessons are hidden in the dusty data. Some doctors have made much of the “nonclassic” symptoms seen in recent anthrax victims, such as drenching sweat and nausea, asserting that these clinical signs are new. In Sverdlovsk, however, many patients felt sick to their stomach; researchers documented gastrointestinal lesions in 39 of the dead. Similarly, in the hours before patient T.T. died in the New Hampshire outbreak, he sweated so much his bed sheets were changed five times. “If you really want to understand anthrax, go to the literature,” advises Lane.

Animal studies lend some sobering perspective as well. Although the medical spotlight shines on just 11 patients, many more may still be at risk of anthrax, says Colonel Arthur Friedlander, senior science adviser at the U.S. Army Medical Research Institute of Infectious Diseases at Fort Detrick, Maryland. He notes that anthrax spores germinate at different rates inside the body. During experiments with monkeys, for instance, fatal anthrax occurred as long as 98 days after exposure.

And those people now taking preventive antibiotics—typically a regimen of drugs twice a day, for 60 days—could be in trouble if they fail to complete the full course. In one primate study done before the Gulf War, experimental animals exposed to anthrax and given prophylactic antibiotics still came down with the disease when the drugs were stopped after 30—and in one case, 58—days. According to CDC scientist Julie Gerberding, the agency is working with health departments and other groups to urge people to finish their medicine, although she notes that compliance with such a long regimen rarely reaches 100%.

## New lessons

What distinguishes this outbreak from earlier episodes is the sheer power of modern medicine. Over the weekend of 19 October, when physicians referred two postal workers to Inova Fairfax Hospital, the emergency room staff immediately ordered chest x-rays—and then chest computerized tomography scans. The CDC had not yet cautioned postal workers about anthrax risk, but at least one of the patients was concerned.

And that concern was merited: In both patients, chest scans showed massive lymph nodes, as well as swelling between the lungs, in a space called the mediastinum—telltale signs of advancing anthrax. Doctors gave the patients triple antibiotics to kill the bacteria: ciprofloxacin, rifampin, and clindamycin. They also drained bloody fluid around the patients' lungs. “We decided to treat the fluid buildup aggressively, and I think that made a big difference,” says Naaz Fatteh, an Inova Fairfax doctor and co-author of a report on the cases in the 28 November Journal of the American Medical Association.

From the new cases, a more coherent clinical picture of inhalational anthrax is emerging. As their disease progressed, the first 10 patients showed striking similarity. All had some abnormality on the chest x-ray. Eight had pleural effusions, or fluid around the lungs—another common finding in Sverdlovsk. Seven of the patients had a widened mediastinum. And seven showed cobweblike fibers, called infiltrates, in their lungs.

By the time the patients arrived at the emergency room, many already had alarming amounts of anthrax bacteria in their bodies. One challenge, says Jernigan, lead author of a paper analyzing the 10 cases in the November-December issue of Emerging Infectious Diseases, is to better recognize this brief window, before the toxic second stage kicks in. “Once toxin production reaches some critical level, treatment is much less likely to succeed,” he notes.

Doctors aren't quite ready to declare the current outbreak over. Just last week, they announced this fall's 11th case of inhalational anthrax: a 94-year-old woman in Oxford, Connecticut, whose route of exposure to the bacteria remains a mystery. “While it's extremely gratifying to see these first survivors,” Inova's Mayer says, “we have to realize that there could be more cases, either now or in the future. The genie is out of the bottle.”

And the inhalational anthrax survivors face an uncertain prognosis. “Will they develop scarring in the middle of their chest or lymph nodes?” asks Fatteh. “And will that decrease lung function?” There's no way to know, because so few people have ever survived inhalational anthrax. These patients, Fatteh says, will need regular lung tests for years to come. Along the way, doctors hope to learn more about anthrax—and prepare for whatever form of bioterror arrives at the emergency room next.

12. PALEONTOLOGY

# TV Dinosaur Team Treads Tricky Mammalian Terrain

1. Andrew Watson*
1. Andrew Watson is a writer in Norwich, U.K.

Walking with Dinosaurs was a smash hit, but researchers were uneasy with the mix of fact and guesswork. Now mammals get the same treatment

NORWICH, U.K.—After dinosaurs, mammals took center stage: in real life and on television. The same production team that made giant reptiles stomp and roar across our screens in Walking with Dinosaurs is continuing where the series left off, 65 million years ago. Like its predecessor, Walking with Beasts—a six-part series that began in the U.K. this month and will air on a single night (9 December) in the United States—blends expert knowledge, computer animation, and mechanized models to present extinct mammals feeding, fighting, and fleeing in a “wildlife documentary” format.

This time around, the BBC programmakers faced new challenges, including a subject less familiar to the public and dominated by twitchy, hairy creatures that are harder to animate than dinosaurs. And the makers have taken the bold step of conjuring our own distant ancestors, inferring behavior from modern apes. “It was a very daring decision,” says paleontologist Michael Benton of the University of Bristol, U.K.

Benton is a vocal supporter of a production that inevitably attracts criticism from experts, just as Dinosaurs did 2 years ago for its blending of fact and inspired guesswork into appealing scenarios (Science, 7 April 2000, p. 29). Because of the success of Dinosaurs, the team stuck to the same recipe for Beasts, but this time around the programmakers provided even more background material using Web sites and interactive TV. “It's all laid out bare for anyone to look at,” says series producer Jasper James. “We hide nothing.”

How the makers of the program fared is dividing the experts. Benton, a dinosaur specialist, proclaims Beasts “a gift to paleontology.” Mammal expert Jerry Hooker of London's Natural History Museum is positive, despite some quibbles. “The animation generally is very good … when [the animals] walk, their feet really do seem to be in contact with the ground; when they tread in the dust the dust goes up.” But, Hooker adds, “I do cringe sometimes at some of the things that are said.”

Andrew Currant, a colleague of Hooker's, declares himself “a little bit disappointed” with Beasts. Some of the creatures' movements, he says, are “very Bambi, very Disney.” But Currant argues that anything that inspires public interest in lost mammals is good.

James admits that mammals were a tough job: Hair alone was a headache. To prevent the hairy animals from “coming out looking like Muppets,” he says, the animators had to develop new techniques to get realistic hair. But mammals have whiskers that move, eyebrows that shift, cheeks that wobble, and ears that twitch, says lead animator Mike Milne: “It was more the extra mobility that mammals have that took us by surprise.”

