# News this Week

Science  09 Apr 2004:
Vol. 304, Issue 5668, pp. 184
1. SCIENCE POLITICS

# White House Rebuts Charges It Has Politicized Science

1. David Malakoff

The White House came out swinging last week against critics who have accused the Bush Administration of systematically manipulating science to advance its political agenda. In a sharply worded rebuttal, White House science adviser John Marburger attempts to turn the tables by accusing the accusers of “errors, distortions, and misunderstandings.”

The critics, however, aren't impressed. “I don't detect that [the White House] has acknowledged any problem … and that's a problem,” says Lewis Branscomb of Harvard University in Cambridge, Massachusetts.

Branscomb, who led the National Bureau of Standards (the predecessor of the National Institute of Standards and Technology) under President Richard Nixon, is one of 60 prominent scientists who in February signed a statement accusing the Bush Administration of suppressing or ignoring technical findings that don't square with its political views (Science, 5 March, p. 1446). The statement accompanied a report by the Union of Concerned Scientists (UCS), a Cambridge-based advocacy group, that listed more than 20 incidents in which it claimed that the Administration had politicized science, including stacking advisory panels with ideological allies, doctoring reports that didn't support its policies, and muzzling government scientists. Marburger, who heads the White House's Office of Science and Technology Policy (OSTP), promised Congress a point-by-point response, and another senior official predicted that it would “shred” every UCS allegation.

The 20-page rebuttal*—released on 2 April—falls short of that high standard, although it does expose errors and incomplete explanations in the UCS report. (Last week the group quietly issued a revised version correcting and clarifying some points.) The OSTP analysis points out that the bases for government actions are rarely clear-cut, and Marburger states in the introduction that scientists shouldn't expect to have the final word in policy battles. “Even when the science is clear—and often it is not—it is but one input into the policy process,” he writes. But “in this Administration, science strongly informs policy.”

Marburger has publicly rebutted many of the UCS charges before. For example, he says that although a discussion of climate change was removed from an Environmental Protection Agency (EPA) report, the deletion occurred because the subject was covered in a White House report on climate change research—a report that was strongly influenced by advice the Administration requested from the National Academies. The OSTP report also plows some new ground, however. For instance:

• OSTP concedes that a preamble to proposed EPA regulations on mercury emissions included language from industry memos. “Such direct use of submitted memoranda should not have occurred,” the report says. But the incident “had nothing to do with the integrity of the science used by EPA” in the regulations themselves.

• Both sides agree that a former U.S. Department of Agriculture (USDA) researcher was blocked from discussing controversial findings that hog farms can produce airborne bacteria that are resistant to antibiotics, a potential health hazard. But although UCS says microbiologist James Zahn was muzzled by USDA on “no fewer than 11 occasions,” OSTP says the right number is five. Zahn “did not have any scientific data or expertise in the scientific areas in question,” the report says, adding that he was allowed to speak on two other occasions.

But Zahn, now in private industry, says 11 is the correct number. A Republican who says he voted for President George W. Bush, Zahn says no one from the White House contacted him to check their claims before “questioning my credibility and expertise.”

• OSTP answers UCS's claim that the Administration applies a political “litmus test” when selecting the members of more than 600 scientific advisory bodies by noting that there are “numerous cases of Democrats appointed to panels at all levels.” Marburger himself, the report adds, is “a lifelong Democrat.” It dismisses as “rare events” the Administration's controversial appointments to advisory panels dealing with lead exposure and ergonomics issues. And OSTP claims that one researcher UCS said had been blackballed was actually named to the ergonomics panel.

• A UCS accusation that the State Department abandoned a science advisory panel on arms control is inaccurate, OSTP says. The panel's charter had expired, and officials have since revitalized it and are currently recruiting new members. But that version of events “is less than a half-truth,” says Richard Garwin, a retired IBM executive who chaired the panel. The report doesn't note that the agency waited more than 2 years to revive the panel, he says. “This is not a matter of ignoring scientific advice but of not seeking it.”

• OSTP says UCS distorted or ignored “vital facts” in claiming that the Administration has weakened enforcement of the Endangered Species Act—a point the group at least partly concedes. Gridlock in listing new endangered species is the result of longstanding budget and legal problems, not flawed science, the two sides appear to agree. And UCS says that OSTP is at least partly correct in attributing controversial changes in the makeup of a scientific panel reviewing conservation efforts along the Missouri River to legal deadlines, not politics.

Marburger hopes the report “will allay the concerns of the scientists who signed the UCS statement.” He'll find out soon: He expects to discuss the matter this month with a group of signers attending the annual meeting of the National Academy of Sciences. And the Senate Commerce, Science, and Transportation Committee, which last month cancelled a hearing on the UCS report, says it still plans to look into the issue.

2. GENETICS

# Two New Asthma Genes Uncovered

1. Jennifer Couzin

A painstaking, decade-long hunt spanning two continents has led a team of Finnish scientists to two new asthma genes. Geneticists don't yet know what role the genes play in asthma, which afflicts about 16 million people in the United States alone. But scientists say that the work, described on page 300, has uncovered some of the most compelling genetic associations yet for the disease.

Although researchers had previously linked three other genes to asthma, those studies involved fewer individuals and mostly found a relatively weak genetic effect in conferring susceptibility. Not only are the current associations strong, but one of the new genes may be a good target for novel antiasthma drugs. “One of the beauties of this is that they came up with a drug target,” says Jack Elias, a pulmonary specialist at Yale University in New Haven, Connecticut.

The work also appears to validate the technique of using geographically isolated populations, called founder populations, to find genes for common complex diseases such as cancer and diabetes. These diseases are tough to trace genetically because they are apparently caused by combinations of genes, along with environmental triggers. “People have said, ‘Oh, you need thousands of patients, and it's so difficult’” to identify genes for asthma and other common disorders, says Scott Weiss, an asthma geneticist at Harvard Medical School in Boston. But the discovery of these genes “shows that you can do this,” he notes.

The first clue to the genes' existence came in the mid-1990s when molecular geneticist Juha Kere of the University of Helsinki and his colleagues linked increased susceptibility to asthma to a massive, 20-million-base swath of chromosome 7. To find the culprit genes, Kere, Helsinki colleague Tarja Laitinen, and others began collecting blood samples from both healthy and asthmatic individuals in families afflicted with asthma—a total of nearly 900 samples. The Finns, he and others reasoned, were ideal for this sort of study: Geographically isolated and genetically fairly uniform, their disease genes might be easier to spot than those in more diverse populations.

By sequencing parts of the suspicious stretch of chromosome 7, Kere, now at the Karolinska Institute in Stockholm, Sweden, and his team found that both the healthy and asthmatic individuals, all of whom lived in the same Finnish province near the Russian border, carried one of seven possible variant DNA sequences, known as haplotypes, in that chromosomal region. Certain haplotypes, many believe, predispose to disease. In the Finnish case, this theory apparently held: Three of the seven haplotypes Kere's group found were associated with asthma, and more than half the asthma patients carried them as opposed to just a third of the healthy people.

To make certain these haplotypes weren't unique to Finns, Kere teamed up with Thomas Hudson, a haplotype expert at McGill University in Montreal, Canada. Hudson had collected nearly 400 DNA samples from asthmatics and nonasthmatics in Quebec. Close inspection revealed the same seven haplotypes in this group as in the Finns and confirmed that three are strongly linked to asthma susceptibility. The hazardous haplotypes appear to boost asthma risk by up to 2.5 times normal.

Further comparisons of the haplotypes helped Kere's team uncover two genes with alterations unique to the high-risk haplotypes. One of these is a complete mystery. It doesn't appear to code for a protein, and the scientists currently don't know what to make of it.

But the other, which the group labeled GPRA, has generated ripples of excitement: It produces a so-called G protein-coupled receptor, which belongs to a class of molecules that have proved their usefulness as drug targets. Kere and his colleagues have launched a company, GeneOS Ltd., and filed for patents on the GPRA protein, in the hope of producing a drug to modulate its activity.

The researchers still need to pin down how a mutated GPRA gene increases asthma susceptibility and whether it's widespread in populations outside Quebec and Finland. But there are indications, Kere says, that its role may not be limited to asthmatics who carry the mutant gene. His team tested bronchial tissue from eight asthmatics with unknown haplotypes; all showed high concentrations of the protein, even though only half would be expected to carry the mutant gene. Tissues from 10 healthy volunteers did not show elevated amounts of GPRA.

There are also hints that the GPRA gene may be linked to allergies. Human tissue samples revealed that the GPRA protein is present in the air passages of normal lungs, although at much lower concentrations than in the lungs of asthma patients, as well as in the outer layers of the skin and the gut lining—all sites commonly associated with allergic reactions.

3. SCHOLARLY PUBLISHING

# U.S. Eases the Squeeze on 'Sanctioned' Authors

1. Yudhijit Bhattacharjee*
1. With reporting by Jeffrey Mervis.

The Treasury Department has reversed a controversial ruling that would have required U.S. scholarly journals to obtain the government's permission to edit papers from countries under a U.S. trade embargo. A new policy directive, spelled out in a 2 April letter to the Institute of Electrical and Electronics Engineers (IEEE), is expected to resolve months of confusion about how to handle manuscripts from those countries.

In 2002, citing uncertainties in trade regulations, IEEE decided to exclude papers from scientists in Iran, Cuba, Iraq, Libya, and Sudan, pending a ruling from the Treasury Department's Office of Foreign Assets Control (OFAC) on the government's position (Science, 19 September 2003, p. 1646). On 30 September OFAC said that a license would be required to perform any editing of manuscripts from Iran (and, by extension, other sanctioned countries) (Science, 10 October 2003, p. 210). But last week, after boning up on the scientific peer-review process, OFAC cleared the way for publishers to follow their normal procedures in editing papers from anywhere in the world.

“Scientific communities in sanctioned countries may publish their works in U.S. scholarly journals,” says OFAC Director Richard Newcomb. In his letter to Nelson Dong, a Seattle attorney representing IEEE, Newcomb explains that IEEE is “not constrained” from having reviewers propose substantive improvements to the manuscript nor from making editorial changes before publication.

The new ruling refers to a 1988 amendment to the Trading With the Enemy Act that exempts “informational materials.” A senior Treasury official explained this week that “peer review and copy editing are permitted because they are exempt under the Berman Amendment.” A spokesperson for Representative Howard Berman (D-CA) called it “a positive step” but warned that “we need to look carefully at how it's applied to other things that publishers do.”

