Hot off the presses! Jun 10 Nature

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Latest Articles Include:

• Putting gender on the agenda
  - Nature (London) 465(7299):665 (2010)
  Nature | Editorial Putting gender on the agenda Journal name:NatureVolume:465,Page:665Date published:(10 June 2010)DOI:doi:10.1038/465665aPublished online09 June 2010 Biomedical research continues to use many more male subjects than females in both animal studies and human clinical trials. The unintended effect is to short-change women's health care. Article tools * 日本語要約 * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Differences in the physiology of males and females, and in their response to disease, have been recognized for decades in many species — not least Homo sapiens. The literature on these differences now encompasses everything from variations in gene expression between male and female mice, to a higher susceptibility to adverse drug reactions in women compared with men. Moreover, hormones made by the ovaries are known to influence symptoms in human diseases ranging from multiple sclerosis to epilepsy. And yet, despite the obvious relevance of these sex differences to experimental outcomes, three articles in this issue (see pages 688, 689 and 690) document that male research subjects continue to dominate biomedical studies. Some 5.5 male animal models are used for every female in neuroscience, for example. And apart from a few large, all-female projects, such as the Women's Health Study on how aspirin and vitamin E affect cardiovascular disease and cancer, women subjects remain seriously under-represented in clinical cohorts. This is despite reforms undertaken in the 1990s, when sex discrimination in human trials was first widely recognized as a problem. Admittedly, there can be legitimate reasons to skew the ratios. For instance, researchers may use male models to minimize the variability due to the oestrous cycle, or because males allow them to study the Y chromosome as well as the X. And in studies of conditions such as heart disease, from which female mice are thought to be somewhat protected by their hormones, scientists may choose to concentrate on male mice to maximize the outcome under study. "Medicine as it is currently applied to women is less evidence-based than that being applied to men." However justifiable these imbalances may be on a case-by-case basis, their cumulative effect is pernicious: medicine as it is currently applied to women is less evidence-based than that being applied to men. The research community can take a number of steps to address this problem. Journals can insist that authors document the sex of animals in published papers — the Nature journals are at present considering whether to require the inclusion of such information. Funding agencies should demand that researchers justify sex inequities in grant proposals and, other factors being equal, should favour studies that are more equitable. Funding agencies and researchers alike should also start thinking seriously about how to deal with the most fundamental sex difference: pregnancy. Pregnant women get ill, and sick women get pregnant. They need therapies, too, even though they are carrying a highly vulnerable fetus and their bodies are undergoing massive changes in hormonal balance, immune function and much else besides. Entering pregnant women in clinical trials is problematic in the extreme, for a host of ethical reasons. But ignoring the problem is not an answer either — the result is that physicians will prescribe drugs whose effects during pregnancy are poorly known. One possible solution is systematic retrospective data collection from women who have had no choice but to take an unproven drug while they were pregnant. More generally, drug regulators should ensure that physicians and the public alike are aware of sex-based differences in drug reactions and dosages. And medical-school accrediting bodies should impress on their member institutions the importance of training twenty-first-century physicians in how disease symptoms and drug responses can differ by sex. Finally, speeding more women into the senior ranks of science, which they still struggle to reach (see page 832), could well have a salutary effect in creating an environment in which all such efforts can thrive. These may be the first steps in the direction of truly personalized medicine — what, after all, is more personal than sex. But they are urgently necessary ones. Additional data
• Unknown quantities
  - Nature (London) 465(7299):665 (2010)
  Nature | Editorial Unknown quantities Journal name:NatureVolume:465,Pages:665–666Date published:(10 June 2010)DOI:doi:10.1038/465665bPublished online09 June 2010 It is in researchers' interests to help funding agencies quantify the economic benefits of their work. Article tools * 日本語要約 * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg When research agencies are pressed by politicians to quantify the economic value of scientific research, it is only natural that they reach for whatever numbers they can find and then repeat them as well-established fact. Natural, but wrong. The reality is that few of those numbers — typically, assertions that each unit of research investment will yield a certain amount of additional economic activity — rest on a secure basis (see page 682). Economists can say with some certainty that basic scientific research plays a substantial role in fostering innovation — by which they mean new technologies, services and business methods. They also have good evidence that innovation is essential for strong economic growth, especially when society faces constraints on key inputs such as labour, capital and materials. Beyond that, they can't predict which disciplines of scientific research will lead to future innovation — that would require a time machine. Nor, thus far, can they trace how additional research investment will influence a society's ability to innovate. The problem is that innovation is not a simple, linear system in which basic research begets technology, and technology begets innovation — although that has always been the easiest model for policy-makers to envisage. Innovation is a complex, highly nonlinear ecosystem, full of interdependencies and feedback loops that aren't even completely mapped yet, never mind ripe for quantification. How do multiple basic-research findings accumulate into useful technology, for example? How do discoveries in one geographical region influence innovation in another? Most of the attempts to count the economic benefits of investment in science have been derived from the efforts of lobbying groups and funding agencies to justify science spending. The few studies that have made a genuine attempt objectively to assess the economic outcomes of research — such as the 2008 UK study Medical Research: What's it Worth? — have highlighted vast swathes of uncertainty. In the United States, there are moves afoot to do better. Physicist John Marburger, when he was running the White House Office of Science and Technology Policy during the George W. Bush administration, launched a number of measures to fill the gap, including an $8-million National Science Foundation programme to fund research into the science of science and innovation policy. But, as Marburger himself admits, this is a long-term research project; when officials claim that such investigations will next year yield useful data about the impact of President Barack Obama's 2009 stimulus package, they are promising more than what economics is likely to deliver. That observation is reinforced by the experience of Europe, where economic competitiveness has been a constant concern for policy-makers, and where assessing the economic outcome of investments in science has been a major priority of research agencies for 20 years. That effort has led to a lot of interesting questions, but no solid guidance for policy-makers wondering how much to spend on research or what exactly they should spend their money on. In time, the innovation ecosystem will be better understood. Meanwhile, researchers should do themselves a favour by cooperating with the good-faith efforts of economists and sociologists to improve that understanding. They should also comply with apparently tiresome demands from funding agencies for more complete information about how they spend their grants, their interactions with colleagues and their 'outputs' such as publications, patents and commercial benefits. And in the public arena, scientists should talk like scientists and desist from using dodgy numbers to bolster the already powerful case for research spending to be maintained, or even increased, during difficult economic times. Additional data
• A question of trust
  - Nature (London) 465(7299):666 (2010)
  Nature | Editorial A question of trust Journal name:NatureVolume:465,Page:666Date published:(10 June 2010)DOI:doi:10.1038/465666aPublished online09 June 2010 The re-auditing of accounts from the closed Sixth Framework Programme is generating hostility. Article tools * 日本語要約 * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg The rules that guide how member nations participate in the multibillion-euro Framework programmes of research run by the European Union (EU) are complex, confusing and in urgent need of simplification — and nothing illustrates that better than the ongoing 're-audit' of the Sixth Framework Programme (FP6). The programme closed in 2006, but in recent months several national research agencies have been accused of cheating by the European Commission, the EU's executive arm, and have been asked to pay back grant money worth millions of euros. The affair began last year, when pressure from the European Court of Auditors led the commission to re-audit sample files from those national research agencies that had handled the largest numbers of FP6 projects. The re-audit unearthed many cases of what the commission insists are irregularities, mainly in personnel costs and overheads. In those cases in which the same irregularities were found in each of the sampled files from an agency, the commission assumed them to be systematic and demanded that the money be returned. For a research agency such as France's CNRS, which handled nearly 900 FP6 projects, the sums being disputed amount to tens of millions of euros. The agencies, however, are incensed. They maintain that they operated with the commission's approval, following an interpretation of the FP6 rules that allowed them to use their own customary national accounting and management practices. Now, they say, the commission has reinterpreted the rules after the fact, and has used the re-audit to impose penalties on the same customary practices it previously approved. The exercise has become expensive for all concerned. The accused research agencies are having to allocate already-scarce resources to a check of all their individual files, trying to prove that they did nothing wrong. And the commission has had to hire extra staff to assist with the unanticipated re-audits — to the point that the enterprise is costing much more money than the commission could ever expect to gain though repayments. Worse is the loss of good will. Some research organizations say that their scientists are becoming wary of involvement with Framework programmes for fear of future unforeseeable bombshells. The mood is not helped by the commission's proclamation that only errors in its own favour are subject to correction — those financial mistakes that would require a repayment to project scientists cannot be reimbursed because the FP6 is officially closed. Both sides had hoped that the rules of participation would be clearer for the Seventh Framework Programme (FP7). The commission had promised to grant 'methodology certificates' to institutes whose customary practices it explicitly accepts. But as that seven-year programme reaches its halfway point, virtually no certificates have been awarded because the commission still lacks a confident understanding of its own rules — and that leaves the FP7 as vulnerable as the FP6. The FP6 re-audit debacle will probably be concluded behind closed doors; neither the commission nor the research agencies seem to want a public scandal. In the meantime, there is — at last — broad political will to simplify the rules of the Eighth Framework Programme (FP8), set to launch in 2014. This simplification must be accompanied by an infusion of institutional trust. On the whole, scientists are not potential criminals; they need to be attracted to, not repelled by, Framework programmes. Like all public funds, Framework money must be accounted for appropriately — but heavy-handed, after-the-fact meddling is not the best way to do it. Straightforward audits that are based on clear, consistent and agreed rules are a much better approach. It is too late for the FP6, and perhaps too late for the FP7. But the FP8 should treat researchers with the trust and dignity they need to do their best work. Additional data
• Animal behaviour: Mongoose traditions
  - Nature (London) 465(7299):668 (2010)
  
• Metabolism: Obese cells 'self-undereat'
  - Nature (London) 465(7299):668 (2010)
  
• Neuroscience: Drug shrinks brain
  - Nature (London) 465(7299):668 (2010)
  
• Nanotechnology: Aquatic speakers
  - Nature (London) 465(7299):668 (2010)
  
• Biochemistry: Picture protein
  - Nature (London) 465(7299):668 (2010)
  
• Genomics: Genetic editing
  - Nature (London) 465(7299):668 (2010)
  
• Virology: Back-up resistance
  - Nature (London) 465(7299):669 (2010)
  
• Biophysics: Molecular carnival ride
  - Nature (London) 465(7299):669 (2010)
  
• Biomaterials: Surgical solution
  - Nature (London) 465(7299):669 (2010)
  
• Animal cognition: Colder is cleverer
  - Nature (London) 465(7299):669 (2010)
  
• Journal club
  - Nature (London) 465(7299):669 (2010)
  
• News briefing: 10 June 2010
  - Nature (London) 465(7299):670 (2010)
  The week in science. This article is best viewed as a PDF. Policy|People|Research|Awards|Events|Business|Business watch|The week ahead|Number crunch|Sound bites Medical personnel on the payroll of the Central Intelligence Agency (CIA) participated in experimentation on detainees — to refine torture techniques such as waterboarding — during interrogations, according to Physicians for Human Rights (PHR), an advocacy group in Cambridge, Massachusetts. The actions during the administration of former president George W. Bush, which included gauging prisoners' pain tolerance, contravened the Nuremberg Code of research ethics for human experimentation and other accepted standards, says the PHR. The White House, the CIA and the Department of Justice had not responded to comment requests as Nature went to press. See go.nature.com/5ZxcYY for more. The European Chemicals Agency revealed last week that nearly a quarter of the companies it inspected last year were not fully complying with Europe's stringent REACH legislation on chemical safety. The agency inspected 1,543 firms in 25 European Economic Area states between May and December 2009 and found 378 breaches such as not providing appropriate safety data. The food-safety research programme at the US Food and Drug Administration is "fragmented and poorly managed", lacks strategic planning and is badly coordinated, according to a report from the Institute of Medicine. The report was requested by Congress in the wake of several contamination incidents, including melamine in pet food in 2007 and Escherichia coli in cookie dough in 2009. Subra Suresh has been nominated by the US government to lead the US$6.9-billion National Science Foundation (NSF) in Arlington, Virginia. Suresh is currently dean of the engineering school at the Massachusetts Institute of Technology in Cambridge. Until he is confirmed by the Senate, the NSF will be led by acting director Cora Marrett. See page 673 for more. KYODO Japan's prime minister, Yukio Hatoyama, resigned on 2 June. Hatoyama's Democratic Party of Japan (DPJ) instigated controversial but largely popular budgetary reforms and a frugality in scientific budgets (see Nature 462, 258–259; 2009). But his popularity has plummeted since he backtracked in May on election promises to remove a US military base on Okinawa. The future of those reforms, and plans to overhaul the Council for Science and Technology Policy, will depend on upper-house elections this July and on the performance of his successor Naoto Kan (pictured, centre, with DPJ party members), Japan's sixth prime minister in four years. Results of the pilot phase of the Global Earthquake Model were announced at a meeting in Washington DC on 3–4 June. Funded by partners including national governments and the World Bank, the project aims to predict the risk from earthquakes to different communities. The model is expected to be fully working by the end of 2013. See go.nature.com/CY2Qj4 for more. The city of Cambridge, Massachusetts, has approved construction of a billion-dollar biotechnology complex. The Binney Street Project in East Cambridge, near the Massachusetts Institute of Technology, will be developed by California-based firm Alexandria Real Estate Equities, which specializes in providing life-science laboratory space. The firm says that the complex will include 160,000 square metres of office and lab space. This year's three prizes, each worth US$1 million, have been shared between eight scientists. Jerry Nelson of the University of California, Santa Cruz, Roger Angel of the University of Arizona in Tucson, and Ray Wilson, formerly of the European Southern Observatory in Garching, Germany, share the astrophysics prize. In neuroscience, the prize goes to Thomas Südhof, Richard Scheller and James Rothman of Stanford University School of Medicine, Palo Alto, California, biotech company Genentech, based in San Francisco, California, and Yale University, New Haven, Connecticut, respectively. The nanoscience prize was awarded to Donald Eigler of IBM, headquartered in Armonk, New York, and Nadrian Seeman of New York University, New York. Commercial space-flight company SpaceX successfully sent its flagship Falcon 9 rocket into Earth orbit on 4 June from Cape Canaveral, Florida, carrying a mock-up version of its Dragon cargo capsule. Its launch bodes well for NASA science-mission managers, who desperately need a cheap supplier of medium-sized rockets (see Nature 465, 276–277; 2010). Satellite-phone operator Iridium of McLean, Virginia, has awarded what is currently the world's biggest commercial space-flight contract to Thales Alenia Space, headquartered in Cannes, France. Thales Alenia will build 81 satellites for the telecommunications network: 72 for launch and 9 ground-based spares. Iridium says that the development, manufacture and launch of these satellites will cost US$2.9 billion. The London-based drug company GlaxoSmithKline (GSK) announced last week that it has settled litigation related to its diabetes drug Avandia (rosiglitazone), which was due to go to trial in Philadelphia on 1 June. GSK still faces thousands more cases alleging that Avandia caused heart attacks and strokes, but analysts say that its costs may eventually be less than the $4.85 billion incurred by pharmaceutical firm Merck to settle lawsuits related to the painkiller Vioxx. The former head of research at Sequenom, a biotech company based in San Diego, California, has pleaded guilty to conspiring to defraud the company's shareholders. Elizabeth Dragon was charged by the US Securities and Exchange Commission on 2 June with lying about the accuracy of the company's prenatal test for Down's syndrome. She acknowledged that she publicly stated that the test was nearly 100% accurate while knowing that this was not the case on three occasions between June 2008 and January 2009. S. GUPTA/EPA/CORBIS A court in Bhopal, India, has sentenced seven men to two years each in jail after finding them guilty of criminal negligence over an accident that spewed tonnes of poisonous gas from a chemical plant run by Union Carbide India in 1984, killing thousands. These are the first convictions related to the Bhopal gas accident, which continues to spark protests (pictured). Activist groups say they will appeal to higher courts for larger penalties for the seven and push for the extradition of Warren Anderson, head of Union Carbide at the time, from the United States. Oil giant BP's share price has been hit hard as the company fights to stem the flood of oil from its ruptured well in the Gulf of Mexico. Since the explosion on the Deepwater Horizon oil rig on 20 April, the company's shares have lost about 30% of their value. SOURCE: GOOGLE FINANCE Just before the accident, shares were riding at a four-year high of 653 pence, but had fallen to 495p by the end of May as the situation worsened. The biggest plunge came on 1 June, after the company confirmed that its 'top kill' operation to staunch the oil had failed (see graph). On 2 June, global financial-services company Credit Suisse estimated that clean-up costs alone could rise to US$23 billion, with lawsuits adding $14 billion to BP's spill bill. However, shares rallied briefly on 7 June after the news that BP was siphoning about 1.67 million litres of oil per day from the leak, 55–90% of the total official estimated flow. BP is not the only oil-industry company to take a plunge on the stock markets. Transocean, the offshore-drilling contractor which owned the rig, has seen its shares lose about half of their value in the past six weeks, and the share prices of other oil companies dropped after the 27 May announcement by the administration of US President Barack Obama of a six-month moratorium on deep-water oil exploration. Researchers convene at the Genetics Society of America's biennial meeting in Boston, Massachusetts, to discuss model organisms and human biology. → www.mohb.org/2010 Japan's Hayabusa space probe, which may be carrying a sample grabbed from the asteroid Itokawa, should land somewhere in south Australia. → http://hayabusa.jaxa.jp/e The committee that is independently reviewing the way the Intergovernmental Panel on Climate Change produces its reports holds its second meeting at McGill University in Montreal, Canada. → go.nature.com/onAkRb Preclinical drug development is the theme at the 9th Annual World Pharmaceutical Congress in Philadelphia, Pennsylvania. → www.worldpharmacongress.com San Francisco, California, hosts the 8th annual meeting of the International Society for Stem Cell Research, co-sponsored by the California Institute for Regenerative Medicine. → www.isscr.org/meetings The length of time a six-man crew will spend locked away in Moscow on a simulated mission to Mars. The crew entered their craft on 3 June and won't leave until November 2011. Source: European Space Agency The Bavarian environment minister's spokesperson comments on the discovery of trace-level contamination of crops around Germany with a banned genetically modified variety of maize. Source: Süddeutsche Zeitung There are currently no comments. This is a public forum. Please keep to our Community Guidelines. You can be controversial, but please don't get personal or offensive and do keep it brief. Remember our threads are for feedback and discussion - not for publishing papers, press releases or advertisements.
• Flu experts rebut conflict claims
  - Nature (London) 465(7299):672 (2010)
  Reports throw unsubstantiated suspicion on scientific advice given to the World Health Organization. Conspiracy theories have fuelled protests against influenza vaccination.D. Cheskin/PA "Drug firms 'encouraged world health body to exaggerate swine flu threat'," screamed Britain's Daily Mail newspaper on 4 June. "2 European reports criticize WHO's H1N1 pandemic guidelines as tainted," headlined The Washington Post the next day. To judge from media coverage last week, a major scandal had been exposed in the handling of the H1N1 flu pandemic by the World Health Organization (WHO). But nothing could be further from the truth. The news articles reported on two investigations: one by journalists at the BMJ and the Bureau of Investigative Journalism, a non-profit body in London launched in April; the other by the health committee of the Parliamentary Assembly of the Council of Europe (PACE) — a human-rights body based in Strasbourg, France, independent of the European Union. Both reports allege that the WHO might have been unduly influenced by the pharmaceutical industry in declaring H1N1 flu a pandemic, and in backing widespread vaccination and stockpiling of antiviral drugs, a claim often made by conspiracy theorists. They also complain that a 2004 WHO pandemic-preparedness document did not reveal that some of its authors had been paid for work by pharmaceutical companies — although the scientists had declared their competing interests elsewhere. Suspicious minds Paul Flynn, a UK Labour Member of Parliament and rapporteur of the PACE report (see go.nature.com/G9CvVL), and Fiona Godlee, editor-in-chief of the BMJ, presented their reports at a press conference together in Paris on 4 June, with Flynn asserting that "this was a pandemic that never really was". Afterwards, he wrote on his blog: "One of the joys today was giving evidence with the editor of the splendid British Medical Journal. We have never met before but we cooed in harmony and just avoided saying it was the Pharmas that did it." Both reports say that it is suspicious, for example, that the WHO has kept secret the names of its Emergency Committee, an expert group that advises the WHO on the status of international public-health emergencies, including the declaration of a flu pandemic. Gregory Hartl, a spokesman for the WHO, says that the secrecy of the Emergency Committee's membership is maintained to buffer its deliberations from outside pressure. The WHO says that it will make public the names, and any competing interests, once the pandemic is declared over. "A key question will be whether the pharmaceutical companies, which had invested around $4 bn (£2.8bn, €3.3bn) in developing the swine flu vaccine, had supporters inside the emergency committee, who then put pressure on WHO to declare a pandemic," says the article in the BMJ (D. Cohen and P. Carter Br. Med. J. 340, c2912; 2010). "It was the declaring of the pandemic that triggered the contracts." This is false. Many countries — including the United Kingdom, France, Belgium, Finland, Canada, the Netherlands and Switzerland — had already placed large orders for H1N1 vaccine weeks before the WHO declared H1N1 a pandemic on 11 June 2009. The United States, for example, ordered US$649 million of pandemic H1N1 influenza vaccine antigen and $283 million of adjuvant on 22 May 2009. So the Emergency Committee could not have influenced these in any way. "You are absolutely right," conceded the authors of the articles in the BMJ when challenged with this timeline. Both reports also seize on the WHO's April 2009 revision of its criteria on what constitutes a pandemic, which removed the need for an assessment of the 'severity' of the disease, based on estimates of future mortality. Flynn speculated in the Daily Mail that this was suspicious: "In this case, it might not just be a conspiracy theory, it might be a very profitable conspiracy." Neither report provides any evidence to substantiate its implication that the WHO rushed to declare a pandemic to boost pharmaceutical company sales. Moreover, the WHO says that the revisions were finalized in February 2009, before pandemic H1N1 was on the horizon. Scientists interviewed by Nature early on in the pandemic noted that severity is impossible to pin down until many months after it starts. Also, pandemic viruses can mutate or reassort to become more severe, so initial estimates are in any case of limited use. Clear firewall The BMJ also notes that three scientists who were involved in the preparation of a 2004 WHO document, WHO Guidelines on the Use of Vaccines and Antivirals during Influenza Pandemics, had received payments from pharmaceutical companies, including research funding, or consultancy or speaker fees. The scientists told the BMJ that they had declared these competing interests to the WHO, although the WHO had not included these in its report. Michael Osterholm, director of the University of Minnesota's Center for Infectious Disease Research and Policy in Minneapolis, points out that the 2004 document was based on input from an international panel of 22 scientists and public-health officials, in response to the threat of the deadly H5N1 avian flu virus. "To suggest that the three scientists were able to direct and control the final recommendations is naive, and stated without a single shred of evidence," he says. The BMJ also claims that industry funding of the European Scientific Working Group on Influenza (ESWI), a group of flu scientists that provided advice to the WHO, presented a "potential conflict of interest". It notes that several ESWI scientists also receive industry funding directly. One of those scientists is Albert Osterhaus, a virologist at Erasmus Medical Centre in Rotterdam in the Netherlands, who chairs the ESWI. He says that the body has a "clear firewall" with its funders, and that it informs all partners about any of its competing interests — its sources of funding are also listed on its website. Private–public partnerships are essential in tackling pandemics, and excluding flu researchers with industry links would deprive advisory panels of world-class expertise, he says. "The critical thing is transparency," says Osterhaus. "I have always declared my own competing interests." ADVERTISEMENT The BMJ acknowledges that the researchers had declared their interests elsewhere. But it takes issue with the WHO's not having included them in its pandemic-planning documents. David Ozonoff, an epidemiologist at the Boston University School of Public Health in Massachusetts, says that the reports "smear" the scientists involved in pandemic planning by "insinuating" that they would have offered different advice had they not had a relationship with drug companies. "This is a pretty serious charge," he says. "We think this is the researcher's reading into it, not necessarily ours," the BMJ authors respond. Marc Lipsitch, an epidemiologist at Harvard School of Public Health in Boston, Massachusetts, says that the WHO's advice on the pandemic has been sound, and has reflected the state of scientific opinion. Comparing the situation with the ongoing Deepwater Horizon oil spill, Lipsitch says that "it is ironic, as we watch for the second time in five years the catastrophic results of 'best-case scenario planning' in the Gulf of Mexico, to have the WHO coming under criticism for planning for, and raising awareness of, the possibility of a severe pandemic. That is what public-health agencies should do, and what most did in this instance, and they should be commended for it." There are currently no comments. This is a public forum. Please keep to our Community Guidelines. You can be controversial, but please don't get personal or offensive and do keep it brief. Remember our threads are for feedback and discussion - not for publishing papers, press releases or advertisements.