A particularly exacting task was depicting Australopithecus, a 3-million-year-old upright hominid. Beasts charts a troop after the death of a lead female, the subsequent tensions in the troop, and the troop's search for a new home. The animators worked hard with Robin Crompton of the University of Liverpool, U.K., to give Australopithecus a justifiable and believable movement, between ape and human. They based the hominid's behavior on bonobos, a species similar to chimpanzees. Even though the TV crew ducked some contentious questions, such as whether Australopithecus had tamed fire, James expects this element to attract the harshest scrutiny. “It's a hot subject,” he says.

But on the whole, mammals simply aren't as sexy as dinosaurs—to the public or to the research community. Paleontologists are keen to point out that this is not from a lack of fossils. “The quality of the fossil record of mammals in the last 65 million years on the whole is better than that of the dinosaurs,” says Benton. He notes that some sites, such as the Messel oil shales near Frankfurt, contain mammalian fossils showing hair and gut contents with “astonishing preservation.”

Although more may be known about what ancient mammals ate and looked like, scientists remain uncomfortable about inferring behavior, as was necessary for Beasts. Besides the hot-button hominids, one example occurs when an early horse, Propalaeotherium, is shown eating rotting grapes on the forest floor. Its senses dulled by alcohol in the grapes, the diminutive creature becomes an easy meal for Gastornis, a flightless bird that looks like a cross between a parrot and a Tyrannosaurus rex.

Far-fetched? Depends on whom you ask. Hooker says the evidence is “absolutely positive” that Propalaeotherium ate grapes because a fossil of the horse found in the Messel beds had grape pits in its stomach. Elephants and other modern animals get drunk when they eat rotten grapes. But Hooker thinks putting two and two together is “pushing it a bit.” Whereas some researchers shy from making the connection, Benton says it's fair game, arguing that TV needs a compelling story.

British viewers with access to digital television will be able to make up their own minds. If anything appears puzzling, “they can press a button and there will be fossils and a scientist telling them how we know that,” says James. This is the first time that interactive TV has been used for a documentary in the U.K. The BBC has also put together two 50-minute “science of” programs, to be screened during the Beasts series, and a Web site with animation sequences, mammalian family trees, and explanations of how the programmakers decided on the appearance and behavior of every star creature (www.bbc.co.uk/beasts). The U.S. production will air as a 3-hour-long program on the Discovery Channel, Walking with Prehistoric Beasts, in which animation is interspersed with talking heads who provide scientific background directly.

Paleoanthropologist Leslie Aiello of University College London, a consultant to Beasts, admits she is uneasy about the show's tendency to stray into “paleofantasy,” but overall she declares it “an exciting, good series.” Now it's up to U.S. viewers to give their own thumbs-up—or thumbs-down—to the latest prehistoric diorama come to life.

13. GAMMA RAY BURSTS

# Cosmic Mystery Objects Start to Yield Secrets

1. Govert Schilling*
1. Govert Schilling is an astronomy writer in Utrecht, the Netherlands. His book Flash! The Hunt for the Biggest Explosions in the Universe will be published by Cambridge University Press in March 2002.

Powerful deep-space explosions remain a puzzle, but new data are giving important clues about their structure, origins, and numbers

Almost 5 years after astronomers first discovered their telltale afterglows, the brief, ultrabright flashes of high-energy radiation known as cosmic gamma ray bursts (GRBs) are still among the most mysterious phenomena in the universe. Most researchers agree that the most common GRBs—those that last between about a second and a minute—signal the catastrophic collapse of massive, rapidly rotating stars into black holes. The details of their origin, however, are unknown, and the nature of bursts that wink out in less than a second is anybody's guess (see bottom sidebar, p. 1817).

Nevertheless, astronomers have made enormous progress in understanding these events, the biggest explosions in the universe, and they hope that satellites such as NASA's High-Energy Transient Explorer 2 (HETE-2; see top sidebar, p. 1817) will help unravel the remaining mysteries. At a recent workshop,* researchers presented evidence that GRBs are surprisingly frequent—one pops off somewhere in the universe every single minute—and that the bursts may be closer kin to normal supernova explosions than scientists had assumed. Soon, experts hope, these bright beacons might be used to study the first generation of stars, the origin of the large-scale structure of the universe, the history of the cosmic star-formation rate, and the chemical evolution of the universe. “The promise of gamma ray bursts as cosmological probes is enormous,” says Donald Lamb of the University of Chicago.

The first GRBs were serendipitously discovered by military satellites in the late 1960s. During the 1990s, NASA's Compton Gamma Ray Observatory detected some 400 bursts per year, but their precise sky positions, distances, and energy yields remained unknown until 1997. That year, the small Italian-Dutch satellite BeppoSAX discovered the first afterglows: the fading embers of the explosions, faintly glowing at x-ray and optical wavelengths, that last for weeks or months (Science, 23 May 1997, p. 1194).

Analysis of the radiation from the afterglows showed that GRBs occur in extremely remote galaxies. That presented a puzzle: If GRBs spew out high-energy gamma rays in all directions, the tiny fraction reaching Earth over such vast distances implied that they are producing more energy than any known natural mechanism could explain. Instead, astronomers had to assume that at least some GRBs concentrate their energy in two oppositely directed jets and become visible only when one jet happens to point toward Earth. Without knowing how tightly focused the jets are, however, they could not estimate the total energy output of a burst or what proportion of GRBs they could see.

Now, a group of astronomers led by Dale Frail of the National Radio Astronomy Observatory in Socorro, New Mexico, has determined the strength of the so-called beaming effect. By carefully analyzing the fading of 17 afterglows at a variety of wavelengths, they deduced that most jets are just a few degrees wide. In that case, Frail and colleagues conclude in a paper accepted for publication in Astrophysical Journal Letters, a typical GRB gives off only slightly more energy than a regular supernova, the terminal explosion of a massive star. What's more, that total energy output appears to be remarkably similar for all GRBs—a result that raises the possibility that GRBs might serve as “standard candles” for astronomical measurements. According to team member Re'em Sari of the California Institute of Technology in Pasadena, the strong beaming effect also means that for every observed GRB, there must be some 500 unobserved bursts whose jets do not point in our direction. “If a gamma ray burst signals the birth of a black hole,” Sari says, “this means a birthrate of one black hole per minute.”