OFAC's decision appears to vindicate IEEE; critics had warned that its query could force OFAC to act on a matter that it might otherwise ignore. A number of organizations refused to alter their procedures, citing the First Amendment clause on free speech, whereas the American Chemical Society temporarily suspended its review of manuscripts by authors from the sanctioned countries. A few, including the American Society for Microbiology, applied for a license. Last month Science reported that OFAC was planning to relax its policy (Science, 19 March, p. 1742).

“Perhaps we invited some of [the controversy], so it was incumbent upon us to get a resolution,” says Arthur Winston, president of IEEE, which convened a February meeting with scientific publishers on the issue and met last month with OFAC officials. Since the September ruling, IEEE has published manuscripts from the embargoed countries without making any stylistic or editorial changes. “Effective immediately,” Winston said, “IEEE is returning to its normal publishing process for all authors.”

4. BIOETHICS

# White House Panel Issues Its Final Word on Reproductive Technology

1. Constance Holden

The President's Council on Bioethics has come up with an idea that it hopes will break the stalemate in Congress over anticloning legislation: a measure that doesn't have the word “cloning” in it. The council unveiled this solution last week in its third major report, Reproduction and Responsibility: The Regulation of New Biotechnologies, presented at its meeting in suburban Washington, D.C. But a Catholic Church leader says the council is just playing with words.

The report (at http://www.bioethics.gov/) attempts to set ethical parameters for the booming assisted-reproduction industry, which, noted council chair Leon Kass, “is the gateway to all the new technologies,” from preimplantation diagnosis to genetic manipulation. In addition to proposing government-led research, data collection, and monitoring, the council would prohibit fertilization of a human egg with animal sperm or vice versa, ban research on embryos more than 2 weeks after fertilization, and prohibit attempts to conceive a child from fetal tissues. “No child should be able to say [its] mother or father was a fetus, an embryo, or a stem cell,” said Kass.

To that end, the council calls for a law against “attempts to conceive a child by any means other than the union of egg and sperm.” The council reasoned that some lawmakers who oppose bills to ban reproductive (but not research) cloning object that such bills implicitly mandate the destruction of cloned embryos. So they might object less to a law that makes no mention of clones at all—an approach that Dartmouth College neuroscientist Michael Gazzaniga, a council member, called “sort of a cloning version of ‘Don't ask, don't tell.’”

Will it fly on Capitol Hill? William Doerflinger, spokesperson for the U.S. Conference of Catholic Bishops, says no: “It doesn't ban cloning.” Anthony Mazzaschi of the Association of American Medical Colleges agrees, saying, “I don't see how the Kass report will change” the standoff. Anticloning legislation has been stalled in the Senate because those who want to ban all forms, including research cloning, lack the votes to overcome procedural obstacles.

The meeting marked the first time the newly reconstituted council has sat down following the dismissals of cell biologist Elizabeth Blackburn of the University of California, San Francisco, and ethicist William May of Southern Methodist University in Dallas, Texas (Science, 5 March, p. 1447). “The council has become imbalanced,” declared council member Daniel Foster, a physician at the University of Texas Southwestern Medical Center in Dallas, who said scientists are unhappy about it. Indeed, Gazzaniga was the only member to urge the council to push for both the creation of new federally supported human embryonic stem cell lines and research cloning.

Although scientists say the council has a conservative tilt, most observers give Kass good marks for fairness. Early drafts of the reproduction report carried ideologically laden terminology—referring, for example, to embryos as “nascent human life,” says Pamela Madsen of the New York-based American Infertility Association. But, she says, “all that ideological language is gone.” She says the council also agreed to drop a recommendation for a mandatory registry of all children born through in vitro fertilization. Now, she says, “I think overall the council got the meat of their work right. And they really listened to stakeholders.”

5. DRUG SAFETY

# IOM Recommends Action on Dietary Supplements

Dietary supplements can be bought off the shelf, purportedly to treat almost any human ailment, but some have hidden dangers. Ephedra, for example, flared on the national radar screen last year when a young pitcher for the Baltimore Orioles died after using this “fat-burning” compound. The U.S. Food and Drug Administration (FDA) has banned sales of ephedra, starting next week, but this is the first time it's taken such a tough step. The main holdup, concludes a new report* from the Institute of Medicine (IOM), is that FDA is legally hamstrung in its efforts to determine the safety of such substances. The report suggests a way to evaluate supplements but urges that the law should be changed to improve the process.