• Engineer set to run NSF
  - Nature (London) 465(7299):673 (2010)
  MIT's Subra Suresh poised to take top job. Subra Suresh is in line to head the US National Science Foundation.D. Coveney/MIT News Office As an engineer, Subra Suresh has made a career of studying stress and fatigue: from aluminium alloys in planes and silicon wafers in chips, to the walls of cells infected with malaria. As the man nominated by Barack Obama's administration to head the US National Science Foundation (NSF), Suresh may soon find himself as challenged as the materials in his lab, as the agency competes for cash in an increasingly austere budget climate. The 54-year-old dean of engineering at the Massachusetts Institute of Technology (MIT) in Cambridge, Suresh was named on 3 June to lead the $6.9-billion NSF, which funds research in the non-medical sciences. If confirmed by the Senate, he would succeed nuclear engineer Arden Bement, who completed his six-year term at the end of May. Suresh — who would be the first Indian American to direct the NSF — would take over just as a $3-billion infusion of economic stimulus money given to the agency early last year begins to run out. "Morale is going to be low," says Samuel Rankin, chair of the Coalition for National Science Funding, an advocacy group based in Washington DC. "He needs to take advantage of the fact that he's new and push for more funding." Suresh studied mechanical engineering at the Indian Institute of Technology Madras in Chennai and received a doctorate from MIT in 1981. His research career has ranged widely, from the macroscopic study of alloys to the microscopic study of thin films such as those used in silicon chips. Now, he focuses on the biomechanics of diseased human cells. Colleagues say he has tried to foster such interdisciplinary work at MIT, and has boosted the number of researchers who hold joint appointments at two departments. "Usually interdisciplinary means non-disciplinary," says Ben Freund, a mechanical engineer at Brown University in Providence, Rhode Island, who in 1983 recruited Suresh as an assistant professor there. "But he's truly made himself proficient in a number of areas and joined them in ways that have had an impact." Ares Rosakis, who met Suresh as a graduate student at Brown, sees similarities between Suresh and another US agency head: Steven Chu at the Department of Energy. Like Chu, Rosakis says, Suresh is an academic shape-shifter who enthusiastically devours new topics. And, like Chu, who directed the Lawrence Berkeley National Laboratory in California before he came to the capital, Suresh has maintained an active research lab after becoming an administrator. "You have to share some of the excitement of the process of research in order to actually implement the right changes," says Rosakis, now an aerospace and mechanical engineer at the California Institute of Technology in Pasadena. In another parallel with Chu, Suresh will arrive in Washington DC as a relative newcomer. "He may not have as much policy experience in Washington as some of his predecessors have had," says Eric Grimson, who works with Suresh as head of MIT's Department of Electrical Engineering and Computer Science. But Suresh has proved himself adroit at securing backing from fellow academics for endeavours such as revamping the engineering curriculum at MIT, his colleagues say. "I have no idea if his exposure to politics is enough," says Rosakis. "But academic politics can be very nasty too." ADVERTISEMENT If confirmed, Suresh is likely to encounter politics in spades. The stimulus funding that was a windfall for the NSF last year came alongside a whopping 6.2% rise in regular agency funding for fiscal year 2010. As a result, the NSF made a record number of research grants, using stimulus funds to boost the acceptance rate for a backlog of qualified proposals. But that rate will drop as the stimulus money peters out, says Rankin. Congress is unlikely to set the NSF's budget until after the midterm elections in November, which means funding could be frozen at 2010 levels well into next year. With concerns about the national debt rising, observers say that when Congress does act, the agency will be lucky to get half of the 8% budget increase that was requested by the White House in February. There are currently no comments. This is a public forum. Please keep to our Community Guidelines. You can be controversial, but please don't get personal or offensive and do keep it brief. Remember our threads are for feedback and discussion - not for publishing papers, press releases or advertisements.
• High hopes for Brazilian science
  - Nature (London) 465(7299):674 (2010)
  As President Lula prepares to leave office, researchers expect that innovation will invigorate the economy. It is rare that a head of state ends a second term with approval ratings of around 80%. But when Brazilian President Luiz Inácio Lula da Silva took to the stage last month at a science-policy conference, his popularity was clear: more than 3,000 scientists, administrators and industrialists stood to applaud him and to cheer his science minister of five years, Sérgio Rezende. Brazil's President Luiz Inácio Lula da Silva wants scientific investment to continue after his departure.G. MIRANDA/AGÊNCIA O GLOBO/NEWSCOM With a government convinced that science is an essential part of a growing economy, Brazilian researchers have never known better times, and the 4th National Conference on Science, Technology and Innovation in Brasilia on 26–28 May was brimming with optimism for an even sunnier future. At the conference, Lula signed a series of bills that will help to sustain his legacy of science investment after he and Rezende leave office on 1 January 2011. The bills, if enacted by the National Congress, will increase funding for postdocs and establish three new biodiversity research centres, with the overall goal being to further reduce the country's brain drain and perhaps even reverse it. The conference will deliver a consensus statement from Brazil's top scientific brass on where its research programme should focus over the next decade. The document is likely to be influential, says Luiz Davidovich, a director of the Brazilian Academy of Sciences and a physicist at the Federal University of Rio de Janeiro. "The conference is the first time that those at the heart of science, and those tangentially involved, have all been brought together — and at a point when things are really taking off," adds Carlos Henrique de Brito Cruz, the scientific director of FAPESP, São Paulo's state research foundation. The consensus statement, due to be published in two months' time, will be sent to all of the presidential candidates. One prominent suggestion expected to be in the statement is the fostering of centres of excellence. "We need to look after our Pelés as well as build more football pitches," says de Brito Cruz. "The current focus of funding is on new centres, but there is no specific programme to fund research stars." Another proposal is to provide more incentives for multinational companies to conduct research and development in Brazil. These policies would build on a well-funded foundation. The Brazilian Ministry of Science and Technology says that after Lula took office in 2003, total public and commercial funding for science and technology soared from 21.4 billion reais (US$11.4 billion) to 43.1 billion reais in 2008 (or from 1.26% to 1.43% of Brazil's growing gross domestic product; GDP) — due in part to Lula, and to policies implemented by former president Fernando Henrique Cardoso. Publications by Brazilians in peer-reviewed science journals have leapt from 14,237 in 2003 to 30,415 in 2008, according to data analysts Thomson Reuters. This is impressive not only in the context of Latin America but also compared with Russia, India and China, for example. In 2000, Brazil generated 43% of Latin America's peer-reviewed publications. Scientific output has since improved across the region, but in 2008, Brazilian publications made up 55% of the total. Brazil has particular strengths in agricultural science; for example, in 2000, a consortium based in São Paulo became the first in the world to sequence the genome of a plant pathogen, the bacterium Xylella fastidiosa, which destroys citrus crops. Brazil spends significantly more per researcher than China or Russia, according to its science ministry. "I believe we have reached a point where the sector will grow organically," says Rezende. "So the next person in charge will not have to do much." Science is also doing well at the level of individual states, which provide a significant source of public funding, although efforts to boost science are patchy. Many states are looking to emulate wealthy São Paulo, which has the strongest scientific tradition. "There is an article from 1947 in the constitution of the state of São Paulo," explains de Brito Cruz. "It says that 1% of all revenues of the state go towards research. No other science-funding agency in possibly the whole world has that kind of financial security and autonomy [from the federal government]." The benefits of having significant funding separate from federal sources were felt most keenly in the 1990s, when Brazil's government struggled with economic stresses such as hyperinflation. Science funding dried up elsewhere in the country, but researchers in São Paulo experienced much less disruption. Recently, other states have copied this legislation. In addition, São Paulo's three large state universities receive 9.57% of the state's income from its lucrative sales tax, giving them a unique boost. But even in São Paulo, the growth in published research has not been matched by growth in patented research, which is crucial if science is to invigorate the economy and provide a better quality of life for Brazil's 193 million inhabitants. Most scientists at the May conference agreed that solving this problem is probably the biggest challenge facing Brazilian science. Early in its tenure, Lula's administration made it legal for the government to fund research by private companies, and afforded tax breaks to firms that invest in innovation. But the number of patented inventions coming out of Brazil has risen only slightly since these measures were passed. "The problem is that company directors have the option of putting money in the hands of their heads of finance to generate a return in the financial markets, or in those of their head of research and development, which is risky and expensive," says Eduardo Viotti of Columbia University in New York, who advises the Brazilian senate on science policy. "In the past, at least, it has seemed less risky to them to bet on the financial markets." ADVERTISEMENT Commercial research and development is being boosted by the discovery in 2007 of large oil deposits off the coast of São Paulo and Rio de Janeiro. When oil does start flowing, Lula has promised that a proportion of the riches will be siphoned towards science. The exact percentage is still being debated, but it will be set before Lula and Rezende leave office. The chances are good that scientists will get much of what they ask for on their consensus wishlist, even after Lula's departure. The frontrunners in October's presidential election are José Serra, a former governor of science-friendly São Paulo, and Lula's former chief of staff Dilma Rousseff, who is backed by Lula and is expected to continue his policies. These may include his plan to raise science spending to 2% of GDP by 2020. There are currently no comments. This is a public forum. Please keep to our Community Guidelines. You can be controversial, but please don't get personal or offensive and do keep it brief. 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• Endangered-porpoise numbers fall to just 250
  - Nature (London) 465(7299):674 (2010)
  Time is running out for vanishing vaquitas. The diminutive vaquita is found only in the Gulf of California.F. Nicklin/Minden Pictures/FLPA At the northern end of the Gulf of California, where the Baja peninsula joins the rest of Mexico, the world's most endangered marine mammal is inching closer to extinction. With adults only 1.5 metres long, the vaquita (Phocoena sinus), a rare porpoise found only in these waters, epitomizes the plight of small cetaceans, which bear the brunt of pollution, ship traffic and fishing because they live in rivers and coastal areas. In China, the Yangtze river dolphin (Lipotes vexillifer) was last seen in 2007 and is now considered extinct. The vaquita — vulnerable to gill nets used by local fisherman — could be the next to go. On the basis of data gathered in 2008 during an acoustic survey1 researchers now estimate that only 250 individuals of the species remain, a drop of 56% in just over a decade. The finding was presented this week at a scientific meeting of the International Whaling Commission in Agadir, Morocco. "This information shows we don't have a lot of time to save the vaquita," says Timothy Ragen, executive director of the Marine Mammal Commission in Bethesda, Maryland, which part-funded the survey. First documented in 1958, the vaquita is an elusive and poorly understood species. Genetic analyses suggest that its ancestors were Southern Hemisphere porpoises that migrated north during the last ice age. Individuals travel in small groups and rarely attract attention by leaping or splashing. In 1997, Tim Gerrodette, a marine biologist at the Southwest Fisheries Science Center in La Jolla, California, led the first comprehensive survey of the vaquita, estimating the population to be 567 individuals2. A decade later, another analysis3, based on porpoise population rates and numbers of vaquita caught by fishermen, suggested that the number had dropped to 150. Fearing that the porpoise's population might become too small to survive, Lorenzo Rojas-Bracho, a marine biologist at the National Institute of Ecology office in Ensenada, Mexico, teamed up with Gerrodette and others in 2008 to undertake a new abundance analysis. The team used the research ship David Starr Jordan, operated by the US National Oceanic and Atmospheric Administration in Washington DC, and a small sailing boat, the Vaquita Express, sponsored by the Intercultural Center for the Study of Deserts and Oceans, in Tucson, Arizona, to count vaquita. The vessels ran coordinated transects in different water depths, tallying sightings and, on some transects, towing hydrophones to catch the porpoise's distinctive communicative clicks. "Coordinating the paths of a sailboat dependent on the wind and a motorized ship was a bit tricky," says Rojas-Bracho. The team combined the occurrence of vaquita clicks with the total area covered to estimate population size. Although the results show a precipitous decline in numbers since 1997, the findings are better than the earlier prediction. "We are encouraged, as it is not as bad as we feared," says Gerrodette. He and his colleagues were also encouraged by the sight of several newborn vaquitas. "But clearly, the number is not good news." Now the challenge is to protect the surviving group. In 2005, Mexico created a reserve and later followed this with a ban on gill nets in the area, which covers nearly 2,000 square kilometres in waters near San Felipe off the northern Baja peninsula. Vaquita easily become entangled in the nets and drown. ADVERTISEMENT Rojas-Bracho hopes to introduce alternative methods of fishing that do not rely on the nets. He and his colleagues also plan to deploy an array of 60 acoustic devices on the sea floor to detect population changes on the basis of the frequency and pattern of clicks. But because of the potential for vandalism by fishing supporters, the locations of these hydrophones cannot be marked with floating buoys. Instead, the team needs to devise an underwater system for locating and releasing the hydrophones. If successful, the system could serve as a model for monitoring other cetaceans. A more immediate challenge is to expand the protected area. "We need to get all the gill nets out of the water," says Ragen. But a broader ban would be a difficult economic and political challenge, pitting the vaquita against the livelihoods of local fishermen. * References * Dalton, R. Nature456, 431 (2008). | Article | PubMed | ChemPort | * Jaramillo-Legorreta, A., Rojas-Bracho, L. & Gerrodette, T. Marine Mammal Sci.15, 957-973 (1999). | Article * Jaramillo-Legorreta, A. et al. Conserv. Biol.6, 1653-1655 (2007). This is a public forum. Please keep to our Community Guidelines. You can be controversial, but please don't get personal or offensive and do keep it brief. Remember our threads are for feedback and discussion - not for publishing papers, press releases or advertisements.
• Glaciers' wane not all down to humans
  - Nature (London) 465(7299):677 (2010)
  Natural climate swings have had a major role in eroding Alpine ice. The Great Aletsch Glacier lost about 10 km3 of ice between 1890 (top) and 2005 (bottom).Roger Viollet Collection/Getty Images; G. Fischer/Photolibrary The Great Aletsch Glacier is ill. Over the course of the twentieth century, the largest Alpine glacier, in Valais, Switzerland, receded by more than two kilometres, and Switzerland's 1,500 smaller glaciers are not faring any better. Is it all down to man-made global warming? Not according to a recent study, which finds that about half of the glacier loss in the Swiss Alps is due to natural climate variability1 — a result likely to be true for glaciers around the world. "This doesn't question the actuality, and the seriousness, of man-made climate change in any way," says Matthias Huss, a glaciologist at the University of Fribourg in Switzerland, who led the study. "But what we do see is that current glacier retreat might be equally due to natural climate variations as it is to anthropogenic greenhouse warming." "This is the first detailed attribution of known climate forces on glacier behaviour," says Georg Kaser, a glaciologist at the University of Innsbruck in Austria, who was not involved in the study. "Given the importance of glaciers to local water supply, this is essential information." Researchers have long suspected that glaciers respond sensitively to natural climate swings such as those caused by the rhythmic rise and fall of North Atlantic sea surface temperatures by up to 1 °C roughly every 60 years. This Atlantic multidecadal oscillation (AMO), driven by changes in ocean circulation, is thought to affect phenomena including Atlantic hurricanes and rainfall in Europe. "The idea that glacier retreat is the solely due to increased air temperature is overly simplistic." In most places, historical records of glacier retreat and local climate are too sparse for researchers to separate the effect of this natural cycle from that of man-made warming. In the relatively well-monitored Swiss Alps, however, Huss and his team managed to gather some 10,000 in situ observations that had been made over the past 100 years, and constructed three-dimensional computer models of 30 glaciers. By comparing a time series of daily melt, snow accumulation and ice and snow volume readings of the glaciers with a widely used index of the AMO, they teased out the impact of natural climate variability. Although the mass balance of individual glaciers varied, the long-term overall trend followed the pulse of the AMO. Since 1910, the 30 glaciers have lost a total of 13 cubic kilometres of ice — about 50% of their former volume. Brief periods of mass gain during cool AMO phases in the 1910s and late 1970s were outweighed by rapid losses during warm phases in the 1940s and since 1980, when temperatures rose and more precipitation fell as rain than as snow. The scientists believe that these changes are due to the combined effects of the natural cycle and anthropogenic global warming, which now seems to have a greater role than early in the twentieth century. Subtle mix Natural climate variability is likely to have driven twentieth-century glacier shrinkage and thinning in other parts of the world, says Kaser. For example, his own research on the glaciers of Mount Kilimanjaro in Tanzania suggests that their dramatic recession is mainly due to multidecadal fluctuations in air moisture2. "The widespread idea that glacier retreat is the sole consequence of increased air temperature is overly simplistic," he says. "Glaciologists have known for more than 50 years that glaciers are sensitive to a variety of climate variables, not all of which can be attributed to global warming." Questions about the effect of global warming on glaciers hit the headlines earlier this year, after an error was found in the latest assessment report from the Intergovernmental Panel on Climate Change (IPCC), based in Geneva, Switzerland, which wrongly stated that most Himalayan glaciers could disappear by the year 20353. The resulting furore put the IPCC's credibility under scrutiny, and has triggered an independent review by the InterAcademy Council in Amsterdam, which represents 15 national academies of science. But scientists don't expect the latest findings on Swiss glaciers to rekindle the controversy. "Without studies like this, climate science would actually be less credible than it is," says Martin Beniston, a regional climate modeller at the University of Geneva in Switzerland, who was not involved in the study. "Problems related to global warming are caused by a subtle mix of human activity and natural changes, and these new findings are a rare opportunity to illustrate this complexity in a comprehensible way. It is a question of scientific honesty to admit that not all the effects of climate change are solely the result of increased greenhouse gases." ADVERTISEMENT Beniston adds that recognizing the role of natural climate shifts doesn't diminish the problem. "Even if greenhouse gases contribute just 50% to glacier retreat, this is anything but negligible." Although Himalayan glaciers may not be as vulnerable as the IPCC report originally suggested, the European Alps, where most glaciers are already in decline, could lose up to 90% of their glaciers by the end of the century, says Kaser. The authors of the latest study cautiously suggest that a phase shift in the AMO might give a reprieve to Great Aletsch and other Alpine glaciers in the next decades, but Beniston is doubtful. "We may see a temporary slowdown, but I fear in the long run the still fairly modest greenhouse effect will outweigh any Atlantic relief." * References * Huss, M., Hock, R., Bauder, A. & Funk, M. Geophys. Res. Lett.37, L10501 (2010). | Article * Kaser, G., Hardy, D. R., Molg, T., Bradley, R. S. & Hyera, T. M. Int. J. Climatol.24, 329-339 (2004). | Article * Schiermeier, Q. Nature463, 276-277 (2010). | Article | PubMed | ChemPort | This is a public forum. Please keep to our Community Guidelines. 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• US students pay for downturn
  - Nature (London) 465(7299):678 (2010)
  Tuition fees have risen, but public universities still face a shortfall, and students are feeling the squeeze. The global recession has produced some surprising winners. Take the companies that make 'clickers' — hand-held electronic gadgets that allow students to answer pop quizzes, discussion questions or straw polls during class. As university budgets are squeezed, class sizes are ballooning — and so is the demand for clickers, which help instructors to retain some interactivity in lecture classes of several hundred students. "Big classes are something we benefit from," confirms Kevin Owens, spokesman for Turning Technologies, a clicker maker based in Youngstown, Ohio. Few others can make that claim. Standing room only: university class sizes have exploded as funding has dropped.E. RISBERG/AP PHOTO Nearly three-quarters of the United States' 17 million undergraduates attend state-funded universities, according to the Association of Public and Land-grant Universities (APLU) in Washington DC. And those universities are suffering in the recession. When the economic crisis struck in autumn 2008, many state governments rescinded their budgets and made immediate cuts. The 2009 budget cycle brought more pain. A survey of 87 of the APLU's member institutions conducted late last summer found that 85% had had their state funds cut and that 50% had seen a drop in financial resources despite tuition hikes and increasing enrolment. The result? More than half the universities surveyed admitted that the cuts were affecting their undergraduates' education. Classes are swollen into the hundreds; there are fewer teaching assistants; hands-on laboratory sections and some whole courses have disappeared, programmes and departments have vanished (see 'Feeling the pinch'). And it's likely that things will get worse before they get better. Economic recovery has been slow and state tax revenues will necessarily lag behind. Meanwhile, federal stimulus funds approved by Congress in 2009 are set to run out this autumn. "Most undergraduates will experience larger classes and shorter hours of availability for labs and libraries." "Most undergraduates will experience larger classes and shorter hours of availability for labs and libraries," says David Shulenburger, vice-president for academic affairs at the APLU. "Some students simply won't be able to get into courses." Many of Europe's universities have also seen large budget cuts since the downturn. France and Germany have defied the trend by pumping more money into higher education, but funding has dropped in the Baltic States, the United Kingdom, Italy, Ireland and Romania, according to a report by the European University Association in Brussels. In the United States, science departments are tending to fare better than their counterparts in other fields, and in the United Kingdom special funds have been designated to boost science, technology, engineering and maths education. And Shulenburger says that at US state universities, science courses are less likely to be cut than low-enrolment humanities courses. Click for a larger version.SOURCE: APLU But many science undergraduates are also feeling the squeeze, especially at universities where departmental budgets are declining even as demand for courses increases. Cathy Koshland, vice-provost for academic planning and facilities at the University of California, Berkeley, says that although money for undergraduate science is tighter than at any time in recent memory, students are crowding into life-science classes. They are lured by an expected boom in health-care employment as the US population ages and by the growing prominence of biotechnology, bioenergy and biomedicine. "The hot fields at the moment have a strong biological component," says Koshland. The domino effect This affects more than just biology departments, she adds. "Students have to have a certain amount of math and chemistry before they take their first biology course," so crowding is increasing in classes for those subjects too. The result is a domino effect that can carry across years of a university programme. To cope with the crowding, departments at Berkeley have reduced the frequency or doubled the size of discussion sections. In some cases, lectures have been decoupled from labs, so that students don't have to take them during the same semester. Similar stories of shrinking resources and crowded classes are playing out across the country. At the University of Washington in Seattle, introductory biology courses have grown to 700 students and introductory chemistry classes have had half of their lab sessions cancelled. Meanwhile, the College of Southern Nevada in Las Vegas is squeezing extra classes in around the clock — even offering classes at midnight. Susan Elrod leads Project Kaleidoscope, which is funded, in part, by the National Science Foundation and aims to improve learning environments for undergraduate science, technology, maths and engineering. She worries about the effects large classes may have. "Faculty are teaching more students, and that takes away momentum that a department might have gained as far as creating more engaging, interactive courses. It is tempting to just put up the PowerPoint slides and lecture to students, even though we know that this is not very effective." The answer, at least until budgets improve, might be clickers, coupled with group work and "good conceptual questions" in classes and tutorials, Elrod says. Meanwhile, colleges are charging students more for less. Even as class sizes have increased, the Missouri University of Science and Technology in Rolla has raised tuition fees and, as many public universities were doing even before the recession began, looked to private donors to make up for lost state funds. "In the good old days we got about 60% of our operating budget from the state, and now I get about 27%," says Missouri's chancellor John Carney. "You can't have a quality academic programme with smoke and mirrors. You need revenue." One bright spot is the University of Michigan at Ann Arbor, which has for years been pursuing a conservative money-management strategy — a response to rocky times in the car industry that forms the state's economic base. The University of Michigan is set to hire 100 new faculty members by mid-2012. "Michigan has been in a challenging environment for so many years that we have gotten very good at saving money," says spokeswoman Kelly Cunningham. But even at Michigan, tuition fees have gone up. In the United States, fees at public universities have been increasing by nearly 5% above inflation every year since 1999, according to the College Board. The average annual tuition fee for an in-state student at a US four-year public university is $7,020 in the 2009–10 school year, putting pressure on students, parents and college aid programmes. "We've seen a dramatic increase in the demand for financial aid and a dramatic increase in the number of students who are eligible for student aid," says Haley Chitty, spokesman for the National Association of Student Financial Aid Administrators. The Federal Pell Grant Program promises aid to all who qualify, so federal spending on these grants has "exploded", according to Chitty. But universities can't make up holes in their budget just by raising tuition fees and raiding the Pell Grant fund through students. The highest Pell award for 2010–11 is $5,550. "The Pell grant increase isn't going to be nearly enough to cover the tuition increase," says Chitty. According to Shulenburger, "Students' dollars are being substituted for state dollars". Diane Auer Jones, former assistant secretary for postsecondary education at the Department of Education and current head of the Washington Campus, which provides policy training for business students, thinks budget pain should make smaller state schools rethink their research ambitions (see Nature 465, 32–33; 2010). "You look at a state like Maryland where there are these university systems," she says. "In the old days the flagship and one other were the research universities. All the other campuses in the system were teaching colleges. The problem is that in the past 15 years the non-flagship comprehensive undergrad institutions have all decided that they too need to be research universities." The result, Auer Jones says, is that administrators have been spending too much on programmes besides undergraduate education. Faculty members are rewarded on the basis of their research portfolios, and teaching gets mere lip service. Auer Jones's opinions resonate with many in higher education, but, not surprisingly, the institutions contacted by Nature all felt that her criticisms were more applicable to other universities. "You would not be able to continue here if you weren't a good teacher," says Diane Allen, the provost at Salisbury University, a small Maryland state institution that Auer Jones attended. But, she adds, "all of our faculty are expected to do some kind of scholarly work. They need to stay current in their field." Into the field John Banks, who teaches environmental science at the University of Washington in Tacoma, says he still sees the value to his students of coming along on his fieldwork trips in Costa Rica and East Africa. They learn things they couldn't in the classroom, he says. "Everything from linear statistics to negotiating entry to a national park with a ranger with a machine gun at 5'o'clock in the morning." ADVERTISEMENT Yet although the recession has added stress and cost to the undergraduate education system, most administrators argue that an undergraduate degree remains good value. "I don't apologize for the tuition we have to charge because when our students graduate they are making 50,000–70,000 [dollars] a year," says Carney. "It is a tremendous bargain." Shulenburger agrees. "There's still no better investment, long run, than getting that degree," he says. All those in agreement, press your clickers now. There are currently no comments. This is a public forum. Please keep to our Community Guidelines. You can be controversial, but please don't get personal or offensive and do keep it brief. 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• Ecology: Emergency medicine for frogs