Most models imply that GRBs whose beams don't point at Earth still produce observable afterglows, because the afterglow radiation is emitted in all directions. Several searches for such “orphan afterglows” are under way. Already, astronomers at the Sloan Digital Sky Survey may have detected one on images obtained in March 1999 and May 2000. “I can't completely rule out that [this variable source] is a nearby dwarf nova,” says Daniel Vanden Berk of the Fermi National Accelerator Laboratory in Batavia, Illinois, “but all non-afterglow explanations are really unlikely.” Tomonori Totani of the National Astronomical Observatory of Japan is planning a search using a sensitive camera at Japan's 8.2-meter Subaru Telescope on Mauna Kea, Hawaii. With a field of view almost as large as the full moon—very wide for a big telescope like Subaru—Totani hopes the camera will catch an average of one orphan afterglow on six random exposures.

Other clues to the nature of GRBs may come from faint “fossil remnants” of the bursts that remain in the sky long after the afterglows have faded. Ralph Wijers of the State University of New York, Stony Brook, believes such fossil remnants may already have been found in the form of soft x-ray transients: binary systems in which gas from a low-mass star glows with x-rays as it falls into a companion black hole. Models show that in such a system, the black hole probably started out as a rapidly spinning massive star, just the sort of object needed to produce a GRB. If the soft x-ray transients that have been discovered in our Milky Way galaxy are indeed the descendants of GRBs, Wijers says, it may become possible to conduct detailed postmortems on GRBs. Such studies could reveal the mass of the black hole, the “kick velocity” the explosion imparted to it, and the changes in chemistry the exploding star wrought on its smaller companion.

Meanwhile, other astronomers are peering backward in time to study the oldest bursts of all. In the early universe, theorists believe, GRBs must have been much more common than they are today. Astrophysical models show that first-generation stars probably were very massive and contained almost no heavy elements—both properties that make them likely to collapse into black holes, triggering a GRB. Because the radiation takes time to reach Earth, the oldest GRBs are the most distant. By studying these luminous beacons, which can easily be observed over distances of billions of light-years, astronomers hope to learn how many stars were being born at different stages of the universe's history.

Some astronomers believe they have already spotted such ancient GRBs. John Heise of the Space Research Organization Netherlands in Utrecht thinks that the numerous x-ray flashes observed by BeppoSAX may be far-off bursts, their gamma ray emissions stretched into longer wavelength, lower energy x-rays by the expansion of the universe. The stretching would also make a burst appear to last longer. Indeed, the most recent x-ray flash, detected on 30 October, lasted over 8 minutes—the longest on record.

Unfortunately, the distance of the October x-ray flash is still unknown—a nagging information gap that also plagues the majority of GRBs. The reason, in the case of GRBs, is that gamma rays give no information about redshift, the change in an object's spectrum that astronomers use to measure distance. To find the redshift of a GRB, astronomers need to observe its afterglow in optical light. The first optical afterglow, however, was not discovered until 1997, and for many GRBs no afterglow has been found at all, possibly because cosmic dust blocks their light. Consequently, of the nearly 3000 GRBs detected in the past 30 years, fewer than 1% have known distances.

But help may be on the way. Some astronomers hope to calculate a burst's true luminosity from subtle properties of its gamma rays themselves: a slight difference in arrival time between high- and low-energy gamma photons of the burst, and the rate at which the burst flickers. Then, by comparing the true luminosity to the observed brightness of the gamma rays, they will be able to determine the distance to the source.

According to Bradley Schaefer of the University of Texas, Austin, the various provisional distance indicators agree fairly well. When they can be calibrated more precisely, he says, observations of luminosity and redshift may help astronomers deduce whether the expansion of the universe has sped up or slowed over time. “Astronomers who used distant supernovae [as cosmological probes] have made enormous progress in the past 10 years,” Schaefer says. “We could do the same with gamma ray bursts. In the future, both techniques may be combined.”

To make such visions a reality, astronomers will need a large sample of GRBs with known distances. Within a few years, a wealth of data should arrive from satellites such as NASA's Swift mission, an orbiting GRB observatory due to be launched in September 2003. By then, says HETE's principal investigator George Ricker of the Massachusetts Institute of Technology, HETE-2 is expected to have pinpointed the locations of some 35 GRBs, about a dozen of which are likely to show optical afterglows. Welcome information indeed—but probably not enough to solve the many mysteries that still surround these cosmic superbombs.

• * Gamma-Ray Burst and Afterglow Astronomy 2001, 5–9 November, Woods Hole, Massachusetts.

14. GAMMA RAY BURSTS

# HETE-2, in Business at Last

1. Govert Schilling*
1. Govert Schilling is an astronomy writer in Utrecht, the Netherlands. His book Flash! The Hunt for the Biggest Explosions in the Universe will be published by Cambridge University Press in March 2002.

When NASA's High-Energy Transient Explorer 2 (HETE-2) went up on 9 October 2000, astronomers who hoped to search for optical afterglows eagerly awaited a stream of gamma ray burst localizations: precise sky positions, nailed down within seconds after a burst. But it took almost a year for the small satellite, an international project led by the Massachusetts Institute of Technology, to get down to business. The first “HETE afterglow” was found only last month.

“It took a long time, but we got there,” says principal investigator George Ricker. According to Ricker, the main bottleneck was labor. “When NASA came up with additional money in May, most of our problems were solved,” he says. But there were also technical hurdles to overcome. Optical filters on HETE-2's soft x-ray cameras—the instruments that provide the most precise sky positions—were destroyed by oxygen atoms in the uppermost layers of Earth's atmosphere, slashing the cameras' observational efficiency by 75%. “This has been our major technical problem,” says HETE-2 team member Joel Villaseñor. In addition, new software had to be written and uploaded to solve a problem with the telemetry. Glitches arose in the tracking system. And there's still an occasional hiccup in the Burst Alert Network, 14 ground stations that disseminate burst localizations around the globe.

Colleagues are philosophical. “Low-cost missions are difficult,” team member John Doty points out. But things are looking up, Ricker says: “Most of the problems are now fixed.”

15. GAMMA RAY BURSTS

# Short-Lived Mysteries

1. Govert Schilling*
1. Govert Schilling is an astronomy writer in Utrecht, the Netherlands. His book Flash! The Hunt for the Biggest Explosions in the Universe will be published by Cambridge University Press in March 2002.

Although most astrophysicists agree that “long” gamma ray bursts (GRBs), those lasting more than about a second, stem from the catastrophic collapse of rapidly rotating massive stars (see main text), the origin of short bursts remains in doubt. The bursts—which make up about one-third of all observed GRBs—differ markedly from long ones not only in duration but also in having a larger proportion of high-energy gamma rays in their energy distribution. Depending on which theorist you ask, they may result either from a special kind of stellar collapse, or from the merger of two compact neutron stars or of a neutron star and a black hole.