About 4000 unregulated products were on the U.S. market when Congress passed the Dietary Supplement Health and Education Act (DSHEA) in 1994. Today FDA estimates that more than 29,000 are available, with sales topping $18 billion a year. The law empowered FDA to regulate supplements if they pose a “significant or unreasonable risk of illness” but also made the task tricky. Supplement manufacturers don't have to provide FDA with any safety data before marketing products or report adverse effects later. “In many cases there are not a lot of data available to address safety,” says Barbara Schneeman of the University of California, Davis, who chaired the panel. In 2000, FDA asked IOM how the agency could evaluate the safety of such compounds under the current law. The bulk of the report describes a basic strategy: When FDA learns of potential trouble—say, from consumer groups or health professionals—it should scour the literature, draft a safety monograph, consult an expert panel, and then take appropriate action. That's tougher than it sounds. For starters, formulations can vary, and people take multiple supplements, sometimes mixing them with prescription drugs. The framework would be much more effective, the panel concludes, if FDA had more data. To that end, the panel recommends changes to the law: Manufacturers should report all available safety data before a product hits the shelves; they should report any serious adverse events after going to market; and any change in formulation or processing should be considered a new ingredient. “This is a first step in trying to remedy some of the tremendous loopholes” in DSHEA, says Kenneth Kaiten, director of the Tufts Center for the Study of Drug Development in Boston. A House bill to expand FDA's authority is currently stalled in conference, as is a Senate bill, and Kaiten says action is unlikely: “My guess is there won't be any legislation for a while.” 6. ARCHAEOLOGY # Burials in Cyprus Suggest Cats Were Ancient Pets 1. Elizabeth Pennisi Not quite man's best friend—unless you happen to be a cat lover—those purring felines that warm our laps have a longer history as pets than many have thought. As reported on page 259, a newly discovered cat skeleton unearthed in southern Cyprus has pushed back the date of our first cat companions by more than 5000 years. The complete cat skeleton was closely associated with a 9500-year-old human burial, suggesting that the 8-month-old feline was tame, says lead author Jean-Denis Vigne, an archaeologist at the Natural History Museum in Paris. Zooarchaeologist Melinda Zeder of the Smithsonian National Museum of Natural History in Washington, D.C., who was not involved with the study, agrees. “In lieu of finding a bell around its neck, this is about as solid evidence as one can have that cats held a special place in the lives and afterlives of residents of this site,” she says. Until this find, the oldest evidence of tame cats came from Egypt, where 4000-year-old remains and paintings document cats' place of honor in that culture. For the past decade Vigne and his colleagues have been analyzing animal bones from an archaeological dig at a town called Shillourokambos in Cyprus. The site had provided detailed evidence of the island's first human visitors, Neolithic cereal farmers who arrived as early as 10,000 years ago, probably from Turkey. “These early inhabitants seemed to have come with a veritable Noah's ark,” notes Zeder, as over the years Vigne and others had found signs of dogs, cattle, goats, sheep, foxes, pigs, and deer brought by the farmers. Vigne had also found cat bones—a humerus and three jawbones—but although these remains showed that cats were present, the bones provided few clues to the felines' relationship with humans. Then in 2001, a colleague working on the 9500-year-old burial of a 30-year-old human found the remains of a cat. The two sets of bones were less than a half-meter apart, buried at the same depth and in the same sediment, with the same degree of preservation, strongly suggesting that they were buried together. The cat bones were articulated, indicating that the animal was intentionally buried and thus protected from scavengers. “We immediately realized that this was an exceptional discovery,” says Vigne, who excavated the cat skeleton. Because there was no evidence of butchery, “the only possibility is that this cat has been buried with a human,” perhaps to accompany its owner to the hereafter. Vigne argues that it was very likely a tame cat, because wild animals, if buried at all at this time, seem to be represented only by isolated bones and antlers. Vigne was not able to date the cat directly because its bones lacked collagen, but he says that the burial circumstances show that it is the same age as the human. The cat was large compared to modern cats, which is typical of felines of that era. Thus the new find puts cat domestication about 3000 years after dogs became man's best friend and very close to the time that wheat and sheep were domesticated. These observations are quite impressive, says Zeder, but not bulletproof because the cat wasn't dated directly. But anthropologist Ofer Bar-Josef of Harvard University in Cambridge, Massachusetts, is convinced that the result will be widely accepted. Indeed, he argues that the cat and other grave offerings make a larger point, suggesting that at least some of the ancient Cypriots warranted special treatment after death. The burials “indicate that the colonization [of Cyprus] was probably led by chiefs, shamans, or other elite members,” he says. And that implies that even at this point in human history, when farming was just coming into its own, a structured society existed. Cat lovers will likely agree. What could be more civilized than being greeted each evening with a big meow? 7. FRENCH PROTEST # Reshuffled Government Hoists the White Flag 1. Barbara Casassus* 1. Barbara Casassus is a writer based in Paris. PARIS—Licking its wounds after a drubbing in regional elections, the ruling Conservative government is seeking to recapture the affections of at least one segment of the public: its aggrieved research community. Earlier this week the government was expected to announce the restoration of 550 research posts and the creation of 1000 university jobs, gestures that are expected to quell a months-long protest movement that had spread across the country and grown increasingly bitter. “Confidence has partially been restored between the government and the scientific community,” says endocrinologist Étienne-Émile Baulieu, president of the Academy of Sciences. In early January the protesters issued three key demands: that the government come up with €200 million owed to science agencies from past budgets, organize a conference on the future of French science, and reinstate 550 permanent jobs in government labs that had been converted this year to 3- to 5-year contracts. Last month they upped the ante, calling for the creation of hundreds of university posts. Although the government agreed to “unfreeze” €294 million from the 2002 and 2003 budgets and reinstate 120 full-time civil service jobs, more than 3500 research chiefs vowed to stop performing administrative duties and late last month threatened to begin shutting down labs (Science, 19 March, p. 1749). But a Cabinet shakeup last week after the electoral hammering appears to have set the stage for ending the standoff. In a 1 April television interview, President Jacques Chirac acknowledged that R&D funding is insufficient and that researcher malaise is justified. He said he had asked the government to reexamine the jobs issue and vowed that the problem “will be settled.” Moving swiftly to turn words into action, the new education and research minister, François Fillon, and junior research minister François d'Aubert have held a series of talks with protest leaders and other senior science figures. Ironically, many French researchers heap much of the blame for their current plight on Fillon and d'Aubert. The last time they were in charge of the research portfolio—Fillon from 1993 to 1995 and d'Aubert from 1995 to 1997—“marked the beginning of the decline in French research,” claims the SNCS, a leading research union. D'Aubert's predecessor, Claudie Haigneré, was widely seen as ineffectual during the crisis; she is now European Affairs Minister in the reshuffled cabinet. It seems that bygones will be bygones: Researchers credit Fillon with moving swiftly to take a hands-on interest in research, a duty that researchers have accused his predecessor, Luc Ferry, of shirking. “This is very good news,” says Alain Trautmann, a cell biologist at the Cochin Institute in Paris and spokesperson for the protesters. He emerged from a meeting with d'Aubert on 5 April grinning broadly and claiming that the government will make significant concessions on jobs. “There is a change in tone [and] a very clear will for dialogue,” Trautmann told Science. He says that if the announcement is delivered as promised, the researchers' resignations will be withdrawn. 8. SPANISH RESEARCH # Win-Win Scenario: More Cash, Fewer Frigates 1. Xavier Bosch* 1. Xavier Bosch is a science writer based in Barcelona. BARCELONA—In a country consumed by the hunt for terrorists, scientific reforms are not the stuff of headlines. But that hasn't deterred Spain's incoming government from putting together an ambitious new agenda for science. Science has learned that the Socialists, set to take power late next week, plan to double the country's €2 billion R&D budget over the next 4 years and create an agency that, in the spirit of the U.S. National Science Foundation, will fund projects primarily according to merit. The reforms are “great news,” says Enric Banda, former director-general of the European Science Foundation and head of the Catalan Research Foundation in Barcelona. He especially welcomes the new funding body, which is expected to control roughly 90% of Spain's R&D budget: “Research has to be evaluated and financed according to good practices.” The fine details are still being hashed out, but Jaime Lissavetzky, spokesperson for the Socialists in the Congress's Scientific and Technological Commission, says that the new government intends to increase Spain's R&D budget by 25% a year until the next election in 2008. In a departure from the outgoing administration, public R&D spending will no longer include funds for military construction—a kind of budgetary voodoo that had infuriated many researchers. In 1999 alone, for example, 54% of the overall R&D budget was spent on the military, largely for the construction of frigates, tanks, and jetfighters, claims Jordi Armadans, president of the Foundation for Peace, a nonprofit organization that launched a campaign in 2003 to persuade scientists to forswear military research. The 25% increase will apply to civilian and military R&D. Yet the prospect of prosperity hasn't made everyone happy. “The main problem of Spanish science is not a lack of money but inbreeding and influence peddling,” asserts Antonio Ferriz-Mas, an astrophysicist at the University of Vigo. The solution, he says, should be “first more reforms and then more money.” The new research funding agency should ameliorate some concerns. The body will grant funds according to quality, peer review, flexibility, and transparency, says Lissavetzky, and will be run largely by scientists. Banda and other observers say they will press to ensure that the agency is established in a way that gives it substantial independence from government decision-makers. 9. CELL THERAPY # Renovating the Heart 1. Jennifer Couzin, 2. Gretchen Vogel* 1. With reporting by Constance Holden. Inspired by reports that cell infusions can heal animal hearts, cardiologists are rapidly moving to test the idea in humans. Even believers can't explain how or why it might work Cardiologist Richard Schatz is tired of watching his patients die. They face daily anguish—as do millions of other people—because their hearts are pocked with dead muscle, undersupplied by blood, and gradually losing strength. Heart transplants can save a fraction of these patients, about 2000 in the United States each year. The rest get along on an imperfect mix of drugs, dietary restriction, and exercise. Some respond; many do not, joining a toll that makes heart disease the number one killer in developed countries. But Schatz does not accept this as inevitable. Based at the Scripps Clinic in La Jolla, California, he's one of a growing number of cardiologists embracing an experimental approach to attacking heart disease, called cell therapy. It involves taking cells, usually from the patient's own body, and delivering them to the ailing heart. Interest is soaring, with more than a dozen clinical studies under way worldwide. The trials, which got started in 2000 in France and 2001 in Germany, include a hodgepodge of techniques, cell types, and patients. In some, doctors shoot cells straight into the heart; in others, they administer potentially risky drugs that force bone marrow to churn stem cells out into the bloodstream. The results so far have been mixed and hard to interpret, but clinicians say they're seeing benefits. They can't explain why, however: Cardiologists began cell therapy thinking that transplanted cells would grow new heart muscle, but this early notion does not appear to be panning out. Some researchers—particularly in the United States—worry about what could transpire if these trials outpace our understanding of the biology. Their worst nightmare is to repeat the errors of gene therapy, such as a trial in which an 18-year-old died after receiving an infusion of viruses carrying “repair genes.” It was a punishing setback for the field. “We all would like to think that [cell therapy] can't be dangerous,” says Timothy Kamp, a cardiologist at the University of Wisconsin, Madison, studying embryonic stem cells. But, he adds, “there clearly can be unanticipated results.” No potential showstoppers have come to light yet. But there have been warning signs. Four subjects in a French cell-therapy trial developed serious arrhythmias, a common problem among patients with heart failure, and Korean volunteers suffered a renarrowing of repaired arteries, forcing an early end to one study. Physicians, researchers, and regulators are now wrestling with how fast to push cell therapy forward and how to design trials with the best shot at success. Last month, the U.S. Food and Drug Administration (FDA) invited experts from around the world to discuss the technique's promises and pitfalls. The agency, which so far has allowed only a handful of U.S. trials, is holding the reins tightly. Some researchers grumble that this is leaving them behind as their counterparts in Europe and Asia surge ahead. Stalling to let knowledge of biology catch up to medical technology, some point out, could also have serious consequences. “If we wait 5 years, we will have lost the chance to treat hundreds of thousands of patients,” says John Martin of University College London. Many of his colleagues on both sides of the Atlantic agree. But there's no denying that the hoped-for gains will come with new risks. “This is a chance to potentially make leaps and bounds in medical therapy,” says Samuel Dudley, a cardiologist at Emory University in Atlanta. “On the other hand, there are a huge number of questions that we've left behind, because it's so easy to see the possibility” of saving lives. ## Stem cell buzz Hints that failing hearts might benefit from new cells emerged from the excitement sparked by isolating highly versatile stem cells from embryos. Cardiac trials have not yet used embryonic stem cells, nor have they attempted transplanting cells from one patient to another. But efforts to exploit the versatility of a patient's own cells got a boost from tantalizing—although still disputed—reports that immature blood cells could become brain cells and that bone marrow cells could form liver (Science, 8 June 2001, p. 1820). As these headline-grabbing reports were appearing, a team of scientists led by Donald Orlic at the National Human Genome Research Institute in Bethesda, Maryland, published a surprising observation: When the researchers injected bone marrow cells into the hearts of mice that had suffered induced heart attacks, the marrow cells, rich in stem cells, apparently multiplied and helped repair damaged heart muscle. Other studies in mice and rats suggested that giving the animals a cocktail of bone marrow cells improved heart function. Heart specialists were among the first to try to adapt these discoveries to the clinic, perhaps because of their tradition of bold experimentation and a remarkable array of surgical tools. “Cardiologists are just animals—extremely aggressive, inquisitive, [with] no rules,” says Schatz. “They want to do everything yesterday and not today.” Inspired in part by reports from Orlic and others, Bodo Eckhard Strauer and his colleagues at the University of Düsseldorf in Germany sought permission from the university's ethics review board to infuse bone marrow cells into heart patients. On 25 March 2001, a 46-year-old heart attack survivor signed on to become the team's first subject. Doctors had already used a catheter to unclog the blocked vessel in his heart, inserted a metal stent, and stabilized his heart with standard drugs. But his prognosis was bleak: Nearly a third of his heart had been starved of oxygen, and the organ was unlikely to recover more than a small percentage of its lost pumping capacity. With a needle, doctors extracted a few tablespoons of bone marrow from the man's hip. The next morning, Strauer and his colleagues snaked a catheter to the patient's heart and inflated a balloon at its tip for 4 to 5 minutes—blocking the blood flow in the damaged area long enough to give infused bone marrow cells a chance to adhere to the injured tissue. During the historic 25-minute experiment, the patient was awake and chatting with his doctors, Strauer says. Ten weeks later, the weakened area in the patient's heart had shrunk by a third and the organ's pumping capacity had improved by a few percent, although it was still far from normal. Strauer and his colleagues went on to treat more than 60 patients with stem cells. By the fall of 2001, two more German teams were testing similar techniques. A group led by Andreas Zeiher at the University of Frankfurt treated 34 patients in an initial trial. And at the University of Hannover, Helmut Drexler launched a trial comparing 30 patients who received bone marrow stem cells with 30 who received standard care. The German experiments exhibited clinical prowess but dismayed some U.S. researchers, who saw them as dangerously premature. The therapies had not been tested beforehand in large animals, such as pigs. “I was shocked,” says Piero Anversa, who directs the cardiovascular research institute at New York Medical College in Valhalla. He had assumed that clinical trials were 5 to 10 years away. Still, Anversa and others concede that German aggressiveness advanced the field dramatically. Early results suggested that patients treated with stem cells recovered between 5% and 30% of their lost pumping capacity. And none of the teams reported any serious complications. The road has been rockier for a second cell-therapy strategy, which transfers a patient's immature thigh muscle cells into the heart. On 15 June 2000, Philippe Menasché and his colleagues at the Hôpital Européen Georges Pompidou in Paris were among the first to test it—on a 76-year-old man undergoing bypass surgery. While his chest was open, they injected muscle cells collected earlier from the man's thigh into damaged regions of the heart. Menasché says that preclinical work in rats, mice, and sheep had suggested that the technique held promise. But initial excitement gave way to anxiety. One after another, four of Menasché's 10 subjects developed arrhythmia, a potentially fatal condition in which the heart fails to beat evenly. Some observers suspect that the thigh muscle cells, with their own electric rhythms, couldn't pulse in concert with their new neighbors. More than a decade of animal research had failed to predict this unsettling effect. All four patients underwent additional surgery to implant defibrillators, which stave off arrhythmia. ## Exuberance Cardiologists familiar with the European results remain enthralled by the possibilities of cell therapy. But some, such as Menasché, caution against premature exuberance: “We cannot make any reliable conclusion” about cell therapy's effectiveness without a placebo group, he says. He's busy recruiting patients for such a trial right now. Menasché also points out that controls are needed as much to rule out harm as to prove effectiveness. For example, the arrhythmias he saw may have been unrelated to the therapy, because such patients are already at risk for them. But without an untreated control group, he can't prove it. Still, individual case reports are having an impact. Emerson Perin, a cardiologist at the Texas Heart Institute in Houston, recently conducted an autopsy on a man from a Brazilian cell-therapy trial he ran. The patient died of a stroke apparently unrelated to the bone marrow stem cells he'd received 11 months earlier, and Perin notes that “we are seeing angiogenesis and myogenesis”: the growth of both new blood vessels and heart muscle. Even wary cardiologists agree that newly infused cells seem to be helping the heart. But how? “To be honest, we don't even know if they beat,” says Chuck Murry, a cardiovascular pathologist at the University of Washington, Seattle. The attitude of many clinicians, he says, is that “if function's improved and [patients] can climb a flight of stairs, who cares?” Experts disagree on how much they need to know about the cells before they're transplanted. “There are too many people jumping in and trying to do clinical trials without understanding what the mechanism of their particular cells are,” says Silviu Itescu, director of transplantation immunology at Columbia University in New York City. One reason for doubt is that the rationale for cell therapy keeps shifting. Early studies persuaded many that bone marrow stem cells would form new heart muscle cells, called cardiomyocytes. That theory has not been borne out in more detailed animal experiments. Last month, two independent groups reported in Nature that studies of mice with diseased hearts found no new myocytes produced from bone marrow stem cells. Some clinicians are increasingly skeptical that current cell-therapy methods are building new muscle mass: “I don't believe we are generating a large number of new cardiomyocytes,” says Drexler. ## Paradigm shift As evidence for myocyte formation fails to materialize, a number of scientists are suggesting that transplanted cells may not have to morph into muscle for cell therapy to work. Many now say that patients could benefit because the cells support existing heart muscle, by either boosting the growth of new blood vessels or churning out growth factors that encourage cell proliferation and survival. Indeed, Zeiher believes that the bone marrow cells he infused into patients promote existing healing processes—the kind that occur in hearts that partially recover from a heart attack. “We are simply enhancing the natural function” by injecting extra cells, he theorizes. The search for clear answers is complicated by the assortment of techniques. Some teams are using a mixture of bone marrow cells, whereas others are attempting to isolate specific cell types. And the jumble of cells in bone marrow is poorly understood. “It's like a witch's cauldron, stirring around,” says Schatz of bone marrow. “You've got to find out which ingredient is key.” Skeletal myoblasts, the immature muscle cells used in the French studies, also remain popular. A U.S. trial funded by Weston, Florida-based BioHeart Inc. is recruiting 15 patients with heart failure. All must already have a defibrillator in place to guard against arrhythmia. Two more cell types are waiting in the wings: mesenchymal stem cells (MSCs), which are precursors to muscle, bone, and other connective tissue, and embryonic stem cells. Among the first to try MSCs is Joshua Hare, who heads the cardiac section in the Institute for Cell Engineering at Johns Hopkins University, using cells provided by Baltimore-based Osiris Therapeutics. He is running tests in pigs who've suffered induced heart attacks and has found that the animals' heart function returns to normal. Embryonic stem cells might also work: In culture, they form nodes of pulsing cells, presumably immature heart muscle cells, that beat in unison. But some worry that renegade stem cells could morph into different tissues. “What we don't want,” says Columbia's Itescu, “is to develop bone in the middle of the heart.” ## Risks reconsidered FDA's effort to prepare for new clinical trials brought animal researchers and clinicians together last month in a hotel conference room in the Maryland suburbs, where they reviewed some of the risks. One veteran of the field, cardiac expert Doris Taylor of the University of Minnesota, Twin Cities, was troubled that her 15 years of work with skeletal muscle cells in animals failed to turn up the arrhythmias seen in humans. “We didn't really expect there to be safety problems,” she said. View this table: Scientists can't agree whether bone marrow stem cells might also carry a risk for arrhythmias. One who is reluctant to gamble is Douglas Losordo, chief of cardiology at St. Elizabeth's Medical Center in Boston and head of one of the few U.S. cell-therapy trials. He's outfitting subjects with vests that monitor heart activity and treat arrhythmias. FDA is weighing other safety issues, mostly theoretical. Some experts worry that cells transplanted to the heart could boost blood vessel development in undetected tumors, helping them grow. And a drug called G-CSF, which a number of investigators are using to force bone marrow to churn out stem cells so they can be collected from blood and reinfused, could promote cardiac inflammation and other problems. Concerns about G-CSF came to a head late last year in South Korea, where scientists were using the drug in a stem cell trial. Although the therapy seemed to enhance heart function, seven of the 10 people receiving G-CSF experienced a renarrowing of previously blocked arteries. All were successfully treated, but clinicians, alarmed by what they were seeing, shut the trial down. Most trials, however, have advanced smoothly. And that is prompting researchers worldwide to press forward. Europeans are starting placebo-controlled trials involving hundreds of patients, while U.S. scientists are launching pilot studies. Competition drives them on. “We will be licked by the Germans or the Japanese if we don't keep our pace going,” says Vincent Pompili, who directs interventional cardiology at Case Western Reserve University in Cleveland, Ohio. His protocol, to use stem cells to grow new blood vessels, is one of a growing number on the desks of FDA reviewers, awaiting a green light. So far, the agency has approved a small number of clinical trials. Losordo's stem cell trial was approved by FDA in December; Itescu has been cleared to treat intractable angina and blocked arteries; and Perin can expand on his early Brazilian work. FDA will also be weighing at least one stem cell study on patients awaiting heart transplants. That could provide invaluable data because cardiologists would be able to scrutinize cell therapy's effects in the hearts after they are removed. Although FDA demands stringent safety data before allowing cell-therapy trials to proceed, the agency also gives scientists “lots of latitude” for speculating on why their approach might succeed, says Stephen Grant, an FDA reviewer in the cell- and gene-therapy division. Europeans, meanwhile, seem more concerned about funding. Researchers have received some government support but complain that cobbling together public funds for expensive phase II trials is nearly impossible. “Financing is a major issue,” says Drexler, who is working with University College London's Martin to raise money for a trial of their bone marrow cell-infusion technique, which will include a placebo group. “There is nothing to patent, so funding from industry is almost nonexistent,” says Drexler. If the team enrolls 400 or 500 patients, costs could easily top$5 million, he says.