  - Nature (London) 465(7299):680 (2010)
  With chytrid fungus rapidly spreading around the world, researchers are testing an extreme approach to saving endangered amphibian populations. Naomi Lubick reports from a rescue site. Download a PDF of this story. On a mid-April afternoon with rain threatening, Jaime Bosch clambered down the carbonate cliffs of northern Mallorca to a pond overlooking the Mediterranean Sea. After removing his shoes and wading into the shallow pond, Bosch quickly netted 30 wriggling tadpoles and dropped them into a bag filled with water. He gently grabbed one in plastic-gloved hands, swabbed its mouth with a cotton-tipped stick, and returned the tadpole to the pond. The important information was in the DNA captured on the end of the stick. Bosch, an evolutionary biologist at Spain's National Museum of Natural History in Madrid, and his colleagues had conducted a back-breaking experiment in 2009 to try to rid this particular pond of the chytrid fungus Batrachochytrium dendrobatidis. The fungus causes a disease called chytridiomycosis that has wiped out amphibian populations around the world. Given that history, Bosch and his team resorted to extreme measures to save the frogs on Mallorca. At this small pond, the researchers removed all the resident tadpoles during the spring and summer, treated them with an antifungal medication and returned them to the water after cleaning the pond. When Bosch returned to the site this spring, he brought his swabbing kit to see whether the frogs were free of the fungus. He hopes that results from the experiment in Mallorca can help other scientists, who are testing similar approaches in Switzerland and California. These and other frog researchers are closely watching the work in Mallorca because the species in the pond, the midwife toad, makes this an important test case. "Midwife toads are a sentinel species because they are so susceptible to chytrid," says Matthew Fisher, an epidemiologist at Imperial College London. Fisher heads a consortium called RACE (Risk Assessment of Chytridiomycosis to European amphibian biodiversity), which is working with Bosch. Funded by the European Union's Biodiversa research project, RACE brings together specialists such as mycologists and herpetologists to find ways to control the spread of chytrid fungus across Europe. When Bosch's team evacuated the Mallorcan tadpoles last summer, its goal was to completely eradicate the fungus, which gained a foothold on the island about 20 years ago when frogs were imported to help boost a native species. "Only four populations are infected, which is why we need to take action very quickly on the island," says Bosch. The local government agreed and pitched in, donating employees' time and funding. Some of that money covers the tests to detect fungal DNA on the tadpoles. More funding comes from the Spanish National Research Council (CSIS) and from the regional government of Madrid — which has also spent about €75,000 (US$90,000) on a small breeding centre for two endangered frog populations in Spain's Peñalara Nature Reserve, the site of the first European outbreak of chytridiomycosis in 1997. Fungal attacks threaten Mallorcan midwife toads.J. BOSCH Last year, several scientists — including researchers from the Zoological Society of London (ZSL) and others affiliated with RACE — evacuated more than 2,000 tadpoles from the Mallorcan pond, which is near the coast, west of Pollença. In four trips between late March and early August, the team carried hundreds of tadpoles at a time in well-cleaned, 2-litre bottles, filled with pond water and rigged with aquaria air pumps to get oxygen to the tadpoles during the three-hour hike out to the nearest road. The researchers then drove for several hours to a lab facility at Marineland, a dolphin tourist attraction across the island. There, the tadpoles completed a week-long regimen of daily 5-minute baths in itraconazole, an antifungal medicine, and were placed in glass aquaria for up to seven months. Meanwhile, Bosch returned to the pond several times, to attempt to dry it out, because it is suspected that chytrid fungus needs moisture to survive. Eventually, he emptied the pond as much as he could with a bucket, and left it to dry in the hot Mallorcan summer. When the pond refilled with rain in the autumn, Bosch's team airlifted the tadpoles across the island to their home, with the hope that they would survive in the now-clean pond. Sweet sounds Signs were good when Bosch arrived back at the pond in April and heard faint, bell-like pings. Tracking the sound, he raced up a jagged cliff and found two healthy-looking adults, one of which had several eggs attached to its posterior. Male midwife toads carry the fertilized eggs and normally spend their days away from the pond, which makes them difficult to find, says Bosch. This male frog would soon be returning to the water to release the eggs. Adult midwife toads are particularly vulnerable to the chytrid fungus because it attaches to keratin, which covers the adults but only the mouths of tadpoles. So finding the adults near the pond lifted Bosch's hopes, as did the hundreds of tadpoles swimming in the water. Bosch chose Mallorca as a test site partly because the dry environment and the widely separated amphibian populations slow the spread of the fungus. That provides researchers with a chance to wipe out the pathogen, says Bosch. By contrast, it would be out of the question to eliminate the fungus from rainforest communities, such as in Central America, where water is plentiful and the fungus is widespread1. In places where complete eradication is impractical, researchers are hoping to take a modified approach, which relies on the fact that some populations of frogs survive chytrid attack. Vance Vredenburg, a biologist at San Francisco State University in California, is searching for such populations in the Sierra Nevada. The mountain yellow-legged frog in this range is one of the most endangered amphibians in the United States and has been hit by many chytridiomycosis outbreaks over the past decade. By analysing how the frogs responded to those infections, Vredenburg and his colleagues have developed a model that can help to identify the populations that are most at risk and which might benefit from limited intervention2,3. Researchers drained this remote Mallorcan pond to try to save midwife toads from chytridiomycosis.N. LUBICK Vredenburg is now adapting Bosch's technique to try to protect communities by treating some individuals. This spring, his group started capturing frogs in cages in the field, washing them in 5-minute antifungal bath each day for a week and then releasing them. In late April, Bosch received the results from colleagues at the ZSL who had completed the analyses on the tadpole swabs taken in Mallorca. Every sample came back positive for B. dendrobatidis, which means that all the tadpoles in the pond probably carry the fungus, says Bosch. But the number of spores detected on each swab was far smaller than the number seen in tests the previous year, suggesting a lower level of infection. Even so, the news stunned Bosch and his colleagues, who are struggling to understand how the pathogen survived in the pond at all. Jon Bielby, an ecologist at the ZSL's Institute of Zoology, who assisted last year in carrying tadpoles out, wonders whether the froglets — a stage between tadpole and adult that temporarily leaves the pond — picked up the fungus elsewhere and brought it back. Other RACE researchers have floated the idea that the fungus may have a life stage outside water. "But the honest answer is we don't know," says Bielby. Reduction not eradication The results on Mallorca, although disappointing, are valuable for other researchers trying to combat outbreaks of B. dendrobatidis (Bd), says Benedikt Schmidt, coordinator for amphibian and reptile conservation with the Swiss frog-protection group KARCH, based in Neuchâtel. "It means that we should focus on reducing prevalence rather than trying to eradicate Bd from our ponds," he says4. Schmidt and his colleagues hope to use a variation of Bosch's technique to bolster local populations infected by the fungus in Switzerland. Working with graduate student Corina Geiger at the University of Zurich, Schmidt will pull some frogs from three ponds, treat them, and reintroduce them. They will compare those ponds with two untreated control populations. This treatment approach may become a tool to help frogs survive if the chytrid situation in Switzerland and other sites across Europe worsens, he says. Schmidt is also focusing on other threats to frog populations, the biggest of which may be the loss or degradation of habitat. Schmidt worries that human-initiated changes to the landscape, from farming practices to creeping urbanization, are stressing amphibian populations and may be making them more vulnerable to chytrid fungus. Schmidt and others say there is still much unknown about how chytrid affects frogs. Researchers with the RACE project and on Vredenburg's team are looking into the genetics of the fungus, trying to determine whether the infectious strains vary every year, perhaps like the flu strains that infect humans. Biologists are also trying to pinpoint frogs' molecular responses to the fungus — which genes get turned on, and which proteins are produced in response to the infection. Although disappointed by his first test in Mallorca, Bosch remains undeterred. The reduced severity of infection may help the frogs survive in the pond, he says. And his team may try another round of antifungal baths this summer, perhaps treating the difficult-to-locate adult frogs as well as the tadpoles. ADVERTISEMENT Bosch also started up fieldwork in Portugal and Hungary earlier this spring, searching for more amphibian populations infected with chytrid fungus. He is hoping to modify the Mallorcan method to treat midwife toad populations first in Peñalara, and then elsewhere in Spain as well as in Eastern Europe. Bosch's first work with frogs was studying their communication and mating habits. His iPhone rings with the calls of six different frog species that he recorded, and he wishes he could resume his research on sexual behaviour. But for the past ten years, Bosch has spent his days working to preserve frog populations before they disappear. "It's nicer," he says, "to work with live animals." Naomi Lubick is a freelance writer based in Zurich, Switzerland. * References * Lips, K. R.et al. Proc. Natl Acad. Sci. USA103, 3165-3170 (2006). * Vredenburg, V. T. , Knapp, R. A. , Tunstall, T. S. & Briggs, C. J.Proc. Natl Acad. Sci. USA107, 9689-9694 (2010). * Briggs, C. J. , Knapp, R. A. & Vredenburg, V. T.Proc. Natl Acad. Sci. USA107, 9695-9700 (2010). * Tobler, U. & Schmidt, B.PLoS ONE5, e10927 (2010). There are currently no comments. This is a public forum. Please keep to our Community Guidelines. You can be controversial, but please don't get personal or offensive and do keep it brief. Remember our threads are for feedback and discussion - not for publishing papers, press releases or advertisements.
• Science economics: What science is really worth
  - Nature (London) 465(7299):682 (2010)
  Spending on science is one of the best ways to generate jobs and economic growth, say research advocates. But as Colin Macilwain reports, the evidence behind such claims is patchy. Download a PDF of this article. President Barack Obama says it. Francis Collins, director of the US National Institutes of Health (NIH), says it. University and research leaders elsewhere are saying it, too. The number one current rationale for extra research investment is that it will generate badly needed economic growth. "Science is more essential for our prosperity, our health, our environment and our quality of life than it has ever been before," said Obama, addressing the National Academy of Sciences in Washington DC last year. Getting down to the details, Collins has recently cited a report by Families USA, a Washington DC-based health-advocacy group, which found that every US$1 spent by the NIH typically generates $2.21 in additional economic output within 12 months. "Biomedical research has generally been looked at for its health benefits, but the case for it generating economic growth is pretty compelling," says Collins. In Britain, senior scientists have called on the government to support science as a means of helping the economy out of recession. Heeding such arguments, governments in Germany, Sweden, Canada and Australia, as well as the United States, have increased research spending as part of stimulus packages designed to aid their struggling economies. Beneath the rhetoric, however, there is considerable unease that the economic benefits of science spending are being oversold. The Families USA study used a model developed by the Bureau of Economic Analysis at the US Department of Commerce to deduce the likely benefits of NIH spending in each state. Collins says he has been advised that the approach is "standard and considered reliable". But some economists question the basic assumption behind such models — that a certain amount of research input will generate corresponding economic outputs — or that those outputs can be quantified. Costs or benefits The problem, economists say, is that the numbers attached to widely quoted economic benefits of research have been extrapolated from a small number of studies, many of which were undertaken with the explicit aim of building support for research investment, rather than being objective assessments. The economics of health research, on which much analysis of costs and benefits has been focused, "has had very little money invested in it", says Martin Buxton, director of the Health Economics Research Group at Brunel University, UK. "And too much of what has been done, has been done as a process of advocacy." "Too much of what has been done, has been done as a process of advocacy." Research leaders acknowledge that they need better tools. Collins says that the NIH held a workshop with economists in May to see whether it should invest some of its funds into economic outcomes. "We're very interested in tightening up the evidence base," he says. Some of that evidence is already being collected. Under the programme STAR METRICS (Science and Technology in America's Reinvestment — Measuring the Effects of Research on Innovation, Competitiveness and Science), implemented after the US stimulus package was introduced, the Obama administration is seeking to trace the effect of federal research grants and contracts on outcomes such as employment, publications and economic activity (see Nature 464, 488–489; 2010). The programme's supporters say it will provide justification for the stimulus money — exactly what research agencies need as they come under pressure to show what recent investments have produced. Economic arguments have always been used to make the case for science spending, particularly when times are tough. In the United States, these were strengthened by the publication of Rising Above the Gathering Storm, an influential 2006 report from the US National Academies, which called for the sharp expansion of publicly funded research and development to stave off competition from China and elsewhere. The 600-page report, written by a panel chaired by Norman Augustine, former chairman of Lockheed Martin, was put together by a large panel of senior scientists in a matter of weeks, to meet a tight congressional deadline. In a section titled "Why are science and technology critical to America's prosperity in the 21st century?" the report reviews the literature that estimates return on investment (ROI) from research (see Table 2). This is illustrated by various graphs, including one showing steep declines in US death rates from heart disease between 1950 and 2000, inferring that this drop can be partly attributed to biomedical research. Innovation drive Gathering Storm recommended that federal investment in basic research should increase by 10% every year for seven years, and led Congress to consider spending increases of that order, mainly in the physical sciences and engineering. When the newly elected President Obama was hurriedly preparing a February 2009 bill aimed at stimulating economic growth, parts of these increases were thrown in, together with extra spending for the NIH. In the end, the American Recovery and Reinvestment Act included a further $21 billion of research spending, all justified by its supporters on the grounds that it would yield speedy economic returns. Yet Stephen Merrill, executive director of the Board on Science, Technology and Economic Policy at the National Academies but who was not involved in the report, concedes that Gathering Storm doesn't, in itself, make a detailed case for the economic benefits of investing in research. For that, one has to look further back in the literature. Economists have agreed for decades that a large component of modern economic growth has to be driven by 'innovation' — that is, the arrival of new ideas and technologies. "We have very good evidence that 50–70% of productivity growth arises from innovation," says Iain Gillespie, head of the Science and Technology Policy Division at the Organisation for Economic Co-operation and Development in Paris. Greater difficulty arises in determining what drives the innovation, though. Is it basic research, often publicly funded, as the science advocates contend? Or are other factors, such as the demands of consumers who buy, say, mobile phones or computer games, also involved? And even if scientific research does drive innovation, will more investment in science necessarily speed up the process? Unfortunately, economists concede, no one really knows. In one of the bedrock papers in this field, Edwin Mansfield, the late University of Pennsylvania economist, estimated that academic research delivered an annual rate of return of 28% (E. Mansfield Research Policy 20, 1–12; 1991). The figure has been widely quoted ever since. But Mansfield reached this estimate by interviewing chief executives, asking them what proportion of their companies' innovation was derived from university research and, in effect, demanding that they come up with a number. "He was asking an impossible question," says Ben Martin, a former director of the Science and Technology Policy Research Unit at the University of Sussex, UK. "Methodologically, this was a dubious thing to do." NIH director Francis Collin is exploring new ways to document the effect of research investment.A. HARRER/BLOOMBERG VIA GETTY Whatever method economists have used since, measuring the ROI from research has proved tough, and has produced a wide range of values (see Table 2). Some look at the 'micro' level, asking things such as: what contribution did a dozen neuroscience grants received by the University of Cambridge in 1972 eventually make to drug development? Such efforts are complicated, however, by the difficulties of attributing credit for any given drug to the numerous research teams involved over time. Policy-makers are more interested in the 'macro' question, measuring the effect of combined research activities on a country's economic growth. According to Merrill, repeated efforts to pin down firm numbers here have also failed. "It is fair to say that this is an analytical dead end," he told attendees at the American Association for the Advancement of Science annual meeting in February. Exceptional returns? Martin says that for much of the literature, "there is some PR, rather than rigorous research involved". This influence derives in part from the activities of US medical research lobbyists. An example is the 2000 report Exceptional Returns: The Economic Value of America's Investment in Medical Research by Funding First, an initiative of the Mary Woodard Lasker Charitable Trust that advocated biomedical research spending. Pointing to work by various economists, the document estimated that the steep decline in cardiovascular deaths in the United States between 1970 and 1990 has an economic value of $1.5 trillion annually, and deduced that one-third of this — $500 billion a year — could be attributed to medical research that led to new procedures and drugs, a finding that was echoed in the Gathering Storm report. A plethora of studies in the United States and Australia followed through with similar claims. Funding First has been disbanded, but Robert Topel, who studies labour economics at the University of Chicago and whose work was cited in the report, distances himself from some of its claims. "Probably only a little of the fall in the cardiovascular death rate has to do with surgery and beta-blockers," he says. "It is very hard to take changes in public health and attribute their cause." Topel also questions the report's implication that publicly funded biomedical research will create thousands of jobs in the pharmaceutical and biotechnology industries. Topel says that Mark Hatfield, the former Republican senator for Oregon who wrote the report's introduction, was constantly fishing for job numbers. "We kept telling Hatfield that jobs are a cost, not a benefit." "It is very hard to take changes in public health and attribute their cause." The price of research A key problem, says Topel, has been economists' inability to measure the costs of research as well as the benefits. These costs include the added expense of caring for elderly patients kept alive by new treatments, the costs of talented people doing research instead of something economically productive (such as running a technology company or an ice-cream van), and the cost of wayward outcomes, such as nuclear clean-up — a long-term 'outcome' of the research and development of nuclear energy and weaponry. Research agencies have no interest in assessing the costs of research fairly, says Barry Bozeman, a science-policy specialist at Georgia Institute of Technology in Atlanta. "Honest clients are in short supply" for research in this field, he says. "Most of them think they already have the answers, and want someone to find the numbers to prove them right." The flaws in the 'exceptional returns' literature were thrown into sharp relief in a November 2008 study called Medical Research: What's it Worth? by the London-based Wellcome Trust and the UK Medical Research Council. In it, some UK health economists attempted to make rigorous estimates of the economic benefits of publicly and charitably funded medical research in Britain. They estimated that every pound invested in cardiovascular disease and mental-health research brought about, through improved health, economic returns of 9% and 7%, respectively. Work in both fields generated an extra return of 30% through 'spillover' effects from research to the broader economy, such as training and industrial activity. But the report said that these findings were "at best tentative", and spelled out a long list of knowledge gaps. Little is known about how long the economic benefits of research take to accrue; nor the extent to which the benefits of research done in one country or region are specific to that area, which is a central question for policy-makers. "Three-quarters of the benefits are in spillover, and that's where the evidence is weakest," says Jonathan Grant, president of RAND Europe in Cambridge, UK, and one of the study's main authors. Grant also questions the way that data on ROI gathered in one sector, such as agricultural research, have sometimes been applied to others. "Most of the empirical evidence in this area is (a) historical, (b) American and (c) from agriculture. How transferable is that? It's a big question in my mind," he says. Efforts to strengthen the evidence are increasing. An $8-million-a-year grants programme at the National Science Foundation (NSF), for example, is supporting investigations by science-policy specialists and economists into various aspects of research economics, including several approaches to measuring the impact of Obama's stimulus package. The programme grew out of an initiative to build "a scientifically rigorous, quantitative basis" for research policy, launched in 2005 by John Marburger, then science adviser to President George W. Bush. As Marburger explains, "We need disinterested people — as opposed to the current situation, where everyone involved has an interest in the outcome." Julia Lane, head of the NSF project, is also directing the related STAR METRICS programme. The first aim of the programme is to build a 'clean' database of all federally funded researchers in the United States — current records are confused, with conflicting information on names and affiliations — and estimate the number of people that they keep in employment. Later on, the plan is to track patents, citations and other metrics of the research's impact. Lane, like Marburger, suggests that researchers' use of the Internet to communicate and publish will enable STAR METRICS to track the creation and transfer of knowledge properly for the first time. "In the past, we haven't had the data infrastructure to do a full analysis," she says. Tobin Smith, vice-president for policy at the Association of American Universities in Washington DC, is confident that the first STAR METRICS results in summer 2011 will help to show doubters that the stimulus-bill money has been wisely spent. "It will certainly help me," he says, "by telling our campuses how many people they are keeping in work — something universities have never been able to do." Like Smith, most research leaders and advocates seem assured that new data will reveal the healthy return on investment they have been touting all along. ADVERTISEMENT Not that this guarantees that the economic growth argument will continue to persuade. There are signs that a backlash against further research spending is already emerging. In May, the US House of Representatives decisively rejected a bill that would have authorized increased research funding for physical sciences agencies, and in Britain, research spending cuts by the newly elected government are widely anticipated. The pressure is building to show what earlier investments have produced. As one former congressional staffer, who didn't want to be named, puts it: "If it turns out that all the stimulus has done is hire a load of foreign postdocs, there's going to be trouble." There are currently no comments. This is a public forum. Please keep to our Community Guidelines. You can be controversial, but please don't get personal or offensive and do keep it brief. Remember our threads are for feedback and discussion - not for publishing papers, press releases or advertisements.