To test these theories, astronomers need to study x-ray or optical afterglows of short bursts. Unfortunately, the wide-field x-ray cameras (WFCs) on the Italian-Dutch BeppoSAX satellite have never detected x-rays from a short burst, although they should be able to do so in principle. Nor has NASA's High-Energy Transient Explorer 2 (HETE-2), advertised as the first spacecraft designed to locate short GRBs. WFC project scientist John Heise of the Space Research Organization Netherlands in Utrecht suspects that x-rays from short bursts are harder to detect than x-rays from longer bursts because their shorter duration makes them less likely to stand out from background noise. But George Ricker, HETE-2's principal investigator, thinks his satellite is up to the job. “We observe a larger part of the sky [than BeppoSAX], and our instruments have a better time resolution,” he says. So far, though, no optical or x-ray afterglow has turned up for the one short GRB the satellite has spotted.

16. COMPUTING RESEARCH

# Microsoft Settles Down Amongst the Dons

1. Tim Burnhill*
1. Tim Burnhill is a writer in Saffron Walden, U.K.

Microsoft hopes Cambridge's academic hothouse will provide fertile soil when it opens its first lab outside the United States next week

CAMBRIDGE, U.K.—When Microsoft began setting up its first research lab outside the United States here 4 years ago, it started off in much the same way as the megacorporation itself. Microsoft was launched—famously—in a garage. “We started off borrowing some space from a friend at the university” and bought five PCs on personal credit cards, says Roger Needham, the former Cambridge University professor who heads the research lab, formally known as Microsoft Research Cambridge. In true Microsoft style, things have moved rapidly: Next week, the lab will open a new $20 million building here for its 65 researchers. The new facility will liberate staff from cramped quarters and provide room for future expansion. But its very existence begs a question: Why is a heavyweight like Microsoft, which many perceive as the epitome of 21st century capitalism, putting down roots alongside the ancient colleges in this tranquil academic town? And what does the university gain by supping with the often-vilified corporation? Microsoft and Cambridge scientists say the attractions are mutual. The Cambridge operation's parent—Microsoft Research (MSR), based in Redmond, Washington—has a tradition of supporting blue-sky research in an academic atmosphere (Science, 27 February 1998, p. 1294). By immersing themselves in the Cambridge milieu, staff members at Microsoft's nascent British outpost have recreated the Redmond atmosphere here. They and their university colleagues attend each other's seminars, while Microsoft staff members supervise Cambridge grad students and poach postdocs. Cambridge benefits, too, says Ian Leslie, head of the university's computer laboratory: “Not only does it give us places to try out ideas and to see what's going on in industry, but it also gives us access to a bigger pool of brain power.” Many outsiders look on with admiration. “They're bringing together the best computer brains,” says Christopher Strachey, director of Oxford University's computer laboratory. Some, however, view the Cambridge-Microsoft partnership with distrust and envy. Microsoft's research arm is the brainchild of former Microsoft Chief Technology Officer Nathan Myhrvold, who persuaded company chair Bill Gates to start the Redmond lab in 1991. From the outset MSR managers fostered a collegiate atmosphere rather than the pressure cooker of a modern corporate office. MSR boasts that its work “touches nearly every product the company ships, whether by transferring algorithms, consulting with product teams, or creating better developer tools.” In the mid-1990s, Microsoft's hunger for innovative software research outstripped MSR's capacity. But beefing up the Redmond shop seemed an unwise course of action. “They found that not every good researcher wanted to go to the Pacific Northwest,” says Needham. For years Myhrvold had his eye on Europe, and Cambridge seemed a logical choice. Alan Turing, widely regarded as the father of computing, was a graduate of Cambridge University, which built the world's first practical stored-program computer, the EDSAC, in 1949. And the Cambridge region became a high-tech magnet in the 1990s, earning the nickname “Silicon Fen.” Conjuring the Redmond magic fell to Needham, a computer security expert who had been at Cambridge since 1962. Needham says his instructions from Redmond were simple: “You know the sort of research Microsoft is in; go and hire the best people there are and get them to do what they are good at.” He and top lieutenants drew up a recruiting list in the group's anticipated core areas: security, communications, and integrated systems. As in Redmond, the Cambridge lab bills itself as a scientist's fantasy: Curiosity and enthusiasm, not corporate strategy, give the marching orders. “The fact that Microsoft Research labs in Cambridge have a university feel is entirely down to Roger Needham,” says Andrew Pitts, deputy head of Cambridge University's computer laboratory. Driven by the interests of individual researchers, MSR Cambridge's research scope has expanded to include fields as diverse as computer graphics and vision, speech recognition, decision theory, data mining, and natural language processing. “We are not like other companies where managers tell other people what to do,” says Microsoft's Lyndsay Williams. Although no one disputes that Microsoft is nurturing creative minds in Cambridge, the financial connections between the company and the university have raised eyebrows, particularly outside Silicon Fen. The new MSR building sits on university-owned land, next to the new$27 million computer department building—itself helped along with a \$17 million donation from the Bill and Melinda Gates Foundation. Microsoft also sponsors graduate students and funds a number of the university's research projects. The company leaves it to the academic researchers it sponsors to decide whether to put the fruit of their labor in the public domain or market it. If the latter, Microsoft insists on free access to the innovation.

“I do not believe that Microsoft's donation compromises our independence in any way,” says Pitts, who manages a project with both government and Microsoft funding. “They haven't changed the direction of what we are doing one iota.”

Although Microsoft's own researchers follow their whims, one of Needham's tasks is to nudge the lab in directions that dovetail with the work in Redmond and at a 3-year-old Microsoft lab in Beijing, China, which specializes in computer recognition and transcription of Chinese text. The nudging doesn't seem to dim the team's enthusiasm. Williams, for instance, is developing a penlike gadget that will record the words it commits to paper and transmit them, for transcription, to a pocket PC, desktop, cell phone, or tablet computer. “Here,” she says, “the idea is that you demonstrate something and get people so excited and interested that they will just go and volunteer to write a piece of software for it.” These days, that's a precious work environment indeed.

17. GEOLOGICAL SOCIETY OF AMERICA

# Life--Potential, Slow, or Long Dead

1. Richard A. Kerr

BOSTON, MASSACHUSETTS-Talk about life—not just rocks—at this month's annual meeting of the Geological Society of America was common. Lively topics included the potential for life on Mars, the pace of life below the sea floor, and how to decipher the evolution of ancient life.