The one thing no cardiologist worries about is a shortage of study subjects. Clinic waiting rooms are overflowing with potential volunteers. Kamp, the Wisconsin cardiologist, receives e-mails daily from desperate patients seeking new therapies. One recent message was just a single line. “Read about your work,” it read. “I'm willing to be a guinea pig in your clinical trial.”

10. ARDEN L. BEMENT JR. PROFILE

# A Gentlemanly Workaholic Fills In as NSF Director

1. Jeffrey Mervis

Already the director of NIST, Arden Bement couldn't say no when a vacancy emerged at NSF. But his stint as acting director could last several months

Arden Bement is acting director of the National Science Foundation (NSF), thanks to a $75 wager with his foreman at a Colorado molybdenum mill more than a half-century ago. Well, maybe it's a little more complicated than that. But if Bement hadn't accepted that bet—from a supervisor who offered to pay his first semester's tuition to get him off the midnight shift on the mill floor—he might never have gone to college. “I could keep the money if I finished the semester, and I could pay him out of my wages if I dropped out and came back to work,” Bement recalls. “I had never thought about college, but I couldn't refuse the challenge.” A distinguished, 50-year career as a materials engineering researcher and science administrator in academia, government, and private industry followed. And in February, when Rita Colwell gave the White House just 10 days' notice that she would be stepping down as director of NSF (Science, 20 February, p. 1116), President George W. Bush asked Bement to take the reins until a new director is appointed. Approaching his 72nd birthday, and despite a full-time job as director of the National Institute of Standards and Technology (NIST), Bement didn't hesitate when the White House called. Six weeks later, his self-effacing manner, ability to soothe rather than create tensions, and familiarity with the U.S. scientific establishment have already won him plaudits in the science policy arena. “I think that's the best answer I've ever heard from a witness,” enthused Representative James Walsh (R-NY), chair of the House spending panel that oversees NSF, after Bement responded to a question last week on the agency's 2005 budget request. Tall and solidly built, Bement grew up in the blue collar town of New Castle, Pennsylvania. His enrollment in the Colorado School of Mines came with a mandatory ROTC stint that led to a 27-year career as an Army reservist officer in the Corps of Engineers. He earned his master's and doctoral degrees in metallurgical engineering, and his career has featured two cycles of seamless transitions from industry to academia to government: conducting research, teaching, and managing research. The list includes stints at General Electric and TRW, faculty appointments at the Massachusetts Institute of Technology and Purdue University, and assignments at Pacific Northwest National Laboratory and the Department of Defense. He's also served a term on the National Science Board, NSF's governing body. Bement seems to have tempered his initial hope to return to NIST “as quickly as possible.” Although White House officials told him that a search for a permanent director “was already under way,” no name has been announced. And he acknowledges that “the chances are pretty good that I'll be here” past the November election. That's good news for NSF, say those who know him. “He's done superb work as a basic research engineer,” says Purdue's Lefteri Tsoukalas, who succeeded Bement as head of Purdue's nuclear engineering department when Bement went to NIST in December 2001. “He works in a consensus mode, he likes people, and he has a mature ego that's not offended by criticism.” Some would also describe him as a workaholic. Bement arrives at NIST headquarters in Gaithersburg, Maryland, around 6:30 a.m. to check in with acting agency director Hratch Semerjian and handle pressing business. Then it's off to NSF's offices in Arlington, Virginia, a 45-minute drive through the Maryland and Virginia suburbs, where he puts in a full day at the helm of the$5.5 billion agency and its 1250 employees. Leaving NSF at 6 p.m., he heads back to NIST for more paperwork before going home.

Bement says he doesn't have his own agenda for NSF, calling himself a “steward [whose] role is to support efforts already under way.” But he has been putting his people skills to work in improving relations with Congress. In 2002, for example, two committees “urged” NSF to change its policies so that all scientists at the Smithsonian Institution could apply for grants, and in March 2003 NSF promised to do so. After congressional aides belatedly discovered that Smithsonian scientists were still being shut out, they raised the issue in a meeting with the new acting director.