• UK scientific societies need support to increase their impact
  - Nature (London) 465(7299):685 (2010)
  British scientists can connect to new Members of Parliament (MPs) through UK professional bodies other than the Campaign for Science and Engineering and the Royal Society (Nature 465, 135; 2010).
• On the occurrence of similar traits in related organisms
  - Nature (London) 465(7299):685 (2010)
  In Eugenie Scott's review of my book How Science Works: Evolution (Nature 465, 164; 2010), she perpetuates the common error of confusing the definition of the biological term 'homology' with its interpretation.
• Adaptive strategy recommended for US ocean planning
  - Nature (London) 465(7299):685 (2010)
  The United States could learn from the experience of other nations in implementing its proposed ocean-management policy for coastal and marine spatial planning (Nature 465, 9; 2010).
• Reward research that benefits society, with kudos or even cash
  - Nature (London) 465(7299):685 (2010)
  Broadening the impact of university research on society (Nature465, 416–418; 2010) should be included in the academic reward structure.The present scientific reward system threatens to imprison academics in their ivory towers.
• Nature Europe site should highlight most productive countries
  - Nature (London) 465(7299):685 (2010)
  Your newly launched website showcasing Nature Publishing Group's European output (http://www.nature.com/regions/europe
• Defeating the merchants of doubt
  - Nature (London) 465(7299):686 (2010)
  As climate scientists battle climate sceptics, they should note that we have been here before, say Naomi Oreskes and Erik M. Conway. History holds lessons for how researchers can get their message across.
• Sex bias in trials and treatment must end
  - Nature (London) 465(7299):688 (2010)
  Gender inequalities in biomedical research are undermining patient care. In the first of three related pieces, Alison M. Kim, Candace M. Tingen and Teresa K. Woodruff call on journals, funding agencies and researchers to give women parity with men, in studies and in the clinic.
• Pregnant women deserve better
  - Nature (London) 465(7299):689 (2010)
  Clinical trials routinely exclude expectant mothers. This is unethical and unscientific, and regulators must mandate change, says Françoise Baylis, in the second of three related pieces on gender bias in biomedicine.
• Males still dominate animal studies
  - Nature (London) 465(7299):690 (2010)
  Many researchers avoid using female animals. Stringent measures should consign this prejudice to the past, argue Irving Zucker and Annaliese Beery, in the third piece of three on gender bias in biomedicine.
• Lessons in carbon trading
  - Nature (London) 465(7299):691 (2010)
  The most extensive evaluation to date finds that the European Union Emissions Trading Scheme is robust and successfully cut the region's emissions in its first three years, explains Michael Grubb.
• Predicting human activity
  - Nature (London) 465(7299):692 (2010)
  We usually assume that we do things for a reason, whether we are obeying the dictates of the unconscious, rational self-interest or our genetic predisposition. Yet such determinism cannot predict the diverse range of human behaviour.
• The crop circle evolves
  - Nature (London) 465(7299):693 (2010)
  A growing underground art movement combines mathematics, technology, stalks and whimsy. Richard Taylor looks forward to a bumper batch of intricate crop patterns this summer.
• Stem cells: Cues from steroid hormones
  - Nature (London) 465(7299):695 (2010)
  The steroid hormones oestrogen and progesterone have a role in sickness and in health. In breast tissue, both roles probably work through a single mechanism: controlling the number and activity of mammary stem cells.
• Nonlinear dynamics: Chaotic billiard lasers
  - Nature (London) 465(7299):696 (2010)
  The chaotic motion of light rays gives microlasers surprising emission properties, enhancing quantum tunnelling by many orders of magnitude and producing highly directional output beams.
• Blood-vessel formation: Bridges that guide and unite
  - Nature (London) 465(7299):697 (2010)
  To form new blood vessels, the endothelial tip cells of two existing vessels come together by the process of anastomosis. But how do they find each other? Macrophages seem to provide a bridge and mediate their union.
• 50 & 100 years ago
  - Nature (London) 465(7299):699 (2010)
  'Self-regulation for children' — In the review of our book "The Free Family" ... Prof. Vernon says that "it is clear that the children experienced considerable difficulty in adapting themselves to the society of children very differently brought up", and he asks for independent evidence for their "spontaneity, poise and stability". Allow me to say that Prof. Vernon could only have gained the first false impression because we describe in detail the kind of conflicts one of our children had in a specific instance.
• Quantum optics: Single-atom transistor for light
  - Nature (London) 465(7299):699 (2010)
  A subtle quantum-interference effect has been used to control the optical response of a single atom confined in a cavity. It could offer a means to develop logic gates for an optical quantum computer.
• Neuroscience: fMRI under the spotlight
  - Nature (London) 465(7299):700 (2010)
  Analysis of a selected class of neuron in the brains of live animals using functional magnetic resonance imaging (fMRI) opens the door to mapping genetically specified neural circuits.
• Fluid dynamics: Saliva at a stretch
  - Nature (London) 465(7299):701 (2010)
  Abstract
• Planetary science: The birth of Saturn's baby moons
  - Nature (London) 465(7299):701 (2010)
  Simulations show that Saturn's nearby moons, after forming on the outskirts of the planet's main rings, get pushed clear of them. This model reproduces the moons' orbital locations and remarkably low densities.
• Plasticity
  - Nature (London) 465(7299):703 (2010)
  Nature | Insight | Review Article Nuclear reprogramming to a pluripotent state by three approaches * Shinya Yamanaka1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Helen M. Blau3 Search for this author in: * NPG journals * PubMed * Google Scholar * AffiliationsJournal name:NatureVolume:465,Pages:704–712Date published:(10 June 2010)DOI:doi:10.1038/nature09229Published online09 June 2010 Abstract * Abstract * Author information * Comments Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg The stable states of differentiated cells are now known to be controlled by dynamic mechanisms that can easily be perturbed. An adult cell can therefore be reprogrammed, altering its pattern of gene expression, and hence its fate, to that typical of another cell type. This has been shown by three distinct experimental approaches to nuclear reprogramming: nuclear transfer, cell fusion and transcription-factor transduction. Using these approaches, nuclei from 'terminally differentiated' somatic cells can be induced to express genes that are typical of embryonic stem cells, which can differentiate to form all of the cell types in the body. This remarkable discovery of cellular plasticity has important medical applications. View full text Figures at a glance * Figure 1: Three approaches to nuclear reprogramming to pluripotency. , Nuclear transfer. In this approach, the nucleus of a somatic cell (which is diploid, 2n) is transplanted into an enucleated oocyte. In the environment of the oocyte, the somatic cell nucleus is reprogrammed so that the cells derived from it are pluripotent. From this oocyte, a blastocyst is generated, from which embryonic stem (ES)-cell lines are derived in tissue culture. If development is allowed to proceed to completion, an entire cloned organism is generated. , Cell fusion. In this approach, two distinct cell types are combined to form a single entity. The resultant fused cells can be heterokaryons or hybrids. If the fused cells proliferate, they will become hybrids, and on division, the nuclei fuse to become 4n (that is, twice the number of chromosomes in a somatic cell) or greater. If the cells are derived from the same species, their karyotype will remain euploid (that is, they will have balanced sets of chromosomes); however, if they are from different species, the! y will be aneuploid, as chromosomes will be lost and rearranged. Heterokaryons, by contrast, are short-lived and do not divide. As a result, they are multinucleate: the nuclei from the original cells remain intact and distinct, and the influence of one genotype on another can be studied in a stable system in which no chromosomes are lost. If the heterokaryons are of mixed species, the gene products of the two cell types can be distinguished. By altering the nuclear ratio in the fusion, and hence the stoichiometry of the regulators provided by each type of cell, the heterokaryon is reprogrammed towards the desired cell type (Fig. 3). Culture medium also has a role and needs to have a composition favoured by the desired cell type. Dashed arrows indicate slower processes (involving multiple rounds of cell division) than solid arrows (no division). , Transcription factor transduction. This approach can be used to form induced pluripotent stem (iPS) cells, which have similar pro! perties to ES cells and can be generated from almost any cell ! type in the body through the introduction of four genes (Oct4, Sox2, Klf4 and c-Myc) by using retroviruses. The pluripotent state is heritably maintained, and vast numbers of cells can be generated, making this approach advantageous for clinical applications. 1n, haploid. * Figure 2: Timeline of discoveries in nuclear reprogramming. Three approaches to nuclear reprogramming are described: nuclear transfer (blue), cell fusion (pink) and transcription-factor transduction (green). These complementary approaches have provided synergistic insights for almost 50 years and continue to inform the understanding of nuclear reprogramming and influence medical advances. EG cell, embryonic germ cell. * Figure 3: Investigating the genes involved in nuclear reprogramming by using mixed-species heterokaryons. Cell fusion leads to nuclear reprogramming towards a specific phenotype, which is dictated by the nuclear ratio of the fused cell types in heterokaryons, which do not divide. When, for example, cells from humans and mice are fused in a skewed ratio (such as 1:3) (), the human cells will generally be reprogrammed towards the mouse cell phenotype (three examples are shown). To uncover which genes are involved in this process at the onset of reprogramming (), genome-wide species-specific gene expression profiling can be carried out on the three types of heterokaryon shown. In this way, the transcripts of human genes that are induced soon after fusion can be identified, and the effects of knocking down these candidate genes (loss of function) or overexpressing them (gain of function) these candidate genes can also be tested45. The function of these genes can then be validated by assays that assess whether they are required for nuclear transfer or for generating iPS cells or indu! ced somatic cells. For example, assays can test whether expression of the genes identified in the heterokaryons with an ES-cell or iPS-cell phenotype (, centre) enhances, or is required for, the generation of iPS cells or for reprogramming by nuclear transfer. The genes identified in the heterokaryons with a somatic cell phenotype (, top and bottom) can be tested to uncover whether they enhance, or are required for, the conversion of iPS cells or ES cells into a particular somatic cell type or the conversion of one type of somatic cell into another type. Such experiments will increase the understanding of the molecular regulators of nuclear reprogramming and therefore improve the safety and efficacy of cells produced for therapeutic purposes. * Figure 4: Applications of iPS cells. To generate iPS cells, fibroblasts (or another type of adult somatic cell) are transduced with retroviruses encoding four pluripotency factors (SOX2, KLF4, c-MYC and OCT4)56, 63. Fully reprogrammed iPS cells have similar properties to ES cells. They are competent to form teratomas on injection into mice and are capable of generating progeny. A patient's cells can be used to derive iPS cells, which can then be induced to undergo differentiation into various types of somatic cell, all with the same genetic information as the patient. For example, dopaminergic neurons could be generated from the cells of a patient with Parkinson's disease and then transplanted to replace those neurons that have been lost. These differentiated cells can also be used in disease models for studying the molecular basis of a broad range of human diseases that are otherwise difficult to study (for instance, those that affect brain cells) and for screening the efficacy and safety of drug candidates fo! r treating these diseases. * Figure 5: Comparison of the advantages of the three approaches to nuclear reprogramming. The three approaches to reprogramming somatic cells differ in their technical difficulty, speed of reprogramming, efficiency of inducing pluripotency, and cell yield. Therefore, each approach is better suited for studies that provide early mechanistic insights (top) or for therapeutic applications (bottom). The greater the intensity of the colour, the more advantageous the technique. For gaining mechanistic insights (top) into the onset of reprogramming, heterokaryons are particularly advantageous, for three main reasons. First, they are quickly reprogrammed to express pluripotency genes (1 to 2 days ). This is also the case for nuclear transfer. By contrast, it takes weeks to generate iPS cells. Second, reprogramming by cell fusion is highly efficient. When mouse ES cells are fused with human fibroblasts, up to 70% of heterokaryons (enriched by fluorescence-activated cell sorting) activate the expression of pluripotency genes within 1 day. It is technically challenging (and! therefore inefficient) to carry out nuclear transfer in mice, so it is difficult to use this approach for large-scale molecular analyses. Furthermore, the efficiency of generating iPS cells by transcription-factor transduction is low, about 0.01–0.1%. Third, cell division does not occur in heterokaryons. It also does not occur after nuclear transfer during the time when pluripotency genes are induced, allowing active mechanisms that induce pluripotency gene expression to be studied because this induction is independent of cell division and DNA replication; passive mechanisms may accompany cell division (for example dilution of DNA methyltransferases). By contrast, many rounds of cell division are required to generate iPS cells. For therapeutic applications (bottom), iPS cells are particularly advantageous, for three main reasons. First, diseases can readily be modelled using iPS cells derived from patients, overcoming the ethical issues and problems with immunological re! jection that are inherent in obtaining human ES cells for stud! ying disease. Skin fibroblasts can be readily obtained from the skin of an individual with a particular heritable disease, induced to become pluripotent in vitro and then induced to undergo differentiation to become the cell type of interest (for example a specific kind of cardiac cell). The pathways underlying a disease state (that is, gene expression and signalling) can thus be studied in cells that are not easily accessible in living humans. Second, drug screening can be carried out in vitro using these iPS-cell-based disease models to determine whether therapeutic drug candidates ameliorate or correct aberrant pathways. Third, for certain diseases, cell therapy might soon be used to regenerate or replace defective tissues, with the caveat that the tumorigenic potential, which is in part due to viral vector integration, must be overcome. Both nuclear transfer (leading to ES-cell production) and transcription-factor transduction (to produce iPS cells) have a high cell yie! ld, which is important for cell therapy applications. Author information * Abstract * Author information * Comments Affiliations * Center for iPS Cell Research and Application, Kyoto University, Kyoto 606-8507, Japan. * Shinya Yamanaka * Gladstone Institute of Cardiovascular Disease, San Francisco, California 94158, USA. * Shinya Yamanaka * Baxter Laboratory for Stem Cell Biology, Department of Microbiology and Immunology, Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, California 94305, USA. hblau2@stanford.edu * Helen M. Blau Competing financial interests The authors declare no competing financial interests. Reprints and permissions information is available at http://www.nature.com/reprints. Additional data
• Nuclear reprogramming to a pluripotent state by three approaches
  - Nature (London) 465(7299):704 (2010)
  Nature | Insight | Review Article Nuclear reprogramming to a pluripotent state by three approaches * Shinya Yamanaka1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Helen M. Blau3 Search for this author in: * NPG journals * PubMed * Google Scholar * AffiliationsJournal name:NatureVolume:465,Pages:704–712Date published:(10 June 2010)DOI:doi:10.1038/nature09229Published online09 June 2010 Abstract * Abstract * Author information * Comments Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg The stable states of differentiated cells are now known to be controlled by dynamic mechanisms that can easily be perturbed. An adult cell can therefore be reprogrammed, altering its pattern of gene expression, and hence its fate, to that typical of another cell type. This has been shown by three distinct experimental approaches to nuclear reprogramming: nuclear transfer, cell fusion and transcription-factor transduction. Using these approaches, nuclei from 'terminally differentiated' somatic cells can be induced to express genes that are typical of embryonic stem cells, which can differentiate to form all of the cell types in the body. This remarkable discovery of cellular plasticity has important medical applications. View full text Figures at a glance * Figure 1: Three approaches to nuclear reprogramming to pluripotency. , Nuclear transfer. In this approach, the nucleus of a somatic cell (which is diploid, 2n) is transplanted into an enucleated oocyte. In the environment of the oocyte, the somatic cell nucleus is reprogrammed so that the cells derived from it are pluripotent. From this oocyte, a blastocyst is generated, from which embryonic stem (ES)-cell lines are derived in tissue culture. If development is allowed to proceed to completion, an entire cloned organism is generated. , Cell fusion. In this approach, two distinct cell types are combined to form a single entity. The resultant fused cells can be heterokaryons or hybrids. If the fused cells proliferate, they will become hybrids, and on division, the nuclei fuse to become 4n (that is, twice the number of chromosomes in a somatic cell) or greater. If the cells are derived from the same species, their karyotype will remain euploid (that is, they will have balanced sets of chromosomes); however, if they are from different species, the! y will be aneuploid, as chromosomes will be lost and rearranged. Heterokaryons, by contrast, are short-lived and do not divide. As a result, they are multinucleate: the nuclei from the original cells remain intact and distinct, and the influence of one genotype on another can be studied in a stable system in which no chromosomes are lost. If the heterokaryons are of mixed species, the gene products of the two cell types can be distinguished. By altering the nuclear ratio in the fusion, and hence the stoichiometry of the regulators provided by each type of cell, the heterokaryon is reprogrammed towards the desired cell type (Fig. 3). Culture medium also has a role and needs to have a composition favoured by the desired cell type. Dashed arrows indicate slower processes (involving multiple rounds of cell division) than solid arrows (no division). , Transcription factor transduction. This approach can be used to form induced pluripotent stem (iPS) cells, which have similar pro! perties to ES cells and can be generated from almost any cell ! type in the body through the introduction of four genes (Oct4, Sox2, Klf4 and c-Myc) by using retroviruses. The pluripotent state is heritably maintained, and vast numbers of cells can be generated, making this approach advantageous for clinical applications. 1n, haploid. * Figure 2: Timeline of discoveries in nuclear reprogramming. Three approaches to nuclear reprogramming are described: nuclear transfer (blue), cell fusion (pink) and transcription-factor transduction (green). These complementary approaches have provided synergistic insights for almost 50 years and continue to inform the understanding of nuclear reprogramming and influence medical advances. EG cell, embryonic germ cell. * Figure 3: Investigating the genes involved in nuclear reprogramming by using mixed-species heterokaryons. Cell fusion leads to nuclear reprogramming towards a specific phenotype, which is dictated by the nuclear ratio of the fused cell types in heterokaryons, which do not divide. When, for example, cells from humans and mice are fused in a skewed ratio (such as 1:3) (), the human cells will generally be reprogrammed towards the mouse cell phenotype (three examples are shown). To uncover which genes are involved in this process at the onset of reprogramming (), genome-wide species-specific gene expression profiling can be carried out on the three types of heterokaryon shown. In this way, the transcripts of human genes that are induced soon after fusion can be identified, and the effects of knocking down these candidate genes (loss of function) or overexpressing them (gain of function) these candidate genes can also be tested45. The function of these genes can then be validated by assays that assess whether they are required for nuclear transfer or for generating iPS cells or indu! ced somatic cells. For example, assays can test whether expression of the genes identified in the heterokaryons with an ES-cell or iPS-cell phenotype (, centre) enhances, or is required for, the generation of iPS cells or for reprogramming by nuclear transfer. The genes identified in the heterokaryons with a somatic cell phenotype (, top and bottom) can be tested to uncover whether they enhance, or are required for, the conversion of iPS cells or ES cells into a particular somatic cell type or the conversion of one type of somatic cell into another type. Such experiments will increase the understanding of the molecular regulators of nuclear reprogramming and therefore improve the safety and efficacy of cells produced for therapeutic purposes. * Figure 4: Applications of iPS cells. To generate iPS cells, fibroblasts (or another type of adult somatic cell) are transduced with retroviruses encoding four pluripotency factors (SOX2, KLF4, c-MYC and OCT4)56, 63. Fully reprogrammed iPS cells have similar properties to ES cells. They are competent to form teratomas on injection into mice and are capable of generating progeny. A patient's cells can be used to derive iPS cells, which can then be induced to undergo differentiation into various types of somatic cell, all with the same genetic information as the patient. For example, dopaminergic neurons could be generated from the cells of a patient with Parkinson's disease and then transplanted to replace those neurons that have been lost. These differentiated cells can also be used in disease models for studying the molecular basis of a broad range of human diseases that are otherwise difficult to study (for instance, those that affect brain cells) and for screening the efficacy and safety of drug candidates fo! r treating these diseases. * Figure 5: Comparison of the advantages of the three approaches to nuclear reprogramming. The three approaches to reprogramming somatic cells differ in their technical difficulty, speed of reprogramming, efficiency of inducing pluripotency, and cell yield. Therefore, each approach is better suited for studies that provide early mechanistic insights (top) or for therapeutic applications (bottom). The greater the intensity of the colour, the more advantageous the technique. For gaining mechanistic insights (top) into the onset of reprogramming, heterokaryons are particularly advantageous, for three main reasons. First, they are quickly reprogrammed to express pluripotency genes (1 to 2 days ). This is also the case for nuclear transfer. By contrast, it takes weeks to generate iPS cells. Second, reprogramming by cell fusion is highly efficient. When mouse ES cells are fused with human fibroblasts, up to 70% of heterokaryons (enriched by fluorescence-activated cell sorting) activate the expression of pluripotency genes within 1 day. It is technically challenging (and! therefore inefficient) to carry out nuclear transfer in mice, so it is difficult to use this approach for large-scale molecular analyses. Furthermore, the efficiency of generating iPS cells by transcription-factor transduction is low, about 0.01–0.1%. Third, cell division does not occur in heterokaryons. It also does not occur after nuclear transfer during the time when pluripotency genes are induced, allowing active mechanisms that induce pluripotency gene expression to be studied because this induction is independent of cell division and DNA replication; passive mechanisms may accompany cell division (for example dilution of DNA methyltransferases). By contrast, many rounds of cell division are required to generate iPS cells. For therapeutic applications (bottom), iPS cells are particularly advantageous, for three main reasons. First, diseases can readily be modelled using iPS cells derived from patients, overcoming the ethical issues and problems with immunological re! jection that are inherent in obtaining human ES cells for stud! ying disease. Skin fibroblasts can be readily obtained from the skin of an individual with a particular heritable disease, induced to become pluripotent in vitro and then induced to undergo differentiation to become the cell type of interest (for example a specific kind of cardiac cell). The pathways underlying a disease state (that is, gene expression and signalling) can thus be studied in cells that are not easily accessible in living humans. Second, drug screening can be carried out in vitro using these iPS-cell-based disease models to determine whether therapeutic drug candidates ameliorate or correct aberrant pathways. Third, for certain diseases, cell therapy might soon be used to regenerate or replace defective tissues, with the caveat that the tumorigenic potential, which is in part due to viral vector integration, must be overcome. Both nuclear transfer (leading to ES-cell production) and transcription-factor transduction (to produce iPS cells) have a high cell yie! ld, which is important for cell therapy applications. Author information * Abstract * Author information * Comments Affiliations * Center for iPS Cell Research and Application, Kyoto University, Kyoto 606-8507, Japan. * Shinya Yamanaka * Gladstone Institute of Cardiovascular Disease, San Francisco, California 94158, USA. * Shinya Yamanaka * Baxter Laboratory for Stem Cell Biology, Department of Microbiology and Immunology, Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, California 94305, USA. hblau2@stanford.edu * Helen M. Blau Competing financial interests The authors declare no competing financial interests. Reprints and permissions information is available at http://www.nature.com/reprints. Additional data