## A Gusher on Mars

The Cerberus Fossae region could soon become a hot spot for planetary scientists and astrobiologists searching Mars for potentially life-sustaining water. At the meeting and in an upcoming paper, researchers describe evidence that water rushed out of the great cracks that make up 1000-kilometer-long Cerberus Fossae and surged across the landscape within the past 10 million years, carrying with it whatever may remain of any possible subsurface life, past or present. The findings make the area a prime candidate for scientific rovers scheduled to touch down in 2004.

This is not the first time Cerberus and water have been linked. In the 1980s, planetary scientists studied images of the area returned by the Viking orbiters. Some thought they saw deposits laid down by running water, whereas others thought the whole area was a dry lakebed. Still others saw nothing but fields of lava flows.

Since Mars Global Surveyor (MGS) went into orbit in 1997 and began returning sharper pictures and strikingly detailed topography, researchers have zeroed in on the true nature of Cerberus. At the meeting, planetary scientists Susan Sakimoto of the Goddard Earth Sciences and Technology Center (GEST) at NASA's Goddard Space Flight Center in Greenbelt, Maryland; Shauna Riedel of GEST; and Devon Burr of the University of Arizona (UA) in Tucson, reported how the improved imagery and topography allowed them to trace lava flows across plains and through valleys in the south, all the way back to fissures in Cerberus Fossae.

Clearly, the Cerberus region is largely lava-covered, not a lakebed. But it also shows signs of massive water flows. In an upcoming Geophysical Research Letters paper, Burr, Alfred McEwen of UA, and Sakimoto will report unambiguous signs that water flowed out of the southernmost fracture of Cerberus Fossae and southward across the plains. The channel system of Athabasca Valles, which stretches southwestward from the western Cerberus Fossae, reminds these researchers of the Channeled Scabland of the northwestern United States, an area scoured by huge floods when an ice dam holding back a glacial lake in present-day Montana busted near the end of the last ice age. In Athabasca Valles, flat-topped mesas 100 meters high, with teardrop-shaped tails hundreds of meters long, point downstream, paralleled by 10-meter-deep grooves. These features speak of catastrophic water flows reaching 1 million to 2 million cubic meters per second, the researchers say, or more than five times the flow of the Amazon River.

Burr and her colleagues suggest that the fissures, lavas, and water flows of Cerberus Fossae are the surface manifestation of deep-seated magma that could have kept the Cerberus subsurface at temperatures hospitable to life for tens of millions of years. The magma rises toward the surface, they suggest, opening vertical cracks before it. On the way it encounters water, which apparently can gush out before or after lava eruptions to create the intermingled channels and flows seen in images and topography, then it sinks into porous lavas and freezes.

And all this activity has been geologically recent. William Hartmann and Daniel Berman of the Planetary Science Institute in Tucson have estimated from the number of impact craters pocking the surface that Cerberus has been active “in the last 10 million years, maybe less,” says Hartmann. That means the magma and its heat are probably still down there, and Cerberus could surge again.

Planetary scientists will soon get a better look at Cerberus. Mars Odyssey could detect ice within a meter of the surface when it starts to make observations from orbit in February. And Athabasca Valles is one of four sites in the NASA competition for landings by one of the two Mars Exploration Rovers scheduled for launch in 2005. Winners won't be decided until this spring, but all the hot, watery hubbub coming out of Cerberus Fossae “makes Mars a lot more attractive from the point of view of astrobiology,” says Hartmann.

## Lazy Deep Life

Bacterial life is often said to be “thriving” under the most difficult of conditions: encased in rock kilometers beneath the surface, buried under millions of years' worth of ocean sediment, or floating in cloud droplets. But if Steven D'Hondt is right, life on the edge is nothing like life in the fast lane. He and fellow oceanographers Scott Rutherford and Arthur Spivack of the University of Rhode Island (URI), Narragansett Bay, reported at the meeting that, by their best estimate, sub-sea-floor bacteria—which some have claimed constitute more than half of all bacteria on the planet—live at an incredibly slow pace. “Either these things are taking very few breaths very rarely, or they're generally inactive,” said D'Hondt.

Determining how frenetic deep life is presents an experimental challenge. Rather than directly measuring the rate at which bacteria break down the organic matter buried in sediments, the Rhode Island group gauged how quickly deep sediments are consuming a key chemical: sulfate. When bacteria break down organic matter to use the energy stored in its chemical bonds, they shuttle electrons through a series of compounds. The favored choice for the last in the line is dissolved oxygen, when it's available. But oxygen disappears deeper than a few centimeters below the sea floor, so bacteria make do with the sulfate ion of seawater, chemically reducing sulfate to sulfide.

To gauge how fast bacteria are consuming sulfate and therefore how fast they're “breathing,” D'Hondt and his colleagues compiled measurements of how quickly the concentration of sulfate decreases down the length of sediment cores retrieved around the world by the Ocean Drilling Program. They then combined each sulfate concentration gradient with the rate at which sulfate can diffuse through sediment. That gave them the rate at which bacteria are consuming sulfate and therefore the rate at which they're producing energy.

Deep-sediment bacteria, it seems, are not all that energetic. Based on counts of bacterial cells in deep-sea cores, each cell on average is metabolizing at a rate one-100,000th that of the least active bacteria in near-shore sediments, the URI group reported. “The cells are either adapted to live at rates 105 times lower than anything observed,” says Spivack, “or some of what's being measured as viable cells is the remnant of dead cells.”

Most researchers are willing to believe the cell counts are OK and deep-sediment bacteria lead incredibly dull lives. “Very low rates seem fine to me,” says geochemist and astrobiologist David Des Marais of NASA's Ames Research Center at Moffett Field, California. “These folks are perking along at a very low rate.” Oceanographer John Parkes of the University of Bristol, U.K., who made the bacterial cell counts, sees ways for bacteria to get along under the stringent constraints from the sulfate analysis. For one, sulfate is probably not the only electron sink in sediments, he says. The iron of sediment minerals, for example, could steal electrons from sulfide over the millennia, which would recycle sulfide back to sulfate for reuse by bacteria and ever so slightly speed up the pace of deep life.

There may also be a way for bacteria to shut down nearly all their metabolic machinery without abandoning the vegetative life and producing spores. Although cells might on average divide only once in 100,000 years, says Parkes, it may be that a tiny fraction of them are far more active—dividing, say, once every 10 years—while the rest drop to a “maintenance” level of metabolic activity, just enough to hold body and soul together but not enough to grow. Microbiologists don't know how low bacteria could go or how they would do it, but those in deep-sea sediments seem to know how to go slow and steady enough, if not to win the race, at least to keep them in the game.