“We explained how important it was to the committee, and how the Smithsonian was a unique entity,” says an aide who attended the meeting. Bement then worked with science board chair Warren Washington on a resolution that the board passed last month telling Bement to negotiate with the Smithsonian to make the change happen (Science, 2 April, p. 26).

That quick resolution of a nagging problem is typical, says Ohio State University physicist Tom Lemberger, an official in the Midwest Superconductivity Consortium, a group of six universities that Bement led in the 1990s while at Purdue. “He's spent a lifetime in the scientific bureaucracy, and he's gotten really good at it.”

The superconductivity consortium may also be Bement's closest brush with controversy. It survived for nearly a decade on political earmarks—funds for programs benefiting specific audiences and not requested by individual agencies—arranged by Representative John Myers (R-IN). Its existence was a thorn in the side of many researchers in the field. Although Bement didn't start the pork barrel rolling, he justifies its continuation by saying that the research was of high quality. After Myers retired from Congress, however, the funding disappeared and the consortium soon folded its tent.

Colleagues say they have no doubt that Bement will do an excellent job at NSF. And some wouldn't mind if “acting” were dropped from his title. “He's a gentleman, and he knows how to work the system,” says Paul Grant, a retired IBM superconductivity researcher who saw Bement in action as an adviser to the Electric Power Research Institute, a nonprofit industry consortium. “I think he'd make a great NSF director.”

11. LUNAR AND PLANETARY SCIENCE CONFERENCE

# 'Mind-Boggling' Martian Gullies Raise Climate Conundrum

1. Richard A. Kerr

HOUSTON, TEXAS—More than 1000 researchers gathered here near NASA's Johnson Space Center from 15 to 19 March to consider all the solid bodies of the solar system, both rocky and icy. The star attraction, however, was Mars.

Water on Mars has been a given since the 1970s, when spacecraft returned images of great flood channels formed in middle Mars history, several billion years ago, as well as delicate filigreed networks of narrow valleys from the planet's earliest days. But decades later, researchers are still trying to figure out how water could have carved those features on a planet that now locks up all its water in ice. At the meeting, researchers introduced yet another water puzzle: a small area that appears to have been scoured by torrential rainstorms during the planet's latter days of extreme cold and ice. “I haven't seen anything like it elsewhere on the planet,” says Mars geologist James Zimbelman of the Smithsonian National Air and Space Museum in Washington, D.C. “The implications are mind-boggling.”

In a presentation that one listener called “compelling and beautiful,” planetary geologists Rebecca Williams, Kenneth Edgett, and Michael Malin of Malin Space Science Systems in San Diego, California, showed images from their camera on the orbiting Mars Global Surveyor of a 55-kilometer impact crater near the martian equator, which looks for all the world as if torrential rains have repeatedly eroded it. Densely branching tributary channels blanketing much of the crater's rim appear to have funneled sediment-laden water to the crater walls, dumping the sediment into fan-shaped, coalescing piles. “That's exactly what I see in the Mojave [Desert] all the time,” says Zimbelman. In fact, Williams and her colleagues have suggested the name Mojave Crater.

The problem is, Mars is not the Mojave Desert, not now and presumably not in the past few billion years since Mars entered its extremely cold and dry later years. And to judge by the number of impact craters on the terrain around Mojave Crater, the crater formed since the planet chilled and dried out. There are signs of geologically recent water flow on Mars in the gullied sediment fans that Malin and Edgett pointed out in 2000 (Science, 28 February 2003, p. 1294). But the Mojave Crater fans, which formed in the past few billion years, are “gullies on steroids,” as planetary geologist Jeffrey Moore of NASA's Ames Research Center in Mountain View, California, puts it. On Earth, it takes hundreds of once-in-a- millennium rainstorms to carry off enough sediment to build such alluvial fans. Melting snow, one proposed mechanism for martian gully formation, doesn't do it in the Mojave Desert, much less groundwater seeping from cliff faces, another possible explanation for martian gullies.

Relatively young alluvial fans startled the Mars die-hards lingering through the last day of the meeting, but there was little consensus on how the fans arose. The water that deposited the fans must have come from the atmosphere as rain or snow, says Williams, not from the ground. But just how the water managed that feat remains unclear. Williams suggested that the impact that created Mojave Crater may have turned water frozen beneath the surface to steam. The steam might then have promptly condensed and rained back on the crater. She was the first to point out, however, that the fans appear as if they may have been built in repeated episodes of heavy water flows, not a single catastrophe. Moore favors melting snows on the crater uplands during a brief warmer and wetter climate, but Williams suspects that melting snow just couldn't have produced enough water fast enough. All in all, she says, “it's a real puzzle.”

12. LUNAR AND PLANETARY SCIENCE CONFERENCE

# Mars Rock Crud Gets in the Way

1. Richard A. Kerr

HOUSTON, TEXAS—More than 1000 researchers gathered here near NASA's Johnson Space Center from 15 to 19 March to consider all the solid bodies of the solar system, both rocky and icy. The star attraction, however, was Mars.

After decades of peering down hundreds of kilometers from orbit and three up-close encounters of Mars landers with rocks, geologists have discovered a lingering millimeter-thick barrier between them and some of the rocks they have been so eager to understand. Two of the three rocks in Gusev Crater that the Spirit rover has studied are covered by a rind of altered material. Similarly weathered rock surfaces may have seriously misled analysts about the nature of large parts of the martian surface.

On the plus side, the discovery does implicate water in the history of Gusev, although not the craterful that the rover was sent to find. “Not that water sloshed around Gusev,” says rover science team member Harry McSween of the University of Tennessee, Knoxville. “We're talking about small amounts of water, perhaps underground.” At times, it might have been nothing more than a slight dampness. Even so, such water-induced chemical alteration may have changed the appearance of much of the surface of Mars.

The first sign of a rock rind came when Spirit ground into a rock named Humphrey. At the meeting, McSween reported that the rover's rock-abrasion tool (the RAT) exposed a cross section of a 0.3-millimeter surface layer. Elemental compositions determined by the rover's alpha-particle x-ray spectrometer (APXS) as the RAT ground progressively deeper showed that the rind was not a coating of omnipresent martian dust but a presumably water-altered form of the volcanic rock beneath. “We may have found evidence of water,” McSween said. “I hope it isn't the only evidence of water we find at Gusev.”

Not much water may have been needed to alter Humphrey's surface. At the meeting, planetary spectroscopist Scott Murchie of Johns Hopkins University's Applied Physics Laboratory in Laurel, Maryland, reported that careful reanalysis of rock colors recorded at visible wavelengths around the 1997 Mars Pathfinder landing site reveals a thin, red-tinted coating on some rocks. The distinctive color signature most likely developed under a more humid martian climate, Murchie said. On rocks in deserts on Earth, dust in a thin surface film of water—nothing more than a barely perceptible dew—can alter to form a similar red “desert varnish.”

McSween and the science team did find signs of more water once Spirit had ground into another rock, one named Mazatzal. There, the RAT uncovered signs of “three different periods of alteration,” McSween said at a 1 April NASA press conference. Two surface rinds—one atop the other—“look superficially like desert varnish.” That would require nothing more than a more humid climate sometime in the past, he said.

But a mineral-filled crack in Mazatzal would have required more water than a desert varnish, McSween said. If the rock had been buried, moistened, and later exhumed, that might have brought in minerals in solution and deposited them in the crack. But the alteration never went very far. “If there were a great deal of alteration,” said McSween, Mazatzal probably wouldn't have any remaining olivine—an easily altered mineral that the rover's Mössbauer spectrometer still sees.

The presence of alteration rinds on martian rocks calls into question earlier interpretations of both rover and orbital rock data. At the Mars Pathfinder site, an APXS on the Sojourner rover had found elevated amounts of silica—the basic building block of volcanic rocks (Science, 1 August 1997, p. 638). That made the Pathfinder rocks andesitic-looking rather than basaltic; andesite was not expected to form on Mars. And rover science team member Philip Christensen of Arizona State University in Tempe and his colleagues had concluded from infrared spectra returned by the orbiting Mars Global Surveyor that the entire northern lowlands of Mars—a quarter of the planet—is covered with andesitic materials, presumably lavas.

Given the proliferating weathering rinds, Christensen is having second thoughts about andesite. Weathering rinds covering basalt, rather than bare andesite, may well be giving the silica-rich signal, he says, both at the Pathfinder site and in the northern lowlands. That's possible because both an APXS and an infrared spectrometer delve only micrometers into rock, not millimeters. Weathering with water could have enriched silica on the surface of basaltic rocks, disguising them. That would eliminate the awkwardness of making andesite while increasing the amount of weathering that some researchers, including Christensen, think Mars has suffered.

New signs of rock weathering do not mean that Mars was ever “warm and wet” for long, Christensen hastens to add. The debate over the climatic history of Mars, and thus the prospects for martian life, has long been cast as a dichotomy: warm and wet—lots of rain, rivers, lakes—versus today's cold, dry, and icy (Science, 22 August 2003, p. 1037). Christensen is sticking with “cold and humid,” given the abundance of unaltered basalt in dark regions of Mars. “I think you can have a cold, frozen Mars,” he says, “and still have a few wet spots.”

13. LUNAR AND PLANETARY SCIENCE CONFERENCE

# Spirit Coming Up Dry at Gusev

1. Richard A. Kerr

HOUSTON, TEXAS—More than 1000 researchers gathered here near NASA's Johnson Space Center from 15 to 19 March to consider all the solid bodies of the solar system, both rocky and icy. The star attraction, however, was Mars.

From orbit, Gusev may have looked like a huge crater once filled to overflowing with water, but after 90 days on Mars the rover Spirit has not found a single sign that water once flowed there. “It's disappointing,” says rover science team member Michael Carr of the U.S. Geological Survey in Menlo Park, California. Spirit's sibling rover Opportunity has found the remains of a salty sea, but “as far as the water story goes,” says Carr, Gusev “hasn't been very rewarding.” So far the lesson learned, says fellow team member Harry McSween of the University of Tennessee, Knoxville, is that “we can't do a great job yet estimating whether [a landing site] is going to be a riveting geologic site.”

Gusev wasn't initially a prime target in NASA's search for signs of past martian water that might have harbored life. Some of the 185 proposed sites offered more geologically enticing targets, but the list began to shrink. Worries about everything from dangerously high winds at some landing sites to too small a landing target at others whittled it down, as did waning interest in many places that simply looked boring to geologists.