• Extrinsic regulation of pluripotent stem cells
  - Nature (London) 465(7299):713 (2010)
  Nature | Insight | Review Article Extrinsic regulation of pluripotent stem cells * Martin F. Pera1 Search for this author in: * NPG journals * PubMed * Google Scholar * Patrick P. L. Tam2, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * AffiliationsJournal name:NatureVolume:465,Pages:713–720Date published:(10 June 2010)DOI:doi:10.1038/nature09228Published online09 June 2010 Abstract * Abstract * Author information * Comments Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg During early mammalian development, as the pluripotent cells that give rise to all of the tissues of the body proliferate and expand in number, they pass through transition states marked by a stepwise restriction in developmental potential and by changes in the expression of key regulatory genes. Recent findings show that cultured stem-cell lines derived from different stages of mouse development can mimic these transition states. They further reveal that there is a high degree of heterogeneity and plasticity in pluripotent populations in vitro and that these properties are modulated by extrinsic signalling. Understanding the extrinsic control of plasticity will guide efforts to use human pluripotent stem cells in research and therapy. View full text Figures at a glance * Figure 1: Stem-cell types derived from mouse embryos around the time of implantation. At implantation, mouse blastocysts comprise three distinct cell types: the trophectoderm; the inner cell mass, which produces the primitive endoderm; and the 'naive' (or early pre-implantation) epiblast. Under appropriate in vitro culture conditions, three types of stem cell with a comparable potential for differentiation to each of the cell types of the blastocyst can be derived: ES cells, trophoblast stem (TS) cells and extra-embryonic endoderm (XEN) stem cells1, 2, 3, 4, 5, 66, 94, 95, 96, 97. Another type of stem cell, FAB-SCs, can be obtained by culturing blastocysts in medium containing FGF2, activin and the GSK3β inhibitor BIO25. And a further stem-cell type, EPL cells, can be derived from ES cells by culturing them in a cell-line-conditioned medium21. TS cells and XEN stem cells are committed to form extra-embryonic tissues only. After implantation, the blastocyst grows into a pre-gastrulation embryo, which comprises the extra-embryonic ectoderm and the early epibla! st, with the visceral endoderm enveloping both tissues. Pluripotent stem cells can be derived from the epiblast of the early post-implantation embryo at 5.5–5.75 days post coitum22, 23. Like ES cells, these epiblast stem cells (EpiSCs) are pluripotent in that they differentiate into the full range of typical germ-layer tissues in vitro and into teratomas in vivo. Epiblast cells are progressively restricted in differentiation potential as they are allocated to the mesoderm and endoderm through cellular ingression at the primitive streak during gastrulation. Despite the onset of tissue commitment at early gastrulation, EpiSCs can still be derived from the late epiblast of the embryo, the cells of which have been experimentally shown by lineage analysis and fate-mapping studies to retain plasticity in cell fate. * Figure 2: Extrinsic signals that affect self-renewal, differentiation and viability of human ES cells. Signalling mediated by members of the transforming growth factor-β (TGF-β) family — such as TGF-β, activin and nodal, growth differentiation factors (GDFs, including myostatin) and bone morphogenetic proteins (BMPs) — converges mainly on NANOG, which maintains ES cells in an undifferentiated state with the ability to self-renew. Signalling activity mediated by the MEK–ERK receptor tyrosine kinase cascade allows self-renewal of ES cells and maintains their viability (through inhibiting apoptosis and anoikis). In addition, insulin-like growth factor 2 (IGF2)-mediated signalling through phosphatidylinositol-3-OH kinase (PI(3)K) inhibits ES cells from differentiating into endodermal lineage cells. WNT-mediated signalling might affect these cell-fate decisions, but its role is controversial at present. NRG1, neuregulin 1; PDGF, platelet-derived growth factor; S1P, sphingosine 1-phosphate. * Figure 3: Interconversion of mouse embryo-derived stem-cell types. Stem cells with different characteristics can be derived from the mouse blastocyst or the early or late epiblast under different culture conditions. From the blastocyst, four types of stem cell have been harvested: ES cells, TS cells, XEN stem cells and FAB-SCs. When FAB-SCs are cultured in medium supplemented with BMP4 and LIF, the cells are converted into ES-cell-like cells. ES cells can be converted into TS cells by culturing them in mouse-embryonic-fibroblast-conditioned medium (MEF-cm) containing FGF4, together with enforcing the expression of the transcription factor CDX2. ES cells can be turned into EPL cells by culturing them in MedII (medium conditioned by the human hepatocarcinoma cell line HepG2), and EPL cells can be converted back into ES cells by culturing them with LIF. Early (pre-gastrulation embryo) and late (gastrulating embryo) epiblast fragments give rise to EpiSCs when they are cultured in medium supplemented with FGF2 and activin. Single cells from diss! ociated epiblasts that are cultured in MEF-cm with LIF and fetal calf serum (FCS) become cultured epiblast (cEpi) cells, which can be converted into 'reversed' ES cells (rES cells), which resemble ES cells. EpiSCs can be converted into ES cells by culturing them with LIF and inhibitors of GSK3β and ERKs or by enforced Klf4 expression. In the converse process, ES cells can be turned into EpiSCs by culturing them with activin and FGF2. EpiSCs differentiate into cells that resemble primordial germ (PG) cells after being cultured with BMP4 and then with noggin and chordin in the presence of activin and FGF2. These 'PG' cells can be converted into pluripotent embryonic germ (EG) cells by culturing them in medium supplemented with LIF, FCS and FGF2. Author information * Abstract * Author information * Comments Affiliations * Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research, Keck School of Medicine, University of Southern California, Los Angeles, California 90033, USA. pera@usc.edu * Martin F. Pera * Embryology Unit, Children's Medical Research Institute, Westmead, New South Wales 2145, Australia. * Patrick P. L. Tam * Discipline of Medicine, Sydney Medical School, University of Sydney, Sydney, New South Wales 2006, Australia. ptam@cmri.org.au * Patrick P. L. Tam Competing financial interests The authors declare no competing financial interests. Reprints and permissions information is available at http://www.nature.com/reprints. Additional data
• Epigenetics as a unifying principle in the aetiology of complex traits and diseases
  - Nature (London) 465(7299):721 (2010)
  Nature | Insight | Perspective Article Epigenetics as a unifying principle in the aetiology of complex traits and diseases * Arturas Petronis1 Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:NatureVolume:465,Pages:721–727Date published:(10 June 2010)DOI:doi:10.1038/nature09230Published online09 June 2010 Abstract * Abstract * Author information * Comments Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Epigenetic modifications of DNA and histones might be crucial for understanding the molecular basis of complex phenotypes. One reason for this is that epigenetic factors are sometimes malleable and plastic enough to react to cues from the external and internal environments. Such induced epigenetic changes can be solidified and propagated during cell division, resulting in permanent maintenance of the acquired phenotype. In addition, the finding that there is partial epigenetic stability in somatic and germline cells allows insight into the molecular mechanisms of heritability. Epigenetics can provide a new framework for the search of aetiological factors in complex traits and diseases. View full text Figures at a glance * Figure 1: Twin-based epigenetic heritability. DNA methylation profiles are presented as black and white keys in the germ line or zygote (one layer) and somatic cells (multiple layers). Black denotes, for example, methylated cytosine, and white denotes, for example, unmethylated cytosine. Monozygotic twins originate from a single zygote, and their initial epigenetic status is more similar than that of dizygotic twins, who develop from two separate zygotes with different epigenetic profiles. The epigenetic modifications in both monozygotic twins and dizygotic twins are subject to stochastic and, to a lesser extent, environmental factors, which induce similar amounts of somatic epigenetic variation in tissues. Owing to epigenetic differences in the original zygotes, however, dizygotic twins have more epigenetic variation in their somatic cells than do monozygotic twins. This could account for the large phenotypic differences (green stars) observed between dizygotic twins compared with monozygotic twins. * Figure 2: Epigenetic interpretation of cases of sporadic disease and familial disease. DNA methylation profiles are presented as black and white keys in the germ line or zygote (one layer) and somatic cells (multiple layers). Black denotes, for example, methylated cytosine, and white denotes, for example, unmethylated cytosine. Red denotes pathological epigenetic marks (or epimutations). , Sporadic disease. An epimutation occurs in the germ line of the second generation (F2). It is transmitted to F3, spreads in the somatic tissues and induces disease (red stars). The epimutation is, however, corrected in the germ line of F3 and is not transmitted to F4. , Familial disease. A germline epimutation occurs in F1, is transmitted to F2 and induces disease. The epimutation fails to be corrected in the germ line of F2, and it is transmitted to F3. It is not known why correction fails, but the failure might be caused by an aberrant configuration of local chromatin (shown as a DNA loop). Another correction attempt fails, and epimutations are transmitted from F3 to F4. I! n each cycle of gametogenesis, the germline epimutation becomes more severe, resulting in epigenetic anticipation: that is, disease is more severe and occurs earlier in younger generations (depicted as an increasing number of red stars). In this way, sporadic and familial patterns of disease may have a similar molecular epigenetic origin but differ because of the differential efficacy of epigenetic reprogramming during gametogenesis and/or after fertilization. Author information * Abstract * Author information * Comments Affiliations * The Krembil Family Epigenetics Laboratory, Centre for Addiction and Mental Health, 250 College Street, Toronto, Ontario M5T 1R8, Canada. arturas_petronis@camh.net * Arturas Petronis Competing financial interests The author declares no competing financial interests. Reprints and permissions information is available at http://www.nature.com/reprints. Additional data
• Brain function and chromatin plasticity
  - Nature (London) 465(7299):728 (2010)
  Nature | Insight | Review Article Brain function and chromatin plasticity * Catherine Dulac1 Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:NatureVolume:465,Pages:728–735Date published:(10 June 2010)DOI:doi:10.1038/nature09231Published online09 June 2010 Abstract * Abstract * Author information * Comments Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg The characteristics of epigenetic control, including the potential for long-lasting, stable effects on gene expression that outlive an initial transient signal, could be of singular importance for post-mitotic neurons, which are subject to changes with short- to long-lasting influence on their activity and connectivity. Persistent changes in chromatin structure are thought to contribute to mechanisms of epigenetic inheritance. Recent advances in chromatin biology offer new avenues to investigate regulatory mechanisms underlying long-lasting changes in neurons, with direct implications for the study of brain function, behaviour and diseases. View full text Figures at a glance * Figure 1: Mechanisms involved in chromatin modifications. Five broad and interrelated mechanisms are known to affect chromatin structure: DNA methylation, histone modification, remodelling by chromatin-remodelling complexes, insertion of histone variants, and the effects of non-coding RNAs (ncRNAs). All five have been shown to be essential contributors to the development and cell-fate determination of tissues, including those of the nervous system, whereas histone modifications and DNA methylation have, so far, been more extensively investigated in the context of adult brain function. Ac, acetyl; Me, methyl; P, phosphate. * Figure 2: Contribution of various chromatin-remodelling events throughout the life of an organism. Chromatin modifications occurring at different time points during the life of an organism have been associated with various short- to long-lasting regulatory events that affect the development and the function of the brain and other tissues. Author information * Abstract * Author information * Comments Affiliations * Howard Hughes Medical Institute, Harvard University, 16 Divinity Avenue, Cambridge, Massachusetts 02138, USA. dulac@fas.harvard.edu * Catherine Dulac Competing financial interests The author declares no competing financial interests. Reprints and permissions information is available at http://www.nature.com/reprints. Additional data
• Measurement of single-cell dynamics
  - Nature (London) 465(7299):736 (2010)
  Nature | Insight | Review Article Measurement of single-cell dynamics * David G. Spiller1 Search for this author in: * NPG journals * PubMed * Google Scholar * Christopher D. Wood2 Search for this author in: * NPG journals * PubMed * Google Scholar * David A. Rand3 Search for this author in: * NPG journals * PubMed * Google Scholar * Michael R. H. White4 Search for this author in: * NPG journals * PubMed * Google Scholar * AffiliationsJournal name:NatureVolume:465,Pages:736–745Date published:(10 June 2010)DOI:doi:10.1038/nature09232Published online09 June 2010 Abstract * Abstract * Author information * Supplementary information * Comments Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Populations of cells are almost always heterogeneous in function and fate. To understand the plasticity of cells, it is vital to measure quantitatively and dynamically the molecular processes that underlie cell-fate decisions in single cells. Early events in cell signalling often occur within seconds of the stimulus, whereas intracellular signalling processes and transcriptional changes can take minutes or hours. By contrast, cell-fate decisions, such as whether a cell divides, differentiates or dies, can take many hours or days. Multiparameter experimental and computational methods that integrate quantitative measurement and mathematical simulation of these noisy and complex processes are required to understand the highly dynamic mechanisms that control cell plasticity and fate. View full text Figures at a glance * Figure 1: Dynamic processes in living cells. The diagram summarizes the sequence of events from signal recognition to cell fate and indicates some of the measurement technologies used for quantifying these processes. The diagram recapitulates the order in which these processes are reviewed here, starting with signals at the cell membrane. Individual methods are further illustrated by examples of time-lapse analysis of cell processes in Fig. 2. FCS, fluorescence correlation spectroscopy; FRET, fluorescence resonance energy transfer; FUCCI, fluorescent, ubiquitylation-based cell-cycle indicator; mRNA, messenger RNA. * Figure 2: Examples of time-lapse imaging of single cells. , Early signalling events. Calcium imaging of pituitary cells (of the GH3 cell line) is shown after treatment with thyrotropin-releasing hormone. Fluctuations in the amount of calcium in the cytoplasm over seconds were visualized by using fluo-4 dye (green) (Supplementary Movie 1). Scale bar, 20 μm. , Transcription-factor translocation. Fluorescent protein imaging of neuroblastoma cells (of the SK-N-AS cell line) treated with tumour-necrosis factor-α is shown. The protein RELA (which is a subunit of the transcription factor nuclear factor-κB) was fused to the fluorescent protein DsRed-Express (red). RELA oscillates between the cytoplasm and the nucleus of cells with a period of about 100 min. Concurrently, the RELA inhibitor IκBα, labelled with enhanced green fluorescent protein (green), shows cycles of synthesis and degradation that have an inverse phase to the cycles of RELA translocation5 (Supplementary Movie 2). Scale bar, 20 μm. , Transcription analysis. Low-light! -level imaging of pituitary cells (of the GH3 cell line) expressing luciferase under the control of the promoter of the human prolactin gene is shown. The substrate of luciferase, luciferin, was added to the medium, and images were taken at 15-min intervals over hours. The colour scale indicates the range of light emission, from low (blue) to high (red). The cycles of transcription are heterogeneous across the cells65 (Supplementary Movie 3). Scale bar, 50 μm. , Cell division. Imaging of epithelial cells (of the HeLa cell line) by using FUCCI technology, over hours, is shown. Cells transiently express FUCCI proteins, depending on their differing stability at different phases of the cell cycle: G1 phase (red), S phase (green); G2 phase (reduced green fluorescence) and M phase (no fluorescent signal)75. Blue labels indicate the cell-cycle phase of a single cell in this image series, and yellow labels indicate that of another single cell. Each of these cells divides over the ! course of the experiment (Supplementary Movie 4). Scale bar, 2! 0 μm. * Figure 3: Luminescent imaging in vivo and in vitro. , A transgenic rat expresses luciferase in the pituitary gland under the natural control of the promoter of the human prolactin gene60. , A cultured intact pituitary gland from a transgenic rat () shows localization of gene expression within the pituitary gland65. Scale bar, 100 μm. , Single primary rat pituitary cells from disaggregated tissue, in culture, showing heterogeneity between individual cells, which show independent dynamic behaviour65. Scale bar, 100 μm. The luminescence intensity is shown in each image on a scale from low (blue) to high (red). These data show how individual cell heterogeneity must be considered when studying overall organ phenotype and whole-animal physiology. * Figure 4: Example of a microfluidic device for single-cell manipulation and long-term observation. , The diagram shows a cell-trapping chamber containing 440 individual cell 'micro-jails'. The total volume of each chamber is less than 20 nl (a single chamber is shown in the magnification on the right). Suspensions of single cells are flowed through the chamber from the top such that individual cells become trapped in the micro-jails, facilitating their long-term observation. , A promyelocytic leukaemia cell (of the HL-60 cell line) trapped in a micro-jail is exposed to staurosporine, an inducer of apoptosis. The medium contains propidium iodide (PI), a fluorescent dye that is normally excluded from viable cells. Over time, the PI-based fluorescence steadily increases, indicating the dynamic progression of the cell into apoptosis. Composite images of the bright-field and fluorescence micrographs are shown on the right. , The cumulative percentage of cells dying over time is plotted for independent micro-jail arrays. Simultaneous monitoring of multiple micro-jails demonstra! tes the use of the microfluidic device to show the stochastic nature of the apoptotic process, as demonstrated by the varying incidence of cell death in the cell population over time (despite cells being exposed to the same environmental conditions). Such microfluidic devices have potential for drug-screening studies, because they allow the controlled delivery of candidate drugs. (Figure reproduced, with permission, from ref. 92.) Author information * Abstract * Author information * Supplementary information * Comments Affiliations * Centre for Cell Imaging, School of Biological Sciences, Bioscience Research Building, Crown Street, Liverpool L69 7ZB, UK. * David G. Spiller * Instituto de Biotecnología, Universidad Nacional Autónoma de México, Avenida Universidad 2001, Cuernavaca, Morelos 62250, Mexico. * Christopher D. Wood * Warwick Systems Biology and Mathematics Institute, Coventry House, University of Warwick, Coventry CV4 7AL, UK. * David A. Rand * Centre for Cell Imaging, School of Biological Sciences, Bioscience Research Building, Crown Street, Liverpool L69 7ZB, UK. mwhite@liverpool.ac.uk * Michael R. H. White Competing financial interests The authors declare no competing financial interests. Reprints and permissions information is available at http://www.nature.com/reprints. Supplementary information * Abstract * Author information * Supplementary information * Comments Movies * Supplementary Movie 1 (7.9M) This movie shows early signalling events. Calcium imaging of pituitary cells (of the GH3 cell line) is shown before and after treatment with thyrotropin-releasing hormone, which was added to the cultured cells one-third of the way into the time series. Fluctuations in the amount of calcium in the cytoplasm over 400 s (at 1 frame s–1) were visualized by using fluo-4 dye (green), which increases in intensity with increasing calcium concentration (see Fig. 2a, which illustrates a short section of the movie after addition of thyrotropin-releasing hormone). * Supplementary Movie 2 (1.5M) This movie shows transcription-factor translocation. Fluorescent protein imaging for 579 min of neuroblastoma cells (of the SK-N-AS cell line) treated with tumour-necrosis factor-α is shown. The protein RELA (which is a subunit of the transcription factor nuclear factor-κB) was fused to the fluorescent protein DsRed-Express (red). RELA oscillates between the cytoplasm and the nucleus of cells with a period of about 100 min. Concurrently, the RELA inhibitor IκBα, labelled with enhanced green fluorescent protein (green), shows cycles of synthesis and degradation that have an inverse phase to the cycles of RELA translocation. * Supplementary Movie 3 (1.5M) This movie shows transcription analysis. Low-light-level imaging of pituitary cells (of the GH3 cell line) expressing firefly luciferase under the control of the stably transfected promoter of the human prolactin gene is shown. The substrate of luciferase, luciferin, was added to the medium, and images were taken at 15-min intervals over 40 h. Luminescence intensity increases from blue to green to yellow to red. The cycles of transcription are heterogeneous across the cells. * Supplementary Movie 4 (3.3M) This movie shows cell division. Imaging of epithelial cells (of the HeLa cell line) by using fluorescent, ubiquitylation-based cell-cycle indicator (FUCCI) technology, over 22 h, is shown. Cells transiently express FUCCI proteins, depending on their differing stability at different phases of the cell cycle: G1 phase (red), S phase (green), G2 phase (reduced green fluorescence) and M phase (no fluorescent signal). Each of the cells in the field of view at the start of the experiment undergoes division. Additional data
• Crystal structure of HIV-1 Tat complexed with human P-TEFb
  - Nature (London) 465(7299):747 (2010)