## Starving in the Caribbean

A massive collection of some 200,000 fossil shells may have solved a long-standing mystery: What caused the transformation of an entire Caribbean sea-floor community a couple of million years ago? The answer: starvation. The snails and clams of the southwestern Caribbean appear to have run short of food, and that in turn led to a makeover of bottom life into the coral reef-dominated ecosystems seen today. The success bodes well for the new ecological approach to fossil collection.

Just what happened in the tropical western Atlantic Ocean and Caribbean Sea as the world slid from the relative warmth of the Pliocene epoch into the ice ages of the Pleistocene has sparked debate for almost 2 decades. Variously described as a mass extinction or a faunal turnover involving both appearances and disappearances of species, the Plio-Pleistocene faunal event seemed to be related to the gradual closure of the seaway across what is now the Isthmus of Panama. About the time the isthmus rose and blocked the westward current linking the Atlantic and Pacific, the waters of the Caribbean seem to have cooled. Judging by the species and genera of snails and clams that disappeared, especially those preserved around Florida, some paleontologists concluded that this cooling weeded out mollusks that couldn't take the cold. Others blamed the food chain: Mollusks that ate phytoplankton (or ate other animals that did) faced lean days, thought these paleontologists, when redirected currents caused a drop in the productivity of the microscopic plants floating in the overlying waters.

Paleontologists Jonathan Todd of The Natural History Museum in London, Jeremy Jackson of the Scripps Institution of Oceanography in La Jolla, California, and colleagues didn't think either side could prove its case on the evidence they had—often museum drawers stuffed with fossil shells. “Unfortunately, museum collections are great storehouses of rare taxa,” says Todd, “but they really underrepresent common taxa.” Paleontologists tend to pick up one of every species they find, he notes, blurring the distinction between rare and common species. Yet scientists can learn far more about why faunal changes occur by studying the changing proportions of rare and dominant species than by the traditional counting of new and extinct taxa, Todd says.

In 1986, Jackson and Anthony Coates of the Smithsonian Tropical Research Institute in Balboa, Panama, began the Panama Paleontology Project to “gather the sort of data that an ecologist wants,” says Jackson, who began his career as an ecologist. “I'm a person obsessed with sampling,” he adds. So far, that has meant going to 463 sites in Panama and Costa Rica (as well as offshore dredge sites) and collecting 202,897 specimens spanning the past 12 million years. Unlike most other collections—made largely by oil geologists, says Jackson, who were not interested in ecology—these included “everything you can get from” the face of a room-size outcrop and in several bags of dirt. Classifying fossils by how they fed and what they fed on “gives us insight to the changing regional ecology through time,” says Todd.

With their new ecological insights, Todd and his colleagues think they have a much clearer picture of what happened in the Plio-Pleistocene event, at least in the southwest Caribbean. A major extinction did strike the mollusks, but it came around 2 million years ago. “That's a million years too late to be a simple, direct response to the closing of the isthmus,” says Jackson. Lots of new mollusk genera appeared in the same interval, so that, unlike in a mass extinction, the extinctions had no effect on the total diversity of molluscan life.

There was a dramatic ecological change, however. The relative abundance of predatory gastropods—snails that feed on clams and other animals—plummeted and the abundance of filter-feeding clams living off the plankton declined, but the abundance of clams and snails with other life habits and diets did not change. That pattern makes Jackson think that a drop in productivity in overlying waters, rather than a cooling, drove the southwestern Caribbean toward present-day ecosystems in which productivity is centered in coral reefs and shallow seagrass meadows.

That looks right to paleontologist Warren Allmon of the Paleontological Research Institution in Ithaca, New York. But he notes that just what caused productivity to change remains unknown, and concurrent extinctions as far north as the coast of Virginia could conceivably have a different cause. A few hundred thousand more specimens may be in order.

18. # Caveolae: A Once-Elusive Structure Gets Some Respect

1. Jean Marx

These membrane “domains” appear to play several important roles in the cell. But do they really perform all the functions attributed to them?

Cell biologists trying to figure out how cells regulate their many activities have traditionally focused on proteins and the genes that encode them. But studies in several labs over the past few years have revealed that proteins get key assists from an underappreciated cast of players. Lipids, including the oft-maligned cholesterol, apparently facilitate the normal operation of many proteins, particularly those located in cell membranes.

This conclusion derives from a new view of how membranes are organized. For years, researchers thought that proteins float more or less uniformly in the lipids that form cell membranes. But growing evidence paints another picture: Many proteins, including those involved in picking up hormonal and other signals from outside the cell and transmitting them to the interior, seem to be corralled in certain regions, or “domains,” which have a different composition from that of the surrounding membrane. These domains, known as rafts and caveolae, are particularly rich in cholesterol and another type of fatty molecule, sphingolipids.

Rafts and caveolae have been linked to several activities, including transporting materials into and through cells, and organizing the cell's numerous signal-transduction pathways into a coherent whole. Moreover, recent findings suggest that perturbations in the functions of these cellular structures may lead to a wide range of diseases—including cancer, cardiovascular illness, and a rare form of muscular dystrophy (see sidebar on p. 1864).

But even as evidence builds that rafts and caveolae play important roles, the field is divided by a controversy over which of these membrane domains does what. Some researchers ascribe far more functions to caveolae than to rafts, whereas others—probably now in the majority—think that many of the signal-transduction functions in particular reside in rafts. As Robert Parton of the University of Queensland in Brisbane, Australia, says of caveolae, “We still don't know exactly what [they do].” Resolving the issue would give cell biologists a better understanding of how cells detect, and in particular integrate, the signals that control virtually all their activities.

The uncertainty persists despite the fact that caveolae were first recognized almost half a century ago. Electron microscopist Eichi Yamada, then at the University of Washington, Seattle, and cell biologist George Palade, then at the Rockefeller Institute in New York City and now at the University of California, San Diego, independently discovered the structures in the mid-1950s as flask-shaped indentations of the cell membrane. To Yamada, the structures resembled small caves, so that's what he called them—in Latin, caveolae.

Because Palade originally detected caveolae on endothelial cells, which line the insides of blood vessels, he proposed that caveolae might pick up materials from the blood and transport them through the cells, a process known as transcytosis. Work by his team, and by cell biologists Nicoli Simionescu and Maya Simionescu at the Institute of Cellular Biology and Pathology in Bucharest, Romania, during the 1970s and 1980s, supported that idea.