Gusev looked pretty boring too, at least to some geologists. The 900-kilometer channel of Ma'adim Vallis appeared to have sent water gushing into the crater eons ago, but critics saw no sign that water had pooled there for any length of time. Even if it had, they said, any lake sediments were likely buried by Ma'adim's last gush of muddy debris or by volcanic ash from nearby volcano Apollinaris Patera. But as other sites fell by the wayside, Gusev got a boost from the recognition that small impacts on its floor have blown holes in surface deposits and scattered once-buried rocks in every direction. Such rock exposures were enough to carry Gusev into the number two spot behind Opportunity's Meridiani Planum landing site.

At the meeting and at the following week's regular rover press conference, team scientists gave up on the hypothesis that impacts had exposed lakebed deposits, at least where Spirit had landed. The rover had turned up nothing but blocks of dark lava—even the pessimists hadn't expected lava—and the yellow-brown dust that has coated every other site visited on the planet. The lava blocks show signs of surface alteration, but a bit of dampness could easily explain that (see p. 196). Nothing about the lay of the land suggests that flowing water has ever been at work there, says McSween, and a rove up to the rim of 200-meter Bonneville Crater revealed nothing but more lava blocks and dusty soil.

McSween finds it difficult to imagine that Gusev never saw water pouring out of Ma'adim Vallis, so the impact-churned lava and windblown dust must be “covering up whatever [layering] may be below, carrying the water history of Gusev,” he says. That revelation could not be made from orbit. “From orbit, we can do a great job estimating rock abundance, terrain flatness, and winds that affect landing safety,” he says. Judging scientific interest, on the other hand, “depends on interpretations of imagery. The scale on the ground is so different, it's hard to guess at the geology” from orbit, he says.

Despite the disappointing story being read by Spirit, no one is giving up on Gusev. Engineers see more life in Spirit, which is now heading for the hills—the Columbia Hills, 100-meter-high mounds a couple of months' drive east of Bonneville Crater. They “are very different from what we've been driving across,” says science team member James Rice of Arizona State University in Tempe. But once again, orbital surveying is proving problematic, he says. There are hints of layers, but “what they are we don't really know.”

14. EUROPEAN EXPANSION

# A Profound Tilt to the East

1. Martin Enserink*
1. With reporting by Gretchen Vogel in Berlin, Richard Stone in Budapest, and Anna-Karin Berg, Fiona Proffitt, and Theres Redeby in Cambridge, U.K.

More than 100,000 researchers, mostly from the former East Bloc, become E.U. citizens on 1 May. Can they compete with their better-funded peers in Western Europe?

PARIS—When Helena Illnerova talks about the future of science in her country, she sounds just like a science manager in London, Paris, or Berlin. The mantras are the same. Participation in Europe-wide funding schemes needs to increase, says the president of the Academy of Sciences of the Czech Republic. Let's make science more competitive. Ramp up international collaboration and stimulate mobility. It's easy to forget that only 15 years ago, Illnerova, an internationally known chronobiologist, had great trouble collaborating with anybody outside the East Bloc, and her mobility was, well, almost zero.

On 1 May the Czech Republic and seven other former communist nations, along with Malta and Cyprus, will join the European Union (E.U.), marking a stunning historic transition that began with the rise of Poland's Solidarity movement in the mid-1980s. Scientists across Europe are hailing this year's May Day as a milestone for science—if only because, like Illnerova, tens of thousands of scientists now have the freedom that is priceless to their work.

But amid the joy there's considerable anxiety. Illnerova and many Eastern and Central European colleagues worry that, despite painful reforms undertaken since the fall of communism, their countries may not be able to compete against vastly better funded and equipped scientists in the West. Heightening that concern are recent debates about the creation of a European Research Council (ERC) that would make quality its main criterion when doling out money, rather than, say, equity among European regions.

At the same time, researchers in the 15 current member states worry that bringing a clutch of cash-strapped countries into the E.U. will siphon money from the top-notch research that could help Europe gain a competitive edge against the United States and Japan. “That would be a disaster for Europe,” says Bart De Strooper, a neuroscientist at the Katholieke Universiteit in Leuven, Belgium, who helped launch a grassroots petition last month to reform European science funding (Science, 2 April, p. 29).

## Membership has its privileges

Once the gala dinners in Brussels are over and the smoke from the fireworks has dispersed, the E.U. will have to come to grips with 116,000 new scientists within its borders—an expansion of the current workforce by about 12%. About half of the newcomers live in Poland, the giant among the new member states (see graphic). The former East Bloc countries bring with them a strong scientific tradition and a population that, according to one recent survey, has more faith than Western Europeans in science's potential to make life better. But many of the new E.U. states can barely afford to pay their scientific workforce, and science is often seen as an unattractive career path.

Researchers and E.U. officials are quick to point out that little will actually change on 1 May. For the scientific world, the expansion occurred in practice in October 2002, when the 10 incoming member states, along with three candidates for membership—Bulgaria, Romania, and Turkey—signed up for the Sixth Framework Programme, the E.U.'s $20 billion funding scheme for 2003 to 2006. In exchange for a fee based on their gross domestic product (GDP)—and with a discount for the first 4 years of membership—researchers in these countries have been able to compete for contracts on an equal footing with Western counterparts. Other perks have already started to materialize as well. Over the next 3 years, each of the 10 countries will receive handsome sums from the E.U.'s so-called structural funds, which aim to boost development in lagging regions. Although not primarily aimed at science, some of the money can be used for innovation. In Hungary, for instance, a total of €100 million will flow to science and technology, increasing the country's public expenditures on science by about 8% per year. “That's extremely important,” says András Siegler, acting director of the newly established National Office for Research and Technology. Lithuania will have some €50 million to spend on science, Latvia about €25 million, but in Estonia, the money is mostly going for development and has little impact on science, says Estonian science minister Toivo Maimets. Many other things have changed in the run-up to E.U. membership. For one, the majority of new member states have overhauled their often sclerotic, centrally directed scientific systems. After a recent reform of its charter, the Polish Academy of Sciences is in the process of “deeply restructuring” some of its 80 institutes and is closing down a few of them altogether, says academy president Andrzej Legocki, a plant chemist. Legocki, who spent 4 years as a researcher at various U.S. universities, says he's also trying to boost scientific output and bring down the average age of the academy's graying corps. Estonia, too, has reformed its funding system, says Maimets, a molecular biologist who got his Ph.D. in Oxford. Quality has become preeminent, and because a tiny country can't be good at everything, there's a narrower focus on promising areas such as biomedicine—the country has entered the pilot phase of a national genetic database that could help trace disease genes (Science, 8 November 2002, p. 1150)—and materials science. “Now,” says Maimets, “we will see whether we have achieved something. Whether we can compete in Europe.” ## Breadth versus depth? That's a question that all 10 new member nations are asking themselves. Although they have a smattering of scientific gems (see sidebar on p. 201), they are lagging behind their counterparts in Western Europe by several indicators—including publications and patents—according to an E.U. survey released last month. And despite offering a wealth of compelling research opportunities—such as drug-resistant tuberculosis in the Baltics (see sidebar on p. 199), the primeval forests of Poland, and the archaeological treasures of Cyprus—the ability of the newcomers to win grants in the first call of the Sixth Framework was discouraging. On average, 13% of non-E.U. scientists saw their proposals make the cut, compared to 19% of scientists from the current member countries. The talent and money gap has prompted Framework critics like De Strooper to worry about what's next. Framework was designed to yoke science to the economic engine of Europe; as such, the program favors applied research and sprawling international lab networks that spread the wealth but create an administrative nightmare. “Framework funding is what scientists call ‘funny money,’” says Ronald Plasterk, who directs the Hubrecht Laboratory for developmental biology in Utrecht, the Netherlands. “There's no clear relation between a project's quality and its chances of getting funding.” Once the new countries become fully-fledged E.U. members, politicians may be tempted to slide even more research money their way, De Strooper warns, further diluting Europe's research strengths. A widespread disaffection with Framework has helped kindle support for the creation of an ERC (Science, 2 January, p. 23), a body that would award grants for basic research based strictly on merit. Leaders need to keep two clear but separate goals in mind, says Robert May, president of the U.K.'s Royal Society. “My worry is if you confuse the need for capacity building with the need for a merit-based European Research Council, … you will doom the possibility of constructing a high-quality ERC,” he says. “You cannot run the risk of a new European Research Council becoming a welfare project.” If the new countries were to flounder in a quality-driven competition, one alternative might be to hold a special funding round just for them, says Frank Gannon, executive secretary of the European Molecular Biology Organization in Heidelberg, Germany. Perhaps surprisingly, many in the new member states reject that idea out of hand. “We're aware that we will pay a price in the beginning,” says Legocki. “But it would be very bad policy to ask for handicapped criteria. We'll just have to get more competitive.” Maimets agrees. “If the quality of science in Estonia is really much worse than in Western Europe, then we'd deserve to lose out,” he says. The European Commission isn't in favor of a two-tier system, adds Fabio Fabbi, an EC spokesperson. Although Framework makes up only 5% of European science funding—national agencies pay the lion's share—the massive program sums up the challenge of weaving the research communities of the member states into a cohesive whole. The process of integration will only get harder with the addition of 10 new states that, of late, have tended to starve their scientific communities. European ministers have pledged to boost R&D spending to 3% of GDP by 2010. Many current E.U. members are nowhere near that number—Spain and Greece spend only 0.96% and 0.67% respectively, and even the prosperous Netherlands manages only 1.94%. With the new countries, the E.U. average edges down from 1.98% to 1.93% (compared to 2.80% in the U.S. and 3.06% in Japan). One advantage of E.U. membership, researchers say, is that the pressure is now on governments to make investment in science a higher priority. But for all the talk about budgets and competition, perhaps the most precious gift of May Day 2004 is the affirmation that young researchers are free to go wherever they can carve out a niche for themselves, Illnerova says—a chance she never had early in her career. “We're finally part of a large scientific family in Europe again.” 15. EUROPEAN EXPANSION # Outwitting TB on the E.U.'s Eastern Frontier 1. Martin Enserink RIGA, LATVIA—A nurse hands an ashen-faced man in a worn black leather jacket a small plastic cup containing a dozen or so garishly colored pills. With a swig of water he gulps them down quickly, signs a sheet on a clipboard, and pockets a food coupon and some cash for the bus ride home. He mumbles a thank-you and leaves. This kind of scene repeats itself day after day in a health care center in a suburb of Latvia's capital, a battleground in the plodding global struggle against multidrug-resistant (MDR) tuberculosis. Ridding a patient's body of Mycobacterium tuberculosis can take up to 2 years if the bug happens to be one of the MDR strains that are rife in Latvia. Health officials and researchers in this Baltic nation, one of three former Soviet republics to join the European Union (E.U.) on 1 May (see main text), have won praise for waging the fight here more aggressively than anywhere else. Riga's State Centre of Tuberculosis and Lung Diseases has become a magnet for experts from around the world to learn how to counter their own MDR TB threats. Last month it became the newest Collaborating Center for the World Health Organization (WHO), a status that heightens prospects for funding and collaboration. “They've become the world experts in MDR TB,” says Charles Wells of the U.S. Centers for Disease Control and Prevention (CDC) in Atlanta. TB control across the former Soviet Union collapsed when the superpower broke up in 1991. Economic hardship created unemployment and homelessness, two risk factors for TB. In 1995 Latvia became one of the first countries to introduce a new treatment strategy advocated by WHO, called Directly Observed Therapy, Short Course (DOTS), in which patients take a combination of four drugs under medical supervision every day for 6 months to ensure compliance. But by then, years of spotty drug availability and a lack of standardized treatment regimens had given the TB microbes a chance to withstand several drugs. The first worldwide survey of MDR TB, carried out between 1994 and 1996, showed that many of the former Soviet countries had resistance levels that were 10 times higher than in the rest of the world—and Latvia, where 14.4% of all new TB cases had MDR TB, had the biggest problem. To address it, the country started treating MDR patients with a more aggressive 18- to 24-month regimen called DOTS-Plus. The technical and logistical challenges were immense, but observers credit a dynamic couple with solving them. The center, which includes a hospital in a forest 20 kilometers east of Riga, is directed by Janis Leimans, who also leads the national TB control program; his wife, Vaira Leimane, is program manager for DOTS-Plus and runs the international training center. “They make a very good team,” says deputy director Gunta Kirvelaite. “She generates the ideas; he is more the practical manager.” Their eye for young talent has created a buzz well beyond the Baltics, adds Wells. Latvia has since accrued a wealth of experience with MDR TB. “It's very impressive,” says Alexander Klochkov, a TB control official from Ukraine who finished 2 weeks of training in Riga last week. (Ukraine has yet to start implementing DOTS.) The training, which includes site visits to drug dispensaries and discussions on how to reform the health system, was developed with funding from the U.S. Agency for International Development, the State Department, and CDC. Meanwhile, a World Bank loan helped renovate the center's laboratory, now well-equipped and spiffy—a far cry from the crumbling walls and chipping paint in the center's hospital. More changes are in the air as Latvia prepares to join the E.U. Leimans says he hopes that E.U. structural funds will help pay for a new ventilation system for the hospital to help protect staff health—hardly a priority under Soviet rule. But Leimane also would like to win E.U. funding for new research, such as genetic fingerprinting techniques that could help determine whether somebody who gets TB a second time is suffering from a relapse or an infection with a new strain. “I could also see them blossoming into a center for clinical trials,” Wells says. All these efforts are not just putting Latvia on the world map; they're also reducing the burden of disease, Leimane says. According to the latest WHO survey, released last month, the percentage of MDR cases among first-time patients in Latvia had fallen to 9.3%. “We've shown that it's possible” to bring down MDR TB, Leimane says, “and others can do it too.” 16. EUROPEAN EXPANSION # A Nanowire Link Between East and West 1. Fiona Proffitt LJUBLJANA, SLOVENIA—One Saturday morning in the nearly deserted Jozef Stefan Institute (JSI) here in Slovenia's capital city, Dragan Mihailović eagerly shows off some recent fruits of his lab's labors: a vial filled with a black powder resembling soot. Unimpressive at first glance, this substance may just be Slovenian gold. Mihailović intends to position the molybdenum-based powder, which he claims contains the world's first superconducting nanowires, as a rival to carbon nanotubes for applications ranging from light sources in flat-panel displays to friction-reducing oil additives. The description of his nanowires, posted recently in the online edition of Nanotechnology, is whetting appetites far beyond Slovenia, a rugged little country east of the Italian Alps and one of the 10 new members of the European Union (E.U.). The novel materials “are very intriguing and interesting,” says physicist Alex Zettl of the University of California (UC), Berkeley. Mihailović, 46, trained abroad, earning a Ph.D. in physics in 1983 at the University of Oxford's renowned Clarendon Laboratory. A big career break came in 1989, when as a Fulbright Fellow he spent a year studying copper-based superconductors and conducting polymers at UC Santa Barbara in the laboratory of physicist Alan Heeger, who won the Nobel Prize in chemistry in 2000 for conductive polymers. Mihailović “is a solid and creative scientist,” says Heeger. “He often does not follow mainstream ideas [but] goes off into new and productive directions.” Like most Slovenes, whose attachment to their culture, family, and country runs deep, Mihailović felt compelled to return home. In 1990 he resumed his work at JSI, Slovenia's premier research institute. His 10-person team specializes in materials that exhibit quantum phenomena—such as superconductivity and magnetism—arising from complex interactions between electrons. Thanks to E.U. funds, Mihailović's complex matter department has a femtosecond spectrometer for recording light-induced changes in materials on the order of 10−15 seconds. His group also knows how to improvise, having saved roughly$2 million by adapting a secondhand electron microscope for fine-scale lithography, a technique used to make nanowires into minuscule sensors and transistors. Mihailović, JSI's chief scientist, hopes the institute can further enrich its facilities by winning a share of the structural funds being given to each new E.U. member nation (see main text).