  Nature | Article Crystal structure of HIV-1 Tat complexed with human P-TEFb * Tahir H. Tahirov1 Search for this author in: * NPG journals * PubMed * Google Scholar * Nigar D. Babayeva1 Search for this author in: * NPG journals * PubMed * Google Scholar * Katayoun Varzavand2 Search for this author in: * NPG journals * PubMed * Google Scholar * Jeffrey J. Cooper2 Search for this author in: * NPG journals * PubMed * Google Scholar * Stanley C. Sedore2 Search for this author in: * NPG journals * PubMed * Google Scholar * David H. Price2 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:NatureVolume:465,Pages:747–751Date published:(10 June 2010)DOI:doi:10.1038/nature09131Received21 January 2010Accepted27 April 2010 Abstract * Abstract * Accession codes * Author information * Supplementary information * Comments Article tools * Full text * 日本語要約 * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Regulation of the expression of the human immunodeficiency virus (HIV) genome is accomplished in large part by controlling transcription elongation. The viral protein Tat hijacks the host cell's RNA polymerase II elongation control machinery through interaction with the positive transcription elongation factor, P-TEFb, and directs the factor to promote productive elongation of HIV mRNA. Here we describe the crystal structure of the Tat·P-TEFb complex containing HIV-1 Tat, human Cdk9 (also known as CDK9), and human cyclin T1 (also known as CCNT1). Tat adopts a structure complementary to the surface of P-TEFb and makes extensive contacts, mainly with the cyclin T1 subunit of P-TEFb, but also with the T-loop of the Cdk9 subunit. The structure provides a plausible explanation for the tolerance of Tat to sequence variations at certain sites. Importantly, Tat induces significant conformational changes in P-TEFb. This finding lays a foundation for the design of compounds that wo! uld specifically inhibit the Tat·P-TEFb complex and block HIV replication. View full text Subject terms: * Structural biology * Drug discovery Figures at a glance * Figure 1: Overall structure of the Tat·P-TEFb·ATP. , , Ribbon representation () and surface representation () of the Tat·P-TEFb·ATP structure. Cdk9 is light orange, cyclin T1 is pale green and Tat is magenta. The side chains of the Cdk9-interacting residues of Tat, Cys 261 of cyclin T1 and ATP analogue are drawn as sticks, and the zinc and magnesium atoms are drawn as cyan and light blue spheres, respectively. The dashed lines represent the missing link between Lys 88 and Gly 97 of Cdk9, and between Leu 252 and Cys 261 of cyclin T1. * Figure 2: Structure of P-TEFb-bound Tat. , Diagram of the regions and functional domains of HIV-1 Tat proteins. , , Stick () and cartoon() representations of the Tat structure in two orientations. The regions of Tat are coloured as in . A close-up view of Zn1 and Zn2 coordination and a portion of 2Fo−Fc Fourier maps (mesh) contoured at 1σ are shown (–). * Figure 3: The Tat-interacting areas of P-TEFb. , , Mapping of the Tat-interacting residues of P-TEFb () to the surface of P-TEFb and () to the ribbon diagram of P-TEFb. The subunits and interacting areas are coloured according to definitions shown in . * Figure 4: Comparison of the crystal structures of P-TEFb·ATP and Tat·P-TEFb·ATP. , Shift of H5′ and disordering of HC in cyclin T1 of Tat·P-TEFb·ATP (orange). , Superimposition of cyclin T1 showing rotation of Cdk9. P-TEFb·ATP: Cdk9 (green), cyclin T1 (blue); Tat·P-TEFb·ATP: Cdk9 (salmon), cyclin T1 (cyan). , Interactions of phospho-Thr 186 in panel . P-TEFb·ATP (green); Tat·P-TEFb·ATP (coloured by atom type). , Superimposition of Cdk9 showing the shift of cyclin T1. , Concerted movement of the cyclin T1 H5 and the β3αC-loop of Cdk9 causing the conformational switch of the Cdk9 β1β2-loop upon Tat binding to P-TEFb. The residues causing sterical hindrance are drawn as sticks. Colours in and are as in . * Figure 5: A conservation-dependent location of Tat's amino acid residues. , Sequence logos of the Tat activation domain. , Location of Tat invariant residues (yellow) and residues with randomly occurring polymorphs (deep salmon); , residues with predominantly functionally equivalent substitutions (green) and , residues with a variety of substitutions (magenta). In panels – Tat is drawn as grey ribbon, and cyclin T1 (pale cyan) and Cdk9 (pale green) are shown in surface representation. The highlighted Tat residues and Cys 261 of cyclin T1 are represented as ball-and-stick diagrams. The zinc atoms are shown as large grey spheres. Accession codes * Abstract * Accession codes * Author information * Supplementary information * Comments Primary accessions Protein Data Bank * 3mi9 * 3mia * 3mi9 * 3mia Author information * Abstract * Accession codes * Author information * Supplementary information * Comments Affiliations * Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, Nebraska 68198-7696, USA * Tahir H. Tahirov & * Nigar D. Babayeva * Biochemistry Department, University of Iowa, Iowa City, Iowa 52242, USA * Katayoun Varzavand, * Jeffrey J. Cooper, * Stanley C. Sedore & * David H. Price Contributions T.H.T. managed the crystallization and structure determination part of the project, solved the crystal structures and prepared the manuscript. N.D.B. obtained the crystals. Diffraction data collection was performed by N.D.B. and T.H.T. Protein cloning, expression, purification and writing the corresponding methods sections were performed by S.C.S., K.V. and J.J.C., respectively. D.H.P. managed the protein production part of the project, and helped generate and edit the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Tahir H. Tahirov (ttahirov@unmc.edu) Atomic coordinates and structure factors for Tat·P-TEFb and Tat·P-TEFb·ATP structures have been deposited in the Protein Data Bank with accession numbers 3mi9 and 3mia, respectively. Supplementary information * Abstract * Accession codes * Author information * Supplementary information * Comments PDF files * Supplementary Information (14.3M) This file contains Supplementary Figures 1-12 with legends, Supplementary Tables 1-2 and References. Additional data
• The recent formation of Saturn's moonlets from viscous spreading of the main rings
  - Nature (London) 465(7299):752 (2010)
  Nature | Letter The recent formation of Saturn's moonlets from viscous spreading of the main rings * Sébastien Charnoz1 Search for this author in: * NPG journals * PubMed * Google Scholar * Julien Salmon1 Search for this author in: * NPG journals * PubMed * Google Scholar * Aurélien Crida2, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:NatureVolume:465,Pages:752–754Date published:(10 June 2010)DOI:doi:10.1038/nature09096Received15 December 2009Accepted15 April 2010 Article tools * Full text * 日本語要約 * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg The regular satellites of the giant planets are believed to have finished their accretion concurrent with the planets, about 4.5 Gyr ago1, 2, 3, 4. A population of Saturn's small moons orbiting just outside the main rings are dynamically young5, 6 (less than 107 yr old), which is inconsistent with the formation timescale for the regular satellites. They are also underdense7 (~600 kg m−3) and show spectral characteristics similar to those of the main rings8, 9. It has been suggested that they accreted at the rings' edge7, 10, 11, but hitherto it has been impossible to model the formation process fully owing to a lack of computational power. Here we report a hybrid simulation in which the viscous spreading of Saturn's rings beyond the Roche limit (the distance beyond which the rings are gravitationally unstable) gives rise to the small moons. The moonlets' mass distribution and orbital architecture are reproduced. The current confinement of the main rings and! the existence of the dusty F ring are shown to be direct consequences of the coupling of viscous evolution and satellite formation. Saturn's rings, like a mini protoplanetary disk, may be the last place where accretion was recently active in the Solar System, some 106–107 yr ago. View full text Subject terms: * Astronomy * Astrophysics * Planetary sciences Figures at a glance * Figure 1: Mass of Saturn's inner moons versus distance. The names and average diameters of the moons are indicated in the insets. Data for Mimas, Enceladus and Tethys are also plotted, for comparison. The vertical dashed line shows the location of the outer edge of Saturn's A ring, at 136,750 km, and the vertical dash–dot line indicates the location of the F ring (a ~1,000-km-wide ringlet located between Prometheus and Pandora). The blue and red lines show simple logarithmic fits to the mass–distance data for the small moons and, respectively, the main moons. Images from the Cassini mission (courtesy of NASA/JPL/SSI). * Figure 2: Time evolution of our model with σ0 = 400 kg m−2. –, The time evolution of the ring's surface density (solid line) and the masses of the moonlets (black points) as functions of the distance from Saturn's centre. As the ring spreads inwards and outwards, its surface density decreases. Ring material crossing the Roche limit (RL = 140,000 km here) is transformed into moonlets. At the end of the simulation, only 1.5 × 1018 kg of the ring material remains in the A ring, whereas 3.5 × 1018 kg is spread below 120,000 km and 1.8 × 1017 kg has been transformed into moonlets. , The time evolution of the location of the ring's outer edge, defined as the place where the surface density drops below 1 kg m−2. When the edge is confined at the location of a moonlet's resonance, the ring's viscous torque (which tends to push material beyond the ring's edge by transferring angular momentum) is perfectly balanced by the satellite's gravitational torque, thus preventing the ring material from spre! ading farther outwards. , Total mass transformed into satellites as a function of time. The dashed line shows the mass of the largest satellite. * Figure 3: Comparing the mass distribution of the moonlets obtained in our simulation with observations. Cumulative mass distributions of moonlets obtained in four simulations with different initial surface densities, fitted with single power-law functions of the form N(>m) ∝ m−α: case A (σ0 = 400 kg m−2), α = 0.31 ± 0.06; case B (σ0 = 1,000 kg m−2), α = 0.19 ± 0.01; case C (σ0 = 5,000 kg m−2), α = 0.22 ± 0.03; case D (σ0 = 10,000 kg m−2), α = 0.17 ± 0.04. The actual population of Saturn's moonlets (Sat, α = 0.27 ± 0.07) is well within the range of masses and number of bodies obtained in the simulations. We note that some of our distributions (A, B and C) show a knee and a shallower slope at smaller sizes, as is observed for Saturn's small moons (Sat). Author information * Author information * Supplementary information * Comments Affiliations * Laboratoire AIM, Université Paris Diderot/CEA/CNRS, 91191 Gif sur Yvette, France * Sébastien Charnoz & * Julien Salmon * Department of Applied Mathematics and Theoretical Physics, University of Cambridge, Cambridge CB3 0WA, UK * Aurélien Crida * Université de Nice Sophia-Antipolis/CNRS/Observatoire de la Côte d'Azur, Laboratoire Cassiopée, BP4229, 06304 Nice Cedex 4, France * Aurélien Crida Contributions S.C. and J.S. designed the code and analysed the results, and A.C. was involved in the analysis of the results and provided critical contributions. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Sébastien Charnoz (charnoz@cea.fr) Supplementary information * Author information * Supplementary information * Comments PDF files * Supplementary Information (171K) This file contains Supplementary Information and Data, Supplementary Figure SI4 with legend and References. Additional data
• Electromagnetically induced transparency with single atoms in a cavity
  Mücke M Figueroa E Bochmann J Hahn C Murr K Ritter S Villas-Boas CJ Rempe G - Nature (London) 465(7299):755 (2010)
  Nature | Letter Electromagnetically induced transparency with single atoms in a cavity * Martin Mücke1, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Eden Figueroa1, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Joerg Bochmann1 Search for this author in: * NPG journals * PubMed * Google Scholar * Carolin Hahn1 Search for this author in: * NPG journals * PubMed * Google Scholar * Karim Murr1 Search for this author in: * NPG journals * PubMed * Google Scholar * Stephan Ritter1 Search for this author in: * NPG journals * PubMed * Google Scholar * Celso J. Villas-Boas1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Gerhard Rempe1 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:NatureVolume:465,Pages:755–758Date published:(10 June 2010)DOI:doi:10.1038/nature09093Received15 March 2010Accepted20 April 2010Published online12 May 2010 Article tools * Full text * 日本語要約 * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Optical nonlinearities offer unique possibilities for the control of light with light. A prominent example is electromagnetically induced transparency (EIT), where the transmission of a probe beam through an optically dense medium is manipulated by means of a control beam1, 2, 3. Scaling such experiments into the quantum domain with one (or just a few) particles of light and matter will allow for the implementation of quantum computing protocols with atoms and photons4, 5, 6, 7, or the realization of strongly interacting photon gases exhibiting quantum phase transitions of light8, 9. Reaching these aims is challenging and requires an enhanced matter–light interaction, as provided by cavity quantum electrodynamics10, 11, 12. Here we demonstrate EIT with a single atom quasi-permanently trapped inside a high-finesse optical cavity. The atom acts as a quantum-optical transistor with the ability to coherently control13 the transmission of light through the cavity. We investigat! e the scaling of EIT when the atom number is increased one-by-one. The measured spectra are in excellent agreement with a theoretical model. Merging EIT with cavity quantum electrodynamics and single quanta of matter is likely to become the cornerstone for novel applications, such as dynamic control of the photon statistics of propagating light fields14 or the engineering of Fock state superpositions of flying light pulses15. View full text Subject terms: * Journals * Peer-review Figures at a glance * Figure 1: Experimental protocol and cavity EIT. 87Rb atoms are quasi-permanently trapped inside a high-finesse optical cavity. The cavity is resonant with the atomic F = 1 ↔ F′ = 1 transition at 780 nm wavelength. The transmission of the atom–cavity system is probed with a weak laser (probe laser–cavity detuning Δ) for three physical conditions. , With atoms shelved in the hyperfine state F = 2, we record the empty cavity transmission as a reference (black data and curve in ). , With atoms prepared in F = 1, we realize a cavity QED situation and observe a spectrum exhibiting a vacuum-Rabi splitting (red data and curve in ). , An additional laser is used to coherently control the optical properties of the atom–cavity system. , Measured transmission spectra for on average 15 atoms coupled to the cavity. A narrow transmission window (linewidth ~900 kHz) observed at the two-photon resonance in the cavity EIT situation (blue data and theory curve) testifies to the existence of a coherent dark state. Experimental! parameters: maximum intra-cavity photon number 0.02, control power 9 µW (equivalent Rabi frequency 1.3κ). Error bars shown are s.d. and are omitted from the empty cavity and two-level measurement for clarity. * Figure 2: Cavity EIT with a single atom. Colour coding same as in Fig. 1d. , Measured transmission spectra for exactly one atom coupled to the cavity and a control laser power of 3 µW (equivalent Rabi frequency, 0.78κ). EIT is observed with a maximum transparency of 96% and a measured transmission contrast of 20% with respect to the control laser switched off. The linewidth (see Methods Summary) is ~1.2 MHz. The red solid curve is a solution of the time-dependent master equation for the finite probing interval. The red dashed curve is the prediction of equation (1) for zero control power. Error bars shown are s.d. Inset, CCD camera image of a single atom trapped in the cavity (image size, 33 µm × 16 µm). , , The linewidth and contrast of the single-atom transparency feature are tunable by means of the control laser power. The values used are (1, 2, 3) µW and correspond to Rabi frequencies (0.45, 0.63, 0.78) κ. The maximum intra-cavity photon number is 0.02. Error bars shown are s.d. for the contr! ast, ±0.03κ for the linewidth and ±10% for the x axis. * Figure 3: Cavity EIT spectra for N = 2 to 5 atoms. , 2 atoms; , 3 atoms; , 4 atoms; , 5 atoms. Changing the number of atoms enhances the visibility of the dark-state resonance owing to improved contrast with respect to the measurements with two-level atoms coupled to the cavity. The vacuum-Rabi splitting starts being resolved at higher atom number. Colour coding and experimental parameters are as in Fig. 2a. Insets, CCD camera images of the trapped atoms, used to precisely determine their number and physical location in the cavity mode. Error bars shown are ±s.d. and are omitted from the empty cavity and two-level measurement for clarity. * Figure 4: Measured transparency, contrast and linewidth of cavity EIT with N = 1 to 7 atoms. , The maximum transparency (blue bars, top) decreases with the number of atoms from 96% (N = 1) to 78% (N = 7). Nevertheless, the on/off contrast (red bars, bottom) at the two-photon resonance steadily increases from 21% (N = 1) to 60% (N = 7) owing to the reduction of transmission with control laser switched off. , For N ≥ 3, the cavity EIT linewidth decreases with the number of coupled atoms as 1/N (guide to the eye, dashed red curve). For N = 1, 2, the linewidth is nearly constant owing to the interplay between the increase in the two-level atom transmission and the definition of the linewidth used (see Methods Summary). Error bars shown are ±s.d. for the contrast and transparency and ±0.03κ for the linewidth. Author information * Author information * Comments Primary authors * These authors contributed equally to this work. * Martin Mücke & * Eden Figueroa Affiliations * Max-Planck-Institut für Quantenoptik, Hans-Kopfermann-Str. 1, D-85748 Garching, Germany * Martin Mücke, * Eden Figueroa, * Joerg Bochmann, * Carolin Hahn, * Karim Murr, * Stephan Ritter, * Celso J. Villas-Boas & * Gerhard Rempe * Departamento de Fisica, Universidade Federal de São Carlos, 13565-905 São Carlos, São Paulo, Brazil * Celso J. Villas-Boas Contributions All authors contributed to the implementation and modelling of the experiment, the interpretation of the results and the writing of the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Eden Figueroa (eden.figueroa@mpq.mpg.de) Additional data
• Daughter bubble cascades produced by folding of ruptured thin films
  - Nature (London) 465(7299):759 (2010)
  Nature | Letter Daughter bubble cascades produced by folding of ruptured thin films * James C. Bird1 Search for this author in: * NPG journals * PubMed * Google Scholar * Riëlle de Ruiter1 Search for this author in: * NPG journals * PubMed * Google Scholar * Laurent Courbin2 Search for this author in: * NPG journals * PubMed * Google Scholar * Howard A. Stone1, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorsJournal name:NatureVolume:465,Pages:759–762Date published:(10 June 2010)DOI:doi:10.1038/nature09069Received21 January 2010Accepted01 April 2010 Article tools * Full text * 日本語要約 * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Thin liquid films, such as soap bubbles, have been studied extensively for over a century because they are easily formed and mediate a wide range of transport processes in physics, chemistry and engineering1, 2, 3. When a bubble on a liquid–gas or solid–gas interface (referred to herein as an interfacial bubble) ruptures, the general expectation is that the bubble vanishes. More precisely, the ruptured thin film is expected to retract rapidly until it becomes part of the interface, an event that typically occurs within milliseconds4, 5, 6. The assumption that ruptured bubbles vanish is central to theories on foam evolution7 and relevant to health8 and climate9 because bubble rupture is a source for aerosol droplets10, 11. Here we show that for a large range of fluid parameters, interfacial bubbles can create numerous small bubbles when they rupture, rather than vanishing. We demonstrate, both experimentally and numerically, that the curved film of the ruptured bubble can! fold and entrap air as it retracts. The resulting toroidal geometry of the trapped air is unstable, leading to the creation of a ring of smaller bubbles. The higher pressure associated with the higher curvature of the smaller bubbles increases the absorption of gas into the liquid, and increases the efficiency of rupture-induced aerosol dispersal. View full text Subject terms: * Applied physics * Engineering * Environmental science Figures at a glance * Figure 1: The daughter bubble cascade, with jets, droplets and daughter bubbles resulting from bursting bubbles. –, When a single soap bubble ruptures on a rigid surface, a ring of many smaller bubbles can form. Similarly, when one of the daughter bubbles pops, even smaller bubbles are created, demonstrating a bubble-bursting cascade. , The dynamics are more intricate when an air bubble ruptures on a deep pool of water. The Laplace pressure inside the bubble dimples the interface to create a cavity. , Once the bubble ruptures, the film rapidly retracts (within a time t = 3.2 ms). , As the cavity re-equilibrates, a jet of liquid is propelled upward. Many smaller daughter bubbles are visible around the jet. –, Within a fraction of a second, these secondary bubbles burst (), a narrow jet forms (), and tiny liquid droplets are dispersed into the atmosphere (, inside pink circles). For more detail, see Supplementary Movies 1 and 2. * Figure 2: Two-step mechanism to form daughter bubbles. , High-speed images of a glycerol–water bubble filled with air popping on a solid surface viewed from the side. The bubble has an initial radius R = 5.3 mm, dynamic viscosity μ = 0.31 Pa s, surface tension γ = 55 mN m−1, and density ρ = 103 kg m−3. , The rupture is simultaneously filmed from below and reveals two concentric tori that break up into daughter bubbles. The red dotted lines in the final images denote the position of the bubble before rupture, and the time from rupture t is reported in milliseconds. For more detail, see Supplementary Movie 3. * Figure 3: Numerical simulations for understanding film folding and air entrapment. , Our numerical simulation reproduces the inward folding behaviour of the collapsing film. The elapsed time t is dimensionalized by . The selected times correspond to those in the three middle images in Fig. 2a with a film thickness of h = 11 μm. , A cross-sectional schematic of the proposed folding during the last stages of the collapse demonstrates how the film can create two concentric rings of air by intersecting with itself and the lower interface. This situation occurs when the capillary number is large Ca ≡ Uμ/γ  1. , Another schematic illustrates how when the rim is unstable, the film traps only one torus of air. This rim instability appears to occur when Ca  1. * Figure 4: Dynamical characterization of the formation of daughter bubbles. After a hemispherical bubble ruptures, there are either no daughter bubbles (red symbols), a single ring of daughter bubbles (blue symbols), or two concentric rings of daughter bubbles (cyan symbols). The transition between these regimes coincides with Ca ≡ Uμ/γ and Re ≡ ρUR/μ of approximately one, as predicted by our scaling arguments. The shapes of the data points correspond to various configurations: bubbles formed on deep pools of pure silicone oil (squares), on thin films of glycerol–water solutions stabilized by SDS surfactant (all triangles), and on deep pools of local river water stabilized by indigenous surfactant (circles and diamonds). The effects of different gases inside the bubbles were investigated using helium (right triangles and diamonds), nitrogen (up triangles), carbon dioxide (down triangles), and air (squares and circles). Author information * Author information * Supplementary information * Comments Affiliations * School of Engineering and Applied Sciences, Harvard University, Cambridge, Massachusetts 02138, USA * James C. Bird, * Riëlle de Ruiter & * Howard A. Stone * Institut de Physique de Rennes, UMR CNRS 6251, Campus Beaulieu, Université Rennes 1, 35042 Rennes, France * Laurent Courbin * Department of Mechanical and Aerospace Engineering, Princeton University, Princeton, New Jersey 08544, USA * Howard A. Stone Contributions J.C.B., L.C. and H.A.S. designed the research; J.C.B., R.d.R. and L.C. performed the research; J.C.B., R.d.R., L.C. and H.A.S. analysed the data; J.C.B. wrote the manuscript and all authors commented on it. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * James C. Bird (jbird@fas.harvard.edu) or * Howard A. Stone (hastone@princeton.edu) Supplementary information * Author information * Supplementary information * Comments Movies * Supplementary Movie 1 (5.7M) This is the original movie of the bubble dynamics shown in Fig. 1E-G. The interfacial bubble ruptures, uncovering a dimple on the water surface. The high curvature of this dimple causes a jet to form; since this jet does not have enough kinetic energy to be propelled into the atmosphere it returns to the interface. Yet, around the jet, there is a ring of smaller bubbles formed during the film retraction. One of these daughter bubbles ruptures at 56 ms (left side of movie). * Supplementary Movie 2 (1.3M) A smaller, daughter bubble created by the rupture of a larger bubble ruptures on a much faster timescale and also forms a jet. This jet breaks up into micron-sized droplets which are propelled into the atmosphere at speeds exceeding 5 m/s. Therefore the rupture of a centimeter-sized bubble can lead to the aerosolization of dozens of fine droplets into the atmosphere. * Supplementary Movie 3 (1.6M) The two-step mechanism to form daughter bubbles (Fig. 2) is presented with simultaneous movies showing side and bottom perspectives. * Supplementary Movie 4 (837K) Numerical simulations of the film retraction capture the folding dynamics of the film * Supplementary Movie 5 (1.9M) The film retraction of a highly viscous bubble (a million times the viscosity of water). This movie corresponds to the far-left point in Fig. 4 * Supplementary Movie 6 (1.3M) The rupture of an air bubble on water shows how the rim of the film becomes unstable as it retracts. This movie is representative of the points on the far-right of Fig. 4. PDF files * Supplementary Information (549K) This file contains Supplementary Material and Methods, a Supplementary Discussion, Supplementary Figures S1-S2 with legends and References. Additional data