The evidence wasn't airtight, however, primarily because researchers at that time couldn't follow caveolae all the way through endothelial cells. Indeed, caveolae received little attention for decades after their discovery, mainly because researchers lacked a handle for isolating the membrane structures and following their activities.

But that situation began to change in the early 1990s when two teams identified a protein, subsequently named caveolin-1, that localizes in caveolae. (One team included Richard Anderson of the University of Texas Southwestern Medical Center in Dallas and John Glenney of the University of Kentucky School of Medicine in Lexington; the other was led by Kai Simons, who was then at the European Molecular Biology Laboratory in Heidelberg, Germany, and is now at the Max Planck Institute of Molecular Cell Biology and Genetics in Dresden.)

The discovery of caveolin-1 was critical, says Michael Lisanti of the Albert Einstein College of Medicine in New York City, because it was a marker protein for the caveolae organelle. Indeed, Simons, working with Parton, showed that caveolin-1 is necessary for formation of the typical flask-shaped caveolae seen in electron micrographs. Since the protein's identification in the mid-1990s, “the field has really exploded,” Parton says.

## Common carrier?

In one prominent line of investigation, researchers are trying to pin down the role of caveolae in transporting materials into and through cells, an endeavor that should be aided by the recent creation of two strains of mice in which the gene for caveolin-1 has been inactivated, or “knocked out.” The work has been described in the past few weeks by two teams, one led by Teymuras Kurzchalia of the Max Planck Institute of Molecular Cell Biology and Genetics, and the other by Lisanti.

The cells of the knockout animals show no signs of typical flask-shaped caveolae. Even so, Lisanti says that although his team's animals have lost the ability to move some proteins through endothelial cells, they can still move others—evidence that caveolin-1 is needed, at least sometimes, for transcytosis. Jan Schnitzer of the Sidney Kimmel Cancer Center in San Diego suggests, therefore, that caveolae might be useful medically for moving drugs, particularly proteins, across tissue barriers, and his team is now exploring that possibility. (The Kurzchalia team's results appeared in the 28 September issue of Science, and those of the Lisanti group are in the 12 October issue of the Journal of Biological Chemistry.)

Evidence from another line of research has also strengthened the case for the role of caveolae in transcytosis. In as yet unpublished work, Schnitzer and his colleagues produced an antibody that specifically recognizes caveolae and then attached it to tiny gold particles, which are electron-dense and thus show up on electron micrographs. When the researchers washed this tracer antibody into rat lungs, or injected it into the animals' veins, they could see that it was picked up by the caveolae of endothelial cells and transported through them.

Caveolae may be involved in other forms of transport as well. In work begun before they found caveolin-1, the Anderson team was looking at the receptor that brings the B vitamin folic acid into cells and unexpectedly found that the folate receptor is located in caveolae and is carried into the cell by those structures. “As often happens in science, we started out thinking we were doing one thing and ended up doing something else,” Anderson says.

Another area that has been receiving attention lately is the possible role of caveolae in cholesterol transport within the cell. If such involvement does occur, disruptions in the system might contribute to atherosclerosis and heart disease.

## Platforms for efficiency

The most active research area now focuses on the possibility that caveolae are key players in signal transduction. Researchers in several labs have found that the structures contain numerous proteins involved in picking up signals from hormones, growth factors, and other molecules. These include the receptors for insulin and epidermal and platelet-derived growth factors (PDGF), for example, as well as for associated proteins—the so-called G proteins and kinase enzymes, among others—that detect when receptors have been activated and then alert other molecules in the cell interior accordingly.

These discoveries led some researchers, including Lisanti and Anderson, to propose that caveolae serve as platforms for integrating the cell's signal-transduction machinery. They reason that it's inefficient for randomly distributed proteins to somehow piece together an ad hoc pathway in response to each incoming signal; instead, pathways are likely to be preassembled. This would also facilitate the “cross talk” that often occurs between different pathways, as the components for such interaction could be located in the same caveolae. But there is a “critical question,” Schnitzer says. “Is the interaction physiologically significant? Molecules can interact in the test tube but not [necessarily] in the cell.”

In some cases, particularly one involving an enzyme called endothelial nitric oxide synthase (eNOS), there is good agreement that the significance test can be met. As its name implies, eNOS makes nitric oxide in endothelial cells in response to a variety of stimuli, including hormones or neurotransmitters (such as bradykinin and acetylcholine) and the stress exerted on blood vessels by high blood pressure. Nitric oxide helps counteract that stress by relaxing the blood vessels, allowing easier blood flow; it has other effects as well, such as fostering new blood-vessel formation and wound-healing. Several researchers, including Anderson, Lisanti, and Schnitzer, and also Thomas Michel of Brigham and Women's Hospital in Boston and William Sessa of Yale University School of Medicine, have shown that eNOS is bound to caveolin-1 in endothelial-cell caveolae and that this binding keeps eNOS inactive.

For example, in work reported in the December 2000 issue of Nature Medicine, Sessa and his colleagues used a fruit fly protein to carry the segment of caveolin-1 that binds eNOS into rings of mouse aorta maintained in culture. When the researchers then added acetylcholine to the cultures, they found that the aortic rings produced much less nitric oxide than control tissue did and also relaxed less. Activation of eNOS had been blocked. In addition, when injected into mice the caveolin-1 peptide blocked inflammation induced in the animals' ears by mustard oil, a result that indicates that it could have medical benefits.

Researchers have found that signals activate eNOS by first causing its dissociation from caveolin-1. This takes several steps, the first being an increase in the number of intracellular calcium ions. These bind to and activate a protein called calmodulin, which in turn binds to eNOS, causing it to release its grip on caveolin-1 and become active. “The other thing that's interesting,” Michel says, “is that many of the receptors that activate eNOS are also in caveolae.” This suggests the presence of preassembled pathways and the potential for cross talk.

## Tumor suppressor

Caveolae and caveolin-1 also appear to be involved in signaling pathways that control cell growth—a function that suggests a link to cancer. “Almost all normal cells have caveolin-1,” Lisanti says, “but when they are transformed by active [cancer-causing] oncogenes, caveolin-1 expression is down-regulated.” The gene's expression is also low in cells derived from human cancers, including breast cancer. When researchers, including Schnitzer and Lisanti, put an active caveolin-1 gene back into the cancer cells, however, it suppresses their abnormal growth and other cancerous behavior.