Mihailović is one of the first Slovenian scientists to have launched a spinoff firm. He got a close-up view of the process in Santa Barbara in 1990, when Heeger co-founded UNIAX to develop light-emitting polymer displays. Inspired by the venture—UNIAX introduced the first polymer-based plastic display and was snapped up by DuPont in 2000—Mihailović last year formed a company, MO6, to commercialize his nanowires.

Cruising around Ljubljana in a kingfisher blue Jaguar, Mihailović clearly enjoys the good life in Slovenia. He and many of his compatriots insist they wouldn't live anywhere else—suggesting that science in this newest corner of the E.U. will only grow stronger with closer ties to the West.

17. # The Roots of Plant-Microbe Collaborations

1. Jean Marx

Genetic studies are decoding the language plants and microbes use to negotiate the symbioses that help feed the world

Bacterial or fungal invasions do not always cause disease. Some invasions—for instance, when microbes break into the cells of plant roots—are decidedly beneficial. By providing essential nutrients, the microbes help both their host plants and the world's agricultural systems.

In one such symbiotic interaction, the roots of many plants are infected by certain fungi that help them acquire phosphate from the soil. In the other key agricultural symbiosis, rhizobial bacteria set up housekeeping in the roots of legumes, such as peas, soybeans, and alfalfa, where they produce the form of nitrogen needed for plant growth. Recently, plant biologists have begun to dissect out the genes needed to establish these life-sustaining relations.

They've found that genes from both plants and microbes contribute to symbiosis. The two partners engage in a complex molecular conversation that allows the microbes to infect the plant cells and then entice the cells to undergo the developmental changes necessary for establishing the symbioses. “You have a bacterium and a plant that are completely different but have to be wired so that they can recognize one another and coordinate their activities,” says Sharon Long, whose lab at Stanford University is among the leaders in studying rhizobia-plant interactions.

In addition to clarifying the molecular underpinnings of these symbioses, the findings are shedding light on plant evolution. The fungal-plant symbioses are much older than rhizobia-legume associations. Detection of fungi in fossilized plants indicates that the associations date back to the first land plants, some 400 million years ago. The symbiosis-forming fungi, which were preserved in the plant root cells, “are among the few fungi in the fossil record,” says Maria Harrison of the Boyce Thompson Institute for Plant Research in Ithaca, New York.

Legumes, however, are a mere 70 million years old. This has long led plant researchers to suspect that rhizobia might have made use of some of the same plant machinery used by the fungi to establish their symbioses—a suspicion supported by the recent molecular findings showing that some of the same plant genes involved in rhizobia invasions are needed for establishment of the fungal infections.

The work might also have practical implications. Ever since gasoline shortages hit the United States 3 decades ago, researchers have hoped that they might be able to induce nonlegume crop plants, such as corn, to form symbioses with rhizobia. If so, the bacteria could “fix” nitrogen—convert the unusable nitrogen of the air to ammonia—for the cereals just as they do for legumes, thus reducing the need for costly and polluting synthetic nitrogen fertilizers. Identifying the genes needed for rhizobial symbiosis means “we can at least begin to think about how it can be done,” says Douglas Cook of the University of California, Davis.

## The plant speaks first

Although the fungal symbioses are the more venerable of the two types of microbe-plant root interactions, researchers are further ahead in their understanding of how rhizobia infect their legume hosts, partly because the energy crises of the 1970s sparked a considerable investment in nitrogen-fixation research. The fungal associations are also harder to study because these microbes grow only on plant roots. Because they can't be cultured independently, mutational studies are much more difficult.

Rhizobial bacteria can live independently, but because they fix nitrogen only after they have entered legume roots and formed structures called nodules, researchers realized early on that in order to make progress, they had to study the two symbiotic partners together. “The basic idea was that if you wanted to scour for genes [expressed] in symbiosis, you didn't want to look at bacteria in a petri dish,” Long recalls.

By the early 1980s, the field had seen two big advances. In one, Frederick Ausubel's team at Harvard University cloned the first rhizobial nitrogen-fixation genes, which encode proteins needed for the chemical conversion of nitrogen to ammonia.

Before that reaction can take place, however, the bacteria have to induce nodule formation. This is a fairly complicated process that begins when the root hairs, small projections from the root surface, curl around the infecting bacteria. Once encapsulated this way, the bacteria burrow into a root hair cell, where they trigger the formation of a so-called infection thread that will carry them into the root proper. There they elicit the formation of a new structure—the nodule—in which they will reside, producing fixed nitrogen while receiving energy from the plant. Nodulation requires a second set of bacterial genes, the nodulation (nod) genes, and Long, then a postdoc in Ausubel's group, cloned the first of those, also in the early 1980s.

Next, Long and other investigators wanted to find out how the nod genes get switched on. In the mid- to late 1980s, they found that the switches are chemicals called flavonoids that are secreted by legume plant roots, with each legume species producing its own particular cocktail of the compounds. In this way, the plant initiates the molecular dialogue with the specific bacterium needed for their symbiotic interaction.