• Electron localization following attosecond molecular photoionization
  - Nature (London) 465(7299):763 (2010)
  Nature | Letter Electron localization following attosecond molecular photoionization * G. Sansone1, 10 Search for this author in: * NPG journals * PubMed * Google Scholar * F. Kelkensberg2, 10 Search for this author in: * NPG journals * PubMed * Google Scholar * J. F. Pérez-Torres3, 10 Search for this author in: * NPG journals * PubMed * Google Scholar * F. Morales3, 10 Search for this author in: * NPG journals * PubMed * Google Scholar * M. F. Kling4 Search for this author in: * NPG journals * PubMed * Google Scholar * W. Siu2 Search for this author in: * NPG journals * PubMed * Google Scholar * O. Ghafur2 Search for this author in: * NPG journals * PubMed * Google Scholar * P. Johnsson2, 5 Search for this author in: * NPG journals * PubMed * Google Scholar * M. Swoboda5 Search for this author in: * NPG journals * PubMed * Google Scholar * E. Benedetti1 Search for this author in: * NPG journals * PubMed * Google Scholar * F. Ferrari1 Search for this author in: * NPG journals * PubMed * Google Scholar * F. Lépine6 Search for this author in: * NPG journals * PubMed * Google Scholar * J. L. Sanz-Vicario7 Search for this author in: * NPG journals * PubMed * Google Scholar * S. Zherebtsov4 Search for this author in: * NPG journals * PubMed * Google Scholar * I. Znakovskaya4 Search for this author in: * NPG journals * PubMed * Google Scholar * A. L'Huillier5 Search for this author in: * NPG journals * PubMed * Google Scholar * M. Yu. Ivanov8 Search for this author in: * NPG journals * PubMed * Google Scholar * M. Nisoli1 Search for this author in: * NPG journals * PubMed * Google Scholar * F. Martín3 Search for this author in: * NPG journals * PubMed * Google Scholar * M. J. J. Vrakking2, 9 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:NatureVolume:465,Pages:763–766Date published:(10 June 2010)DOI:doi:10.1038/nature09084Received16 July 2009Accepted13 April 2010 Article tools * Full text * 日本語要約 * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg For the past several decades, we have been able to directly probe the motion of atoms that is associated with chemical transformations and which occurs on the femtosecond (10−15-s) timescale. However, studying the inner workings of atoms and molecules on the electronic timescale1, 2, 3, 4 has become possible only with the recent development of isolated attosecond (10−18-s) laser pulses5. Such pulses have been used to investigate atomic photoexcitation and photoionization6, 7 and electron dynamics in solids8, and in molecules could help explore the prompt charge redistribution and localization that accompany photoexcitation processes. In recent work, the dissociative ionization of H2 and D2 was monitored on femtosecond timescales9 and controlled using few-cycle near-infrared laser pulses10. Here we report a molecular attosecond pump–probe experiment based on that work: H2 and D2 are dissociatively ionized by a sequence comprising an isolated attosecond ultraviolet pulse! and an intense few-cycle infrared pulse, and a localization of the electronic charge distribution within the molecule is measured that depends—with attosecond time resolution—on the delay between the pump and probe pulses. The localization occurs by means of two mechanisms, where the infrared laser influences the photoionization or the dissociation of the molecular ion. In the first case, charge localization arises from quantum mechanical interference involving autoionizing states and the laser-altered wavefunction of the departing electron. In the second case, charge localization arises owing to laser-driven population transfer between different electronic states of the molecular ion. These results establish attosecond pump–probe strategies as a powerful tool for investigating the complex molecular dynamics that result from the coupling between electronic and nuclear motions beyond the usual Born–Oppenheimer approximation. View full text Subject terms: * Applied physics * Engineering Figures at a glance * Figure 1: Dissociative ionization of hydrogen by an EUV–infrared pulse sequence. , Photoexcitation of neutral hydrogen leads to the excitation of the Q1 (red) and Q2 (blue) doubly excited states and ionization to the 1sσg and 2pσu states, which can be followed by dissociation. R, internuclear distance; a.u., atomic units (0.529 Å). , , Experimental D+ () and calculated H+ () kinetic energy distributions with (from top to bottom) only the isolated attosecond laser pulse present, with only the few-cycle infrared (IR) laser pulse present and for two delays between the EUV and infrared pulses. , , Experimental D+ () and calculated H+ () kinetic energy distributions as functions of the delay between the attosecond pulse and the infrared pulse. Colour scale shows fragment yield in arbitrary units () and calculated probabilities (). * Figure 2: Asymmetry in EUV–infrared dissociative ionization of hydrogen. , Experimentally measured asymmetry parameter (colour scale) for the formation of D+ ions in two-colour (EUV–infrared) dissociative ionization of D2, as a function of the fragment kinetic energy, Ek, and the EUV–infrared delay. A fragment asymmetry is observed that oscillates as a function of the EUV–infrared delay and that strongly depends on the kinetic energy. , Calculated asymmetry parameter for the formation of H+ ions in two-colour EUV–infrared dissociative ionization of H2, as a function of the fragment kinetic energy, Ek, and the EUV–infrared delay, obtained using the close-coupling method described in the text. , Same as in , but for H+ ions. * Figure 3: Mechanisms that lead to asymmetry in EUV–infrared dissociative ionization. , Asymmetry caused by the interference of a wave packet launched in the 2pσu state by direct EUV ionization or rapid ionization of the Q11Σu+ doubly-excited states by the infrared pulse and a wave packet in the 1sσg state resulting from autoionization of the Q11Σu+ states. Blue arrows indicate the effect of the EUV pulse and red arrows that of the infrared pulse; purple lines and arrows signify dynamics that is intrinsic to the molecule. , Close-coupling calculations in which direct photoexcitation to the 1sσg state has been excluded, supporting the notion that the Q1 autoionizing states have an important role in the localization dynamics. , Asymmetry caused by the interference of a wave packet that is launched in the 2pσu state by direct EUV ionization and a wave packet in the 1sσg state that results from stimulated emission during the dissociation process. , Time-dependent asymmetry from a two-level calculation in which the wavefunction of the dissociating molecule ! is considered to be a coherent superposition of the 1sσg and 2pσu states. Author information * Author information * Supplementary information * Comments Primary authors * These authors contributed equally to this work. * G. Sansone, * F. Kelkensberg, * J. F. Pérez-Torres & * F. Morales Affiliations * CNR-INFM, National Laboratory for Ultrafast and Ultraintense Optical Science, Department of Physics, Politecnico of Milan, Piazza L. da Vinci 32, 20133 Milano, Italy * G. Sansone, * E. Benedetti, * F. Ferrari & * M. Nisoli * FOM-Institute AMOLF, Science Park 104, 1098 XG Amsterdam, The Netherlands * F. Kelkensberg, * W. Siu, * O. Ghafur, * P. Johnsson & * M. J. J. Vrakking * Departamento de Química, C-9, Universidad Autónoma de Madrid, 28049 Madrid, Spain * J. F. Pérez-Torres, * F. Morales & * F. Martín * Max-Planck Institut für Quantenoptik, Hans-Kopfermann Strasse 1, D-85748 Garching, Germany * M. F. Kling, * S. Zherebtsov & * I. Znakovskaya * Department of Physics, Lund University, PO Box 118, SE-221 00 Lund, Sweden * P. Johnsson, * M. Swoboda & * A. L'Huillier * Université Lyon 1/CNRS/LASIM, UMR 5579, 43 Boulevard Du 11 Novembre 1918, F-69622 Villeurbane, France * F. Lépine * Grupo de Física Atómica y Molecular, Instituto de Física, Universidad de Antioquia, AA1226 Medellín, Colombia * J. L. Sanz-Vicario * Department of Physics, Imperial College London, South Kensington Campus, SW7 2AZ, London, UK * M. Yu. Ivanov * Max-Born-Institut, Max-Born Strasse 2A, D-12489 Berlin, Germany * M. J. J. Vrakking Contributions G.S. was responsible for the construction of the attosecond pump–probe set-up and the experiments on H2 and D2. F.K. was responsible for the experiments on H2 and D2 and the development of the semi-classical model. M.Yu.I. helped with the semi-classical model. J.F.P.-T. and F. Morales were responsible for the construction of the close-coupling code and the calculations using this code. M.F.K., W.S., O.G., P.J., M.S., E.B., F.F., F.L., S.Z. and I.Z. contributed to the experiments, which were carried out in three prolonged experimental sessions over almost 18 months. M.N. was in charge of the laboratory where the experiments were performed. A.L'H. supervised M.S. and P.J. F. Martin supervised F. Morales and J.F.P.-T. and was in charge of work with the TDSE model. J.L.S.-V. helped with the TDSE model. M.J.J.V. supervised F.K., W.S., O.G. and P.J. and was responsible for the overall coordination of the project Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * M. J. J. Vrakking (vrakking@mbi-berlin.de) Supplementary information * Author information * Supplementary information * Comments PDF files * Supplementary Figures (39K) This file contains Supplementary Figure 1 with legend. Additional data
• The lead isotopic age of the Earth can be explained by core formation alone
  - Nature (London) 465(7299):767 (2010)
  Nature | Letter The lead isotopic age of the Earth can be explained by core formation alone * Bernard J. Wood1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Alex N. Halliday1 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:NatureVolume:465,Pages:767–770Date published:(10 June 2010)DOI:doi:10.1038/nature09072Received29 July 2009Accepted26 March 2010 Article tools * Full text * 日本語要約 * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg The meaning of the age of the Earth defined by lead isotopes has long been unclear. Recently it has been proposed1 that the age of the Earth deduced from lead isotopes reflects volatile loss to space at the time of the Moon-forming giant impact rather than partitioning into metallic liquids during protracted core formation. Here we show that lead partitioning into liquid iron depends strongly on carbon content and that, given a content of ~0.2% carbon2, 3, experimental and isotopic data both provide evidence of strong partitioning of lead into the core throughout the Earth's accretion. Earlier conclusions that lead is weakly partitioned into iron arose from the use of carbon-saturated (about 5% C) iron alloys. The lead isotopic age of the Earth is therefore consistent with partitioning into the core and with no significant late losses of moderately volatile elements to space during the giant impact. View full text Subject terms: * Planetary sciences * Earth sciences * Geology * Geophysics Figures at a glance * Figure 1: Volatile element abundances in the BSE. Abundances in Allende carbonaceous chondrite (white symbols) and in the BSE31 are plotted against temperature of 50% condensation (T50) from a gas of solar composition28. The abundances are normalized to CI carbonaceous chondrite and Mg. Red symbols refer to highly siderophile elements in the BSE , blue symbols refer to lithophile and black symbols refer to weakly and moderately siderophile elements. We note that siderophile S, Bi and Ge are one to two orders of magnitude more depleted in the BSE than lithophile B and F. Pb is about one order of magnitude more depleted than F. * Figure 2: Experimental data for Pb partitioning into liquid Fe coexisting with liquid silicate. KD is defined as [Pb]metal[Fe]silicate/[Pb]silicate[Fe]metal, where values in brackets are weight concentrations. Error bars refer to ±2 standard errors. , Data for PbO(silicate)+Fe(metal) = Pb(metal)+FeO(silicate) are from refs 1 and 12. All experiments were performed in carbon capsules and are carbon-saturated, apart from the open symbol from ref. 12 (MgO capsule). Metal in the latter contained 9 wt% Si. , Liquid metal–liquid silicate partitioning data from this study. Experiments were performed in carbon and MgO capsules. One-atmosphere thermodynamic data for pure liquids32 are corrected for the activity coefficients of Pb in liquid Fe15. The length of arrow indicates the calculated displacement due to carbon saturation of the Fe liquid at 1,860 K (ref. 15). * Figure 3: Calculated and estimated lead isotopic composition of the BSE. The line labelled 'Geochron' is the zero age line for fractionation of Pb from U at the origin of the Solar System. The dotted line labelled 'Consistent with Hf–W' represents the loci of points for which the timing of U–Pb fractionation would agree with Hf–W fractionation using the exponential growth model22. The dashed lines labelled '80 Myr', '110 Myr' and '140 Myr' represent the loci of points calculated for the addition of 10% to the core during a giant impact at the corresponding time after the origin of the Solar System. The first 90% of accretion was assumed to obey the exponential growth model22 under P,T oxygen fugacity conditions constrained by mantle–core partitioning of a number of siderophile elements18. Symbols are published estimates of the BSE composition16. * Figure 4: Rb–Sr model age of the Moon plotted versus the percentage loss of Rb from the Earth at the time of the giant impact. Sr-isotopic equilibration between Earth and Moon was assumed at time of impact. To satisfy the Sr-isotopic composition of the Moon using the current Rb/Sr of the Earth (0.03), the timing of the Moon-forming impact needs to become earlier as the extent of Rb loss from the Earth becomes greater. The two lines are calculated with different values for the initial isotopic composition of the Solar System33. Because a lunar age of 70–110 million years is consistent with W isotopic data for the Moon8 and the age of the earliest crust10, the loss of Rb and similarly volatile elements such as Pb from the Earth during the giant impact must be small. Author information * Author information * Supplementary information * Comments Affiliations * Department of Earth Sciences, Parks Road, Oxford OX1 3PR, UK * Bernard J. Wood & * Alex N. Halliday * Department of Earth and Planetary Sciences, Macquarie University, New South Wales 2109, Australia * Bernard J. Wood Contributions B.J.W. performed all the experiments and all the electron microprobe and laser ICP-MS analyses, and the Pb-isotopic modelling of Fig. 3. A.N.H. performed the Sr-isotope modelling depicted in Fig. 4. Both authors contributed to the writing of the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Bernard J. Wood (berniew@earth.ox.ac.uk) Supplementary information * Author information * Supplementary information * Comments PDF files * Supplementary Information (623K) This file contains Supplementary Tables 1-2, Supplementary Data and Supplementary Figure S1 with legend. Additional data
• Small mammal diversity loss in response to late-Pleistocene climatic change
  - Nature (London) 465(7299):771 (2010)
  Nature | Letter Small mammal diversity loss in response to late-Pleistocene climatic change * Jessica L. Blois1, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Jenny L. McGuire2 Search for this author in: * NPG journals * PubMed * Google Scholar * Elizabeth A. Hadly1 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:NatureVolume:465,Pages:771–774Date published:(10 June 2010)DOI:doi:10.1038/nature09077Received26 October 2009Accepted01 April 2010Published online23 May 2010 Article tools * Full text * 日本語要約 * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Communities have been shaped in numerous ways by past climatic change; this process continues today1. At the end of the Pleistocene epoch about 11,700 years ago, North American communities were substantially altered by the interplay of two events. The climate shifted from the cold, arid Last Glacial Maximum to the warm, mesic Holocene interglacial, causing many mammal species to shift their geographic distributions substantially2, 3. Populations were further stressed as humans arrived on the continent4. The resulting megafaunal extinction event, in which 70 of the roughly 220 largest mammals in North America (32%) became extinct5, has received much attention. However, responses of small mammals to events at the end of the Pleistocene have been much less studied, despite the sensitivity of these animals to current and future environmental change. Here we examine community changes in small mammals in northern California during the last 'natural' global warming event at t! he Pleistocene–Holocene transition and show that even though no small mammals in the local community became extinct, species losses and gains, combined with changes in abundance, caused declines in both the evenness and richness of communities. Modern mammalian communities are thus depauperate not only as a result of megafaunal extinctions at the end of the Pleistocene but also because of diversity loss among small mammals. Our results suggest that across future landscapes there will be some unanticipated effects of global change on diversity: restructuring of small mammal communities, significant loss of richness, and perhaps the rising dominance of native 'weedy' species. View full text Subject terms: * Palaeontology * Earth sciences Figures at a glance * Figure 1: Location map. Modern mammal species richness in California (see the California Department of Fish and Game website, http://www.dfg.ca.gov/biogeodata/cwhr/), with low richness shown in lighter grey and high richness in darker grey. The filled circle indicates the location of Samwell Cave. , Enlarged map of the boxed region in showing the location of Samwell Cave (filled black point) with the approximate collection radius associated with the fossil deposit (Supplementary Discussion) indicated by the open black circle around the point. Major biogeographic regions surrounding the cave are indicated. * Figure 2: Diversity through time based on standardized abundance data from 1,000 subsamples at n = 132. , Evenness; , richness; , turnover (dotted line, Bray–Curtis index; solid line, Jaccard index). Lines connecting points in are for comparison purposes only. Higher turnover values indicate greater differences between communities over time (Supplementary Discussion). On all figures the light grey curve indicates a temperature proxy, δ18O from the North Greenland Ice Core Project ice-core record20, expressed in parts per thousand with respect to Vienna Standard Mean Ocean Water. Vertical dotted lines indicate SCPD level boundaries. Error bars indicate 95% confidence intervals. Author information * Author information * Supplementary information * Comments Affiliations * Department of Biology, Stanford University, Stanford, California 94305, USA * Jessica L. Blois & * Elizabeth A. Hadly * Department of Integrative Biology, University of California, Berkeley, Berkeley, California 94720, USA * Jenny L. McGuire * Present address: Center for Climatic Research and Department of Geography, University of Wisconsin–Madison, 1225 W. Dayton Street, Madison, Wisconsin 53706-1695, USA. * Jessica L. Blois Contributions J.L.B. planned the project, excavated the deposit, identified specimens, analysed the data and wrote the paper. J.L.M. identified Microtus spp., performed radiocarbon dating and wrote the paper. E.A.H. planned the project, helped excavate the deposit and wrote the paper. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Jessica L. Blois (blois@wisc.edu) Fossil specimens are deposited in the University of California Museum of Paleontology as localities V99822 and V99785. Modern specimens are deposited in the University of California Museum of Vertebrate Zoology under accession number 14590. Supplementary information * Author information * Supplementary information * Comments PDF files * Supplementary Information (627K) This file contains Supplementary Tables 1-5, Supplementary Figures 1-4 with legends, a Supplementary Discussion and References. Additional data
• Putting brain training to the test
  Owen AM Hampshire A Grahn JA Stenton R Dajani S Burns AS Howard RJ Ballard CG - Nature (London) 465(7299):775 (2010)
  Nature | Letter Putting brain training to the test * Adrian M. Owen1 Search for this author in: * NPG journals * PubMed * Google Scholar * Adam Hampshire1 Search for this author in: * NPG journals * PubMed * Google Scholar * Jessica A. Grahn1 Search for this author in: * NPG journals * PubMed * Google Scholar * Robert Stenton2 Search for this author in: * NPG journals * PubMed * Google Scholar * Said Dajani2 Search for this author in: * NPG journals * PubMed * Google Scholar * Alistair S. Burns3 Search for this author in: * NPG journals * PubMed * Google Scholar * Robert J. Howard2 Search for this author in: * NPG journals * PubMed * Google Scholar * Clive G. Ballard2 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:NatureVolume:465,Pages:775–778Date published:(10 June 2010)DOI:doi:10.1038/nature09042Received25 January 2010Accepted29 March 2010Published online20 April 2010 Article tools * Full text * 日本語要約 * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg 'Brain training', or the goal of improved cognitive function through the regular use of computerized tests, is a multimillion-pound industry1, yet in our view scientific evidence to support its efficacy is lacking. Modest effects have been reported in some studies of older individuals2, 3 and preschool children4, and video-game players outperform non-players on some tests of visual attention5. However, the widely held belief that commercially available computerized brain-training programs improve general cognitive function in the wider population in our opinion lacks empirical support. The central question is not whether performance on cognitive tests can be improved by training, but rather, whether those benefits transfer to other untrained tasks or lead to any general improvement in the level of cognitive functioning. Here we report the results of a six-week online study in which 11,430 participants trained several times each week on cognitive tasks designed to improve! reasoning, memory, planning, visuospatial skills and attention. Although improvements were observed in every one of the cognitive tasks that were trained, no evidence was found for transfer effects to untrained tasks, even when those tasks were cognitively closely related. View full text Subject terms: * Psychology * Neuroscience Figures at a glance * Figure 1: Benchmarking scores at baseline and after six weeks of training across the three groups of participants. PAL, paired-associates learning; SWM, spatial working memory; VSTM, verbal short-term memory. Bars represent standard deviations. * Figure 2: First and last training scores for the six tests used to train experimental group 1 and experimental group 2. The first and last scores for the control group are also shown. Bars represent standard deviations. Author information * Author information * Supplementary information * Comments Affiliations * MRC Cognition and Brain Sciences Unit, 15 Chaucer Road, Cambridge CB2 7EF, UK * Adrian M. Owen, * Adam Hampshire & * Jessica A. Grahn * King's College London, Institute of Psychiatry, De Crespigny Park, London SE5 8AF, UK * Robert Stenton, * Said Dajani, * Robert J. Howard & * Clive G. Ballard * University of Manchester and Manchester Academic Health Science Centre, Manchester M13 9PL, UK * Alistair S. Burns Contributions A.M.O. co-designed the study, co-designed the training tasks, designed (with A.H.) the benchmarking tests provided by http://www.cambridgebrainsciences.com, co-conducted the statistical analysis, interpreted the data and took overall responsibility for writing each draft of the manuscript. A.H. contributed to the design of the training tasks, designed (with A.M.O.) and programmed the benchmarking tests provided by http://www.cambridgebrainsciences.com, co-conducted the statistical analysis and contributed to each draft of the manuscript. J.A.G. co-conducted the statistical analysis, contributed to the interpretation of the data, co-wrote the first draft of the manuscript and contributed to each subsequent version. R.S. designed the data capture, data checking and data cleaning protocols and was responsible for converting data into a format for analysis and for the delivery of the trial database for statistical analysis. He was part of the project management group and contrib! uted to each draft of the manuscript. S.D. contributed to the design of the study, piloted brain training modules, contributed to the design and implementation of the recruitment and retention strategies, was part of the project management group and contributed to each draft of the manuscript. A.S.B. was chair of the independent trial steering committee and advised on key aspects of study design and implementation in this capacity. He also contributed to each draft of the manuscript. R.J.H. advised on key aspects of general study design, contributed to the design of the training tasks and contributed to each draft of the manuscript. C.G.B. jointly conceived of and jointly designed the study and wrote the first draft of the protocol. He was part of the project management group, co-conducted the statistical evaluation, contributed to the interpretation of the data and contributed to each draft of the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Adrian M. Owen (adrian.owen@mrc-cbu.cam.ac.uk) Supplementary information * Author information * Supplementary information * Comments PDF files * Supplementary Information (0.516M) This file contains Supplementary Figures 1-2 with legends. An earlier version of this paper was published online on 20 April 2010 Additional data