Lisanti suggests that caveolin-1 works in this situation, much as it does with eNOS, by binding to and inhibiting proteins that drive cell division. The Anderson team, among others, has shown that all the proteins needed for cells to respond to PDGF—including the PDGF receptor and certain kinase enzymes that transmit growth signals to the cell interior—are located in caveolae. Lisanti adds that caveolin-1 binds to kinases in their catalytically active sites, thereby blocking their activity.

The Lisanti team also found evidence that caveolin-1 is a suppressor of human tumors. Two years ago, the researchers mapped the caveolin-1 gene and the gene for caveolin-2, a closely related protein also found in caveolae, to a region of the genome that is often deleted in human cancers—a typical sign that the region contains a tumor suppressor.

But perhaps most intriguing, a team led by Kazuhiko Hayashi of the Nagoya School of Medicine in Japan reported in the 15 March issue of Cancer Research that the caveolin-1 gene was mutated in 16% of the 92 human breast cancers they examined. What's more, the mutation hit the particular segment of the protein that binds to other proteins—a location where it could interfere with its binding ability, thus allowing kinases or other proteins involved in growth to be active when they shouldn't.

## A domain of its own?

Work on the knockout mice has supported the idea that increased cell proliferation may be expected when caveolin-1 is absent. In living animals, this was manifested primarily by extra layers of cells in the lungs. Lisanti and his colleagues also found that embryonic fibroblasts from the knockout mice proliferated twice as fast as those from controls. Exactly why this happened is currently unclear, however, as it did not seem to involve the kinase activation previously observed in other cultured cells.

The eNOS work also received support from the caveolin-1 knockouts, as the animals clearly suffered from the types of defects that would be produced by the enzyme if it were overactive. For example, aortic rings from the animals showed a much greater relaxation response to acetylcholine than rings from unaltered animals. “The one phenotype I would have predicted is exactly the one they have,” says Michel.

Although the knockouts support some of the ideas about the function of caveolae, several researchers have expressed surprise that the animals survived at all, given the large number of functions, particularly in the area of signal transduction, attributed to the structures. The knockout “was a real surprise to the field. We imagined it would be lethal to the embryo,” Parton says. He and others suggest that many of the processes thought to be going on in caveolae are going on elsewhere, such as in lipid rafts.

Schnitzer says, for example, that his team found some signaling proteins in caveolae and others in rafts. And Parton adds that others may be in neither. In fact, he maintains that “for most of the signaling proteins originally claimed to be in caveolae, there is now conflicting evidence.”

Indeed, there is a major controversy in the field over both the nature of caveolae and their functions. Rafts were discovered much more recently, largely as a result of work by the Simons team in the late 1980s and 1990s. Like caveolae, they are thought to be rich in cholesterol and sphingolipids, but are flat and do not contain caveolin. Most researchers in the field now think that caveolae are a subset of rafts, formed by acquisition of caveolin-1. That idea also got a boost from the Kurzchalia and Lisanti caveolin-1 knockouts, which totally lack flask-shaped caveolae.

But there are some dissenters from this view, particularly Anderson. For one, he maintains that caveolae can have any shape. To support this, he cites experiments in which his group blocked cell uptake of folate and found that the flask-shaped caveolae were no longer visible. Yet, the researchers were still able to recover them from membranes. “The fact that flask-shaped caveolae disappear doesn't mean they aren't there,” Anderson says.

He even argues that caveolae don't have to contain caveolin, suggesting that some other protein in the knockouts could be pinch-hitting. “To me,” he says, “[the knockouts] are strong evidence that caveolin isn't the only player.”

There's currently little evidence to support the idea that caveolae can exist without caveolin-1, however, and most researchers remain skeptical. Simons, for one, says he wants to see both a flask shape and the presence of caveolin-1 before he will accept a structure as a “little cave.”

What it will take to resolve the controversy is unclear, given how hard it is to isolate membrane structures. But until cell biologists do, they won't have a clear view of how signals get into the cell. In that spirit, Anderson did say one thing with which everyone would agree: “We still have a lot of work to do.”

19. # Caveolin-3 Helps Build Muscles

1. Jean Marx

The cell-membrane structures called caveolae and their associated proteins—the caveolins—play a role not only in transporting materials and signals into cells (see main text) but in building the body's muscle cells. Indeed, defects in muscle caveolin have been linked to a rare form of muscular dystrophy.

Muscle caveolin, also known as caveolin-3, was originally identified in the mid-1990s by two teams, one led by Michael Lisanti at the Albert Einstein College of Medicine in New York City and the other by Robert Parton at the University of Queensland in Brisbane, Australia. The Albert Einstein group had been searching the gene databases for relatives of the one caveolin gene known at the time, Lisanti recalls. The researchers came up with two, and when they looked to see where those genes are active, the one making caveolin-3 turned out to be highly expressed in all three types of muscle—skeletal, cardiac, and smooth—but not in other cell types.

The researchers also began picking up hints that caveolin-3 might be involved in muscle formation. For example, Parton and his colleagues found in 1997 that during development the protein associates temporarily with one of the structural features of muscle cells, the so-called transverse tubules, which form at the ends of the repeating units that make up the cell's contractile machinery. Meanwhile, Lisanti's group found evidence that caveolin-3 associates with dystrophin, a protein mutated in Duchenne muscular dystrophy.

Such findings raised the possibility that caveolin-3 mutations might also contribute to muscular dystrophy; and in 1998 Lisanti teamed up with Carlo Minetti's group at the University of Genoa, Italy, to investigate. The researchers found mutations in the protein's gene in affected members of two families with histories of limb-girdle muscular dystrophy, a mild form of the disease characterized by overdevelopment of the calf muscles and moderate weakness of the muscles in and near the body trunk. At about the same time, Louis Kunkel's group at Children's Hospital in Boston linked changes in the gene to muscular dystrophy cases with similar symptoms.

Although some of the apparent mutations found in caveolin-3 have turned out to be harmless genetic variations, others cause the protein to misfold, leading to its destruction by the cell, Lisanti says. Additional evidence that such loss can cause limb-girdle muscular dystrophy came when his group and that of Yasuko Hagiwara at the National Center of Neurology and Psychiatry in Kodaira, Japan, inactivated the gene in mice. The researchers found that the animals experience muscle degeneration similar to that of human patients. “Now we can conclusively say that loss of caveolin-3 causes the muscular dystrophy,” Lisanti says.

Too much caveolin-3, however, can disrupt muscle structure as well. Lisanti and co-workers found that when they genetically engineered mice to overproduce the protein, those animals developed pathological changes much like those of Duchenne muscular dystrophy. This may occur because excess caveolin-3 interferes with dystrophin's ability to reach its normal location in the cell membrane.