The fact that the various legumes make different flavonoids also contributes to one of the distinctive characteristics of legume-Rhizobium symbioses: A particular rhizobial species has a very limited host range, usually infecting members of only one or a few genera of legumes. For example, Sinorhizobium meliloti infects alfalfa but not peas, whereas Rhizobium leguminosarum bv. viciae infects peas but not alfalfa.

Subsequent work showed that the bacteria also contribute to this specificity. In the early 1990s, two teams in Toulouse, France, coordinated by Jean Dénarié of the INRA-CNRS Laboratory of Plant-Microorganism Interactions, identified the chemical that S. meliloti uses to alert alfalfa to its presence and initiate infection. This turned out to be a carbohydrate consisting of four linked units of the sugar N-acetyl glucosamine with a fatty acid attached on one end and a sulfate group on the other. Since then, researchers in several labs have identified these so-called Nod factors from several rhizobial species. All slightly different, each evokes a nodulation response only in the legumes that host the particular Rhizobium that produces the Nod factor.

## Continuing the conversation

This specificity in rhizobial signal and legume response strongly suggests that legume root cells carry receptors that recognize and bind the appropriate Nod factors. “These molecules carry information for [nodule] development in the plant,” Cook says. “There must be receptors.” Much recent work has focused on identifying those receptors and working out the signaling pathways that spark the developmental changes that enable bacteria to induce nodule formation in the plant roots. Researchers usually go about this by inducing mutations in the plants, looking in particular for those that upset the earliest stages of Nod factor responses and thus rhizobial infection.

In one such study, a collaborative effort carried out about 4 years ago by Cook's and Dénarié's teams, the researchers treated plants of the model legume Medicago truncatula with a chemical mutagen. Analysis of the defects in the resulting plants turned up four genes, all of which are required for early responses to Nod factors.

At this point, the researchers found direct evidence that signaling to the plant by both the rhizobia and the fungi works at least partly through the same path. Three of the genes, called DMI (for Does Not Make Infections) −1, −2, and −3, also turned out to be needed for M. truncatula infection by a fungus that forms an arbuscular mycorrhiza, the technical term for the fungal-plant symbiosis involved in phosphate uptake. “Legumes form symbiosis with bacteria and with completely different fungi, but the same genes are involved in both,” says Martin Parniske of the John Innes Centre in Norwich, U.K.

At that time, however, the plant genes were known only by the effects produced by their inactivation, such as blocking root hair curling and infection thread formation. Then, about 2 years ago, György Kiss and colleagues at the Biological Research Center of the Hungarian Academy of Sciences in Szeged cloned the M. sativa version of DMI2. At the same time, a multinational team led by Parniske and Jens Stougaard of the University of Aarhus, Denmark, cloned the Lotus japonicus version of DMI2.

The gene's structure indicates that the protein it encodes is a so-called receptorlike kinase. These proteins are embedded in the cell membrane with one region extending to the outside of the cell where it can bind signal molecules such as growth factors, and an interior segment that appears to be a kinase, that is, an enzyme that regulates other proteins by adding phosphate groups to them. This raised the possibility that the DMI2 protein might be part of the recognition machinery for Nod factors. In fact, the Szeged researchers originally called what turned out to be the DMI2 gene NORK (for Nodulation Receptor Kinase), and the Parniske-Stougaard team called its Lotus gene SYMRK (for Symbiosis Receptor-Like Kinase).

Some findings countered the idea that DMI2 serves as part of the Nod receptor, however. For one, although the roots of Lotus or Medicago plants carrying SYMRK/DMI2 mutations do not form infection threads or nodules when exposed to rhizobial bacteria, their root hairs become deformed. This suggests that they still sense the presence of the bacteria and their Nod factors. “Root hair swelling and deformation are the first indication that the root hair recognizes the bacterium,” Stougaard says.

Furthermore, the protein DMI2 is also needed for initiation of phosphate-providing arbuscular mycorrhizae, but researchers consider it unlikely that the fungal equivalents of rhizobial bacteria's Nod factors, which have yet to be identified, interact with the same receptors on plant roots. The fungi trigger different responses in the roots; they don't cause root hair changes, for example. And unlike rhizobial bacteria, the fungi show little specificity for their plant targets.

Indeed, about a year later, Stougaard's group and also that of Ton Bisseling at Wageningen University in the Netherlands came up with better candidates for Nod factor receptors. Stougaard and his colleagues looked at Lotus mutants that didn't show the early root hair deformations. They found that mutations in either of two genes, which they called NFR1 and NFR5, could cause that defect.

Both genes encode receptor kinases, but even more intriguingly, the portions of the proteins that would be on the outside on the root hair cell membrane resemble protein segments that bind chemical structures similar to those of Nod factors. Genetic analysis also showed that these putative Nod factor receptors come into play before the SYMRK/DMI2 kinase, as would be expected if they are involved in the first step: recognizing specific rhizobia.

The Bisseling team cloned its Nod receptor candidates, called LYKs, from M. truncatula. Although the LYK structures are similar to those of the NFRs found in Lotus, the proteins may have somewhat different functions. Mutations in the LYK genes don't prevent root hair deformation but do prevent the formation of infection threads and thus entry of the bacteria into roots. Because Nod factors induce many changes in legume roots, nitrogen-fixation researchers have long debated whether they might have more than one receptor. These results suggest that they do, although additional confirmation is needed.

## Oscillating message

Although the function of DMI2 remains unknown, the roles of DMI1 and DMI3 have become clearer with their recent cloning. Some clues already existed from the earlier work in which the genes were identified and also from studies of the physiological changes that take place in root hair cells when they are stimulated by Nod factors.

In the mid-1990s, Long and her colleagues found that within the first few minutes of stimulation by Nod factors, the root hair cell membranes become depolarized, allowing calcium ions to move into the cells. Then about 10 minutes later, a wave of calcium “spiking” occurs, in which the calcium ion concentrations inside the cell rapidly oscillate up and down.

In other systems, calcium spiking has been linked to changes in gene expression. That may be true here as well, because Nod factors activate the expression of many plant genes needed for nodule development. But whatever its role in the plant, calcium spiking has also proved helpful to researchers, serving as a kind of landmark to help them deduce the relative order of action of the genes they've identified.

The earlier studies showed, Dénarié says, that “DMI1 and −2 are absolutely required for calcium spiking.” Thus, their protein products must come into play before spiking. In contrast, DMI3 mutations do not affect spiking, so its product works downstream of the calcium oscillations.

The structure of the DMI1 gene from M. truncatula, which was described in the 27 February issue of Science (p. 1364) by a large research team including Long, Dénarié, and Cook, suggests that it encodes a channel that allows positively charged ions to move into or out of cells. If so, then it might be involved in the movements of potassium or calcium ions that occur in root cells in response to Nod factor stimulation.

In the same Science issue (p. 1361), Dénarié's team, this time in collaboration with Bisseling and his colleagues, described the cloning and structure of DMI3. This gene closely resembles genes encoding a group of kinases known to be regulated by calcium ions in conjunction with another protein called calmodulin. “It appears that DMI3 can interpret the calcium signal,” says Cook. How it does that remains to be established, but functional DMI3 is apparently needed for the gene activity that occurs in response to Nod factor stimulation. Although calcium spiking occurs in DMI3 mutants, it doesn't induce a change in gene activation.

## End of discussion

In addition to getting a better understanding of how nodulation is turned on in legumes, researchers are beginning to learn how it can be turned off. The plants have at least two ways of limiting nodulation. This may be necessary to protect against the severe energy drain that would be imposed by having too many nitrogen-fixing nodules.

About 7 years ago, Cook and R. Varma Penmetsa, who were then at Texas A&M University in College Station, discovered a M. truncatula mutant that develops 10 times the normal number of nodules when inoculated with S. meliloti. Further analysis showed that the mutant plants are insensitive to ethylene, a plant hormone that promotes growth and other plant activities. “The plant is exquisitely sensitive to Nod factor in the absence of ethylene,” Cook says.

The hormone apparently exerts its suppressive effects within the signaling pathway in which the DMI genes operate. Long's group found that ethylene inhibits early Nod factor responses, including calcium spiking.

About 2 years ago, two teams, one led by Stougaard and the other by Masayoshi Kawaguchi of Niigata University in Japan, cloned a gene from Lotus and pea that is also involved in regulating nodule numbers. Although mutations in the gene, which they called HAR1 (for Hypernodulation Aberrant Root 1), also result in a marked increase in nodulation, it's unrelated to the mutant gene conferring ethylene insensitivity. Instead, HAR1's structure showed that it is the Lotus and pea equivalent of a gene called Clavata1, which regulates growth in the model plant Arabidopsis thaliana. Shortly thereafter, Peter Gresshoff's team at the University of Queensland, Australia, identified the soybean equivalent, and Cook and his colleagues came up with the corresponding gene in M. truncatula. “The [regulatory] pathway is highly conserved across the legumes,” Cook says.

Despite the recent progress, researchers have a long way to go to get a complete picture of the genetic interplay involved in rhizobial symbiosis. “We have only a small fraction of the components identified by mutants,” says Long. “But we're getting there.” The genomes of M. truncatula and L. japonicus are expected to be sequenced by 2006 and should be a big help to the effort, just as the Arabidopsis sequence is for researchers studying that plant. Unfortunately for symbiosis studies, Arabidopsis is not a legume, and it's one of the 20% of land plants that do not form arbuscular mycorrhizae.

Studies of these fungal symbioses may soon get a boost as well, as sequencing of one of the fungal partners is under way. In addition, the rhizobial work has surged ahead, partly because researchers were able to identify the chemical signals that legumes and rhizobial bacteria use to communicate. That work provided simpler study systems by making it possible to use Nod factors, rather than the bacteria themselves, to tweak the plants. Those signals haven't yet been identified for mycorrhizal associations, but results reported last month in Plant Physiology by Dénarié, Guillaume Bécard of the University of Toulouse, and colleagues may point to a solution.

They showed that a variety of arbuscular mycorrhiza fungi produce small, diffusible factors that trigger activity of one of the same genes activated by Nod factors in the outer cell layer of M. truncatula roots. This suggests that the fungal factors may play an analogous role, and Dénarié and his colleagues are now trying to purify them. If they do come up with the fungal equivalent of Nod factors, it should help provide a clearer picture of the molecular underpinnings of the fungal associations and how they compare to those involving rhizobia bacteria. Detangling either system could be a boon to the world's breadbaskets.