• Moonlighting bacteriophage proteins derepress staphylococcal pathogenicity islands
  - Nature (London) 465(7299):779 (2010)
  Nature | Letter Moonlighting bacteriophage proteins derepress staphylococcal pathogenicity islands * María Ángeles Tormo-Más1 Search for this author in: * NPG journals * PubMed * Google Scholar * Ignacio Mir1 Search for this author in: * NPG journals * PubMed * Google Scholar * Archana Shrestha2 Search for this author in: * NPG journals * PubMed * Google Scholar * Sandra M. Tallent2 Search for this author in: * NPG journals * PubMed * Google Scholar * Susana Campoy3 Search for this author in: * NPG journals * PubMed * Google Scholar * Íñigo Lasa4 Search for this author in: * NPG journals * PubMed * Google Scholar * Jordi Barbé3 Search for this author in: * NPG journals * PubMed * Google Scholar * Richard P. Novick5 Search for this author in: * NPG journals * PubMed * Google Scholar * Gail E. Christie2 Search for this author in: * NPG journals * PubMed * Google Scholar * José R. Penadés1, 6 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:NatureVolume:465,Pages:779–782Date published:(10 June 2010)DOI:doi:10.1038/nature09065Received07 September 2009Accepted01 April 2010Published online16 May 2010 Article tools * Full text * 日本語要約 * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Staphylococcal superantigen-carrying pathogenicity islands (SaPIs) are discrete, chromosomally integrated units of ~15 kilobases that are induced by helper phages to excise and replicate. SaPI DNA is then efficiently encapsidated in phage-like infectious particles, leading to extremely high frequencies of intra- as well as intergeneric transfer1, 2, 3. In the absence of helper phage lytic growth, the island is maintained in a quiescent prophage-like state by a global repressor, Stl, which controls expression of most of the SaPI genes4. Here we show that SaPI derepression is effected by a specific, non-essential phage protein that binds to Stl, disrupting the Stl–DNA complex and thereby initiating the excision-replication-packaging cycle of the island. Because SaPIs require phage proteins to be packaged5, 6, this strategy assures that SaPIs will be transferred once induced. Several different SaPIs are induced by helper phage 80α and, in each case, the SaPI commandeers a di! fferent non-essential phage protein for its derepression. The highly specific interactions between different SaPI repressors and helper-phage-encoded antirepressors represent a remarkable evolutionary adaptation involved in pathogenicity island mobilization. View full text Subject terms: * Molecular biology * Evolution * Genetics * Genomics Figures at a glance * Figure 1: Induction of SaPIbov1 by different dut alleles. , Southern blot of φ11 mutant lysates, from strains with (JP1794 and JP4125) or without (RN451 and JP4025) SaPIbov1 tst::tetM, as indicated. Samples were isolated 0 or 60 min after induction with mitomycin C, separated on agarose and blotted with a phage- or SaPIbov1-specific probe. Upper band is 'bulk' DNA, including chromosomal, phage and replicating SaPI; lower band is SaPI linear monomers (L) released from phage heads. , SaPIbov1 excision and replication after induction of cloned dut genes from different staphylococcal phages. A non-lysogenic derivative of strain RN4220 carrying SaPIbov1 was complemented with plasmids expressing 3×Flag-tagged Dut proteins. One millilitre of each culture (optical density (OD)540 nm = 0.3) was collected 3 h after treatment with 5 μM CdCl2 and used to prepare standard minilysates, which were resolved on a 0.7% agarose gel, Southern blotted and probed for SaPIbov1 DNA. Lane 1, JP6789; lane 2, JP6790; lane 3, JP6797; lane 4, JP! 6791; lane 5, JP6796; and lane 6, JP6772. In these experiments, because no helper phage is present, the excised SaPI DNA appears as covalently closed circular molecules (CCC) rather than the linear monomers that are seen following helper-phage-mediated induction and packaging (as in , lane 7). , SaPIbov1 excision and replication induced by constitutive expression of cloned dut genes. Lanes 1–6 are as in , above. Lane 7, SaPIbov1 induction after mitomycin C treatment of a φ11 prophage (JP1794). Lane 8, induction by cloned φ11 Dut in a recA mutant (JP6773). The upper panel is a Southern blot probed for SaPIbov1 DNA; the lower panel is a western blot probed with antibody to the Flag tag carried by the proteins. kDa, kilodaltons. * Figure 2: Phage-inducing proteins bind SaPI-encoded Stl proteins. , dUTPase prevents StlSaPIbov1 from binding to the stl–str divergent region. Shown are electrophoretic mobility shift assays in which increasing concentrations of StlSaPIbov1 (0, 0.03, 0.06, 0.12, 0.24 and 0.48 μg; left), dUTPaseφ11 (0, 0.02, 0.04, 0.08, 0.16 and 0.2 μg; middle), or StlSaPIbov1 (0.12 μg) in the presence of dUTPaseφ11 (0, 0.02, 0.04, 0.08, 0.16 and 0.2 μg) or 10 μg BSA (right) were mixed with labelled DNA containing the SaPIbov1 divergent region. , Affinity chromatography of dUTPase using His6–StlSaPIbov1 (left), or affinity chromatography of StlSaPIbov2 using His6–ORF15φ80α (right). E. coli strains expressing the different pairs were isopropyl-β-d-thiogalactoside (IPTG)-induced and, after disruption of the cells, the expressed proteins were applied to a Ni2+ agarose column and eluted. The presence of the different proteins was monitored in the load (lanes E), flow-through, wash and elute fractions by Coomassie staining. Elution frac! tions (lanes 1, 2, 3 and 4) were concentrated 2.5-fold relative to the load. Lane 1, His6–StlSaPIbov1 and dUTPaseφ11 (JP6760); lane 2, dUTPaseφ11 alone (JP6762); lane 3, His6–StlSaPIbov1 and dUTPasePH15 (JP6761); lane 4, His6–ORF15φ80α and StlSaPIbov2 (JP6763). , Derepression of str transcription by dut expression. Top, schematic representation of the blaZ transcriptional fusion generated in plasmid pJP674. Bottom, strains containing pJP674- and pCN51-derivative plasmids expressing dutPH15 (JP5469) or dutφ11 (JP5468) were assayed for β-lactamase activity in the absence of or 5 h after induction with 5 μM CdCl2. Samples were normalized for total cell mass. Data are from an experiment in triplicate. Error bars represent s.d. * Figure 3: The level of SaPIbov1 inducing activity correlates with the central divergent region of Dut. , Alignment of predicted staphylococcal phage dUTPase protein sequences from phages 80α and φ11. Black arrows indicate the two N-terminal variations between 80α and φ11. The bracket indicates the region that was exchanged between 80α and φ11 dUTPases. Asterisks indicate identical residues between phage 80α and φ11. , , SaPIbov induction and replication was measured in a non-lysogenic derivative of strain RN4220 carrying SaPIbov1 and plasmids expressing 3×flag-tagged Dut proteins containing substitutions of two amino acids in the N-terminal region or an exchange of the central region, as indicated. One millilitre of each culture (OD540 nm = 0.3) was collected in the absence of induction () or 3 h after treatment with 5 μM CdCl2 () and used to prepare standard minilysates, which were resolved on a 0.7% agarose gel, blotted and probed for SaPIbov1 DNA or with antibody to the Flag tag. In both panels, lane 1, JP6789; lane 2, JP6794; lane 3, JP6793; lane 4, JP6800; la! ne 5, JP6797; lane 6, JP6795; lane 7, JP6798; lane 8, JP6799. Author information * Author information * Supplementary information * Comments Affiliations * Centro de Investigación y Tecnología Animal, Instituto Valenciano de Investigaciones Agrarias (CITA-IVIA), Apdo. 187, Segorbe, Castellón 12400, Spain * María Ángeles Tormo-Más, * Ignacio Mir & * José R. Penadés * Department of Microbiology and Immunology, Virginia Commonwealth University School of Medicine, Richmond, Virginia 23298-0678, USA * Archana Shrestha, * Sandra M. Tallent & * Gail E. Christie * Departament de Genètica i Microbiologia, Universitat Autònoma de Barcelona, Barcelona 08193, Spain * Susana Campoy & * Jordi Barbé * Instituto de Agrobiotecnología, CSIC-Universidad Pública de Navarra, Pamplona, Navarra 31006, Spain * Íñigo Lasa * Skirball Institute Program in Molecular Pathogenesis and Departments of Microbiology and Medicine, New York University Medical Center, 540 First Avenue, New York, New York 10016, USA * Richard P. Novick * Departamento de Química, Bioquímica y Biología Molecular, Universidad Cardenal Herrera-CEU, Moncada, Valencia 46113, Spain * José R. Penadés Contributions J.R.P. and G.E.C. conceived and designed the study; M.A.T.-M., I.M., S.M.T. and A.S. isolated and characterized SaPI-resistant phage mutants; M.A.T.-M. and I.M. analysed and characterized the different SaPI-repressor/phage-inducer interactions; S.C. and J.B. performed mobility shift assay experiments; J.R.P., G.E.C., R.P.N., M.A.T.-M., I.M. and I.L. analysed the data; J.R.P., G.E.C. and R.P.N. wrote the manuscript; J.R.P. and G.E.C. supervised the research; J.R.P., G.E.C., J.B., I.L. and R.P.N. obtained funding. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * José R. Penadés (penades_jos@gva.es) Supplementary information * Author information * Supplementary information * Comments PDF files * Supplementary Information (2.7M) This file contains Supplementary Tables 1 - 5, Supplementary Figures 1 - 4 with legends and References. Additional data
• Distinct FGFs promote differentiation of excitatory and inhibitory synapses
  Terauchi A Johnson-Venkatesh EM Toth AB Javed D Sutton MA Umemori H - Nature (London) 465(7299):783 (2010)
  Nature | Letter Distinct FGFs promote differentiation of excitatory and inhibitory synapses * Akiko Terauchi1 Search for this author in: * NPG journals * PubMed * Google Scholar * Erin M. Johnson-Venkatesh1 Search for this author in: * NPG journals * PubMed * Google Scholar * Anna B. Toth1 Search for this author in: * NPG journals * PubMed * Google Scholar * Danish Javed1 Search for this author in: * NPG journals * PubMed * Google Scholar * Michael A. Sutton1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Hisashi Umemori1, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:NatureVolume:465,Pages:783–787Date published:(10 June 2010)DOI:doi:10.1038/nature09041Received31 March 2009Accepted19 March 2010Published online26 May 2010 Article tools * Full text * 日本語要約 * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg The differential formation of excitatory (glutamate-mediated) and inhibitory (GABA-mediated) synapses is a critical step for the proper functioning of the brain. An imbalance in these synapses may lead to various neurological disorders such as autism, schizophrenia, Tourette's syndrome and epilepsy1, 2, 3, 4. Synapses are formed through communication between the appropriate synaptic partners5, 6, 7, 8. However, the molecular mechanisms that mediate the formation of specific synaptic types are not known. Here we show that two members of the fibroblast growth factor (FGF) family, FGF22 and FGF7, promote the organization of excitatory and inhibitory presynaptic terminals, respectively, as target-derived presynaptic organizers. FGF22 and FGF7 are expressed by CA3 pyramidal neurons in the hippocampus. The differentiation of excitatory or inhibitory nerve terminals on dendrites of CA3 pyramidal neurons is specifically impaired in mutants lacking FGF22 or FGF7. These presynaptic ! defects are rescued by postsynaptic expression of the appropriate FGF. FGF22-deficient mice are resistant to epileptic seizures, and FGF7-deficient mice are prone to them, as expected from the alterations in excitatory/inhibitory balance. Differential effects of FGF22 and FGF7 involve both their distinct synaptic localizations and their use of different signalling pathways. These results demonstrate that specific FGFs act as target-derived presynaptic organizers and help to organize specific presynaptic terminals in the mammalian brain. View full text Subject terms: * Neuroscience Figures at a glance * Figure 1: Expression of FGF22 and FGF7 in the hippocampal CA3 region during synapse formation at P8. , fgf22 and fgf7 mRNAs are highly expressed in CA3 pyramidal neurons (arrowheads) but not in CA1 pyramidal neurons. The bottom panels are negative controls using FGFKO sections. , fgfr2 mRNA is widely expressed throughout the hippocampus. , FGF22 and FGF7 proteins are localized in CA3 synapse-rich areas. Pictured areas correspond to the boxed area in . Scale bars, 500 μm (, ) and 50 μm (). SR, stratum radiatum; SL, stratum lucidum. * Figure 2: Specific defects in excitatory or inhibitory presynaptic differentiation in CA3 of FGF22KO and FGF7KO mice. , SV2 staining in CA1 and CA3 from P14 WT, FGF22KO and FGF7KO mice demonstrates decreased synaptic vesicle (SV) clustering in CA3 of FGFKO mice. , Normal active-zone formation (bassoon clustering) in CA3 of FGFKO mice. , , Staining in CA3 for VGLUT1 () and VGAT (), showing impaired glutamatergic and GABAergic SV clustering in CA3 of FGF22KO and FGF7KO mice, respectively. , Normal PSD95 and gephyrin clustering in CA3 of FGFKO mice. –, Electron microscopic analysis of asymmetric (excitatory, –) and symmetric (inhibitory, –) synapses in CA3. Synaptic density (×1,000 per mm2; , ), representative synapses (, ), and analysis of SVs within 400 nm from the active zone (AZ; , ) show specific presynaptic defects in FGFKO mice. , Western blotting of CA3 lysates, indicating no overall change in synaptic protein expression in FGFKO mice. Error bars indicate s.e.m. Staining data are from 15–126 fields from 5–14 mice. Electron microscopic data are from 5–20 synapses. Signifi! cant differences from control: asterisk, P < 0.05; two asterisks, P < 0.01; three asterisks, P < 0.001 (analysis of variance followed by Tukey test). Scale bars, 20 μm (, ) and 200 nm (, ). * Figure 3: Target-derived FGF22 and FGF7 selectively promote differentiation of glutamatergic or GABAergic presynaptic terminals in CA3 through distinct localization and signalling pathways. , VGLUT1 and VGAT clustering on FGF-transfected WT hippocampal neurons (labelled with GFP; 7 DIV). , Defects in the clustering of VGLUT1 and VGAT on the dendrites of FGF22KO or FGF7KO CA3 pyramidal neurons (Py-positive; 14 DIV). The number (puncta per mm neurite) and size of puncta were quantified. , The number (×1,000 puncta per mm2) and size of VGLUT1 and VGAT puncta on non-CA3 pyramidal neurons (Py-negative). , The presynaptic defects in FGFKO cultures are rescued by expression of the corresponding FGF in postsynaptic CA3 pyramidal neurons. Data are normalized to WT. , , FGF22–EGFP localizes to glutamatergic synapses (), and FGF7–DsRed to GABAergic synapses () (arrowheads). , Endogenous FGF22 and FGF7 are co-localized with PSD95 or VGAT. , FGF22–EGFP and FGF7–DsRed show differential dendritic localizations. , , The number (×1,000 puncta per mm2) and size of VGLUT1 () or VGAT () puncta after bath application of FGF to WT cultures. , Staining intensity of VG! LUT1 and VGAT in control and FGFR2KO CA3 sections (P8). , Normalized staining intensity of FGFR2KO sections. Error bars indicate s.e.m. Data are from 50–290 neurites or 8–66 fields from at least three experiments. Significant differences from control: asterisk, P < 0.05; two asterisks, P < 0.01; three asterisks, P < 0.001, t-test (, , ) or analysis of variance followed by Tukey test; hash sign, P = 0.073 (). Scale bars, 10 μm. * Figure 4: Altered synaptic transmission and seizure susceptibility in FGFKO mice, and a model for the role of FGF22 and FGF7 in specific presynaptic differentiation. –, Representative whole-cell recordings of mEPSCs () and mIPSCs () from WT and FGFKO hippocampal neurons; frequency and amplitude of mEPSCs () and mIPSCs () in the indicated groups (18–24 neurons per group). , Representative time course of seizure development during kindling experiments. , Percentage of mice kindled when about half of the control mice are kindled (about 21 injections of pentylenetetrazol (PTZ)) from four independent experiments (4–6 mice per experiment). Error bars indicate s.e.m. Significant differences from control: asterisk, P < 0.05; three asterisks, P < 0.001; analysis of variance followed by Tukey test. , Summary model. FGF22 and FGF7 from CA3 pyramidal neurons promote the differentiation of excitatory and inhibitory presynaptic terminals, respectively. FGF22 and FGF7 are localized at corresponding synapses and activate differential signalling pathways for specific presynaptic differentiation. Author information * Author information * Supplementary information * Comments Affiliations * Molecular and Behavioral Neuroscience Institute, University of Michigan Medical School, Ann Arbor, Michigan 48109-2200, USA * Akiko Terauchi, * Erin M. Johnson-Venkatesh, * Anna B. Toth, * Danish Javed, * Michael A. Sutton & * Hisashi Umemori * Department of Molecular and Integrative Physiology, University of Michigan Medical School, Ann Arbor, Michigan 48109-2200, USA * Michael A. Sutton * Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan 48109-2200, USA * Hisashi Umemori Contributions A.T. and H.U. conceived and designed the experiments, performed or participated in each of the experiments and wrote the manuscript. E.M.J.-V. and M.A.S. performed the electrophysiological recordings. A.B.T. participated in the culture and histological experiments. D.J. performed the seizure-related experiments. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Hisashi Umemori (umemoh@umich.edu) Supplementary information * Author information * Supplementary information * Comments PDF files * Supplementary Information (3.8M) This file contains Supplementary Figures 1-13 with legends and References. Additional data
• Global and local fMRI signals driven by neurons defined optogenetically by type and wiring
  - Nature (London) 465(7299):788 (2010)
  Nature | Letter Global and local fMRI signals driven by neurons defined optogenetically by type and wiring * Jin Hyung Lee1, 2, 8 Search for this author in: * NPG journals * PubMed * Google Scholar * Remy Durand2, 8 Search for this author in: * NPG journals * PubMed * Google Scholar * Viviana Gradinaru2 Search for this author in: * NPG journals * PubMed * Google Scholar * Feng Zhang2 Search for this author in: * NPG journals * PubMed * Google Scholar * Inbal Goshen2 Search for this author in: * NPG journals * PubMed * Google Scholar * Dae-Shik Kim3, 4 Search for this author in: * NPG journals * PubMed * Google Scholar * Lief E. Fenno2 Search for this author in: * NPG journals * PubMed * Google Scholar * Charu Ramakrishnan2 Search for this author in: * NPG journals * PubMed * Google Scholar * Karl Deisseroth2, 5, 6, 7 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorsJournal name:NatureVolume:465,Pages:788–792Date published:(10 June 2010)DOI:doi:10.1038/nature09108Received04 September 2009Accepted26 April 2010Published online16 May 2010Corrected online10 June 2010 Article tools * Full text * 日本語要約 * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Despite a rapidly-growing scientific and clinical brain imaging literature based on functional magnetic resonance imaging (fMRI) using blood oxygenation level-dependent (BOLD)1 signals, it remains controversial whether BOLD signals in a particular region can be caused by activation of local excitatory neurons2. This difficult question is central to the interpretation and utility of BOLD, with major significance for fMRI studies in basic research and clinical applications3. Using a novel integrated technology unifying optogenetic4, 5, 6, 7, 8, 9, 10, 11, 12, 13 control of inputs with high-field fMRI signal readouts, we show here that specific stimulation of local CaMKIIα-expressing excitatory neurons, either in the neocortex or thalamus, elicits positive BOLD signals at the stimulus location with classical kinetics. We also show that optogenetic fMRI (ofMRI) allows visualization of the causal effects of specific cell types defined not only by genetic identity and cell body l! ocation, but also by axonal projection target. Finally, we show that ofMRI within the living and intact mammalian brain reveals BOLD signals in downstream targets distant from the stimulus, indicating that this approach can be used to map the global effects of controlling a local cell population. In this respect, unlike both conventional fMRI studies based on correlations14 and fMRI with electrical stimulation that will also directly drive afferent and nearby axons, this ofMRI approach provides causal information about the global circuits recruited by defined local neuronal activity patterns. Together these findings provide an empirical foundation for the widely-used fMRI BOLD signal, and the features of ofMRI define a potent tool that may be suitable for functional circuit analysis as well as global phenotyping of dysfunctional circuitry. View full text Subject terms: * Neuroscience * Methods * Materials * Physiology Figures at a glance * Figure 1: ofMRI: optogenetic excitation of CaMKIIα neocortical cells drives local positive BOLD. , Transduced cells (triangles), light and location (1…9) of coronal slices. , Confocal images of ChR2–EYFP expression in M1 (left); higher magnification (right). , Optrode recordings during 473 nm optical stimulation (20 Hz/15 ms pulse width; blue); spiking is significantly elevated (error bar indicates ± s.d., two-sample t-test; *** indicates P < 0.001; n = 3). Pre, pre-stimulation; Stim, during stimulation; Post, post-stimulation. , BOLD activation observed with AAV5-CaMKIIα::ChR2-EYFP but not with saline injection (P < 0.001; asterisk, optical stimulation). , Left, ofMRI haemodynamic response (averaged across activated voxels in motor cortex) during 20 s (top) and 30 s (bottom) optical stimuli. Right, mean over stimulus repetitions; baseline, mean pre-stimulation signal. Top panels, n = 3; bottom panels, n = 8. * Figure 2: Nonlocal mapping of the causal role of cells defined by location and genetic identity. , AAV5-CaMKIIα::ChR2-EYFP injection and optical stimulation in M1. Slices in : '1' and '2'. , Fluorescence/bright-field: ChR2–EYFP in thalamus (left); confocal image shows expression limited to axons. , Thalamic ofMRI during M1 optical stimulation (top); superimposed on the Paxinos atlas (bottom). , ofMRI-HRF summary. , M1 optrode and thalamic electrode. , Thalamic spiking follows M1 optical stimulation; delay consistent with BOLD. , Typical M1 and thalamus spikes with M1 optical excitation. , M1 and thalamus spiking summary (error bar indicates ± s.d., two-sample t-test; *** indicates P < 0.001; n = 5). , Spike-frequency time histograms. * Figure 3: Control of cells defined by location, genetic identity and wiring during ofMRI. , M1 injection of AAV5-CaMKIIα::ChR2-EYFP and optical stimulation of the thalamus. Coronal slices shown in marked as '1…6' and '7…12'. , ChR2 expression pattern confirming expression in cortical neurons (left) and cortico-thalamic projections (right; see also Supplementary Fig. 5). , BOLD ofMRI data obtained in thalamus (above) and cortex (below). , ofMRI-HRF for cortical (grey) and thalamic (black) BOLD signals elicited by optical stimulation of cortico-thalamic fibres in thalamus. Both ofMRI-HRFs ramp slowly by comparison with intracortical results in Fig. 1. * Figure 4: Recruitment of bilateral cortices by the anterior thalamus. , Thalamic injection of AAV5-CaMKIIα::ChR2-EYFP and posterior/anterior optical stimulation. Coronal slices marked 'A1…A12' and 'B1…B12'. , Fluorescence overlaid onto bright-field (left) and confocal image (right) illustrating transduction in the thalamus (left) and cortical projections in the internal and external capsule (right). , Posterior thalamus stimulation-evoked ofMRI signal in the ipsilateral thalamus and somatosensory cortex. , ofMRI-HRFs. Excited volumes: 5.5 ± 1.3 mm3 (thalamus); 8.6 ± 2.5 mm3 (somatosensory cortex) (n = 3). , Anterior thalamus stimulation-evoked ofMRI signal in the ipsilateral thalamus and bilateral motor cortex. , ofMRI-HRFs. Excited volumes: 1.5 mm3 (thalamus); 10.1 mm3 (ipsilateral cortex); 3.7 mm3 (contralateral cortex). Change history * Change history * Author information * Supplementary information * CommentsCorrected online 10 June 2010Minor corrections were made to Fig. 1d and to the text of the paragraph beginning, 'To assess fMRI signals ...' Author information * Change history * Author information * Supplementary information * Comments Primary authors * These authors contributed equally to this work. * Jin Hyung Lee & * Remy Durand Affiliations * Department of Electrical Engineering, Psychiatry and Biobehavioral Sciences, Bioengineering, and Radiology, University of California, Los Angeles, California 90095, USA * Jin Hyung Lee * Department of Bioengineering, Stanford University, Stanford, California 94305, USA * Jin Hyung Lee, * Remy Durand, * Viviana Gradinaru, * Feng Zhang, * Inbal Goshen, * Lief E. Fenno, * Charu Ramakrishnan & * Karl Deisseroth * Department of Electrical Engineering, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 305-701, Republic of Korea * Dae-Shik Kim * Department of Anatomy and Neurobiology, Boston University School of Medicine, Boston, Massachusetts 02118, USA * Dae-Shik Kim * Howard Hughes Medical Institute, Stanford University, Stanford, California 94305, USA * Karl Deisseroth * CNC Program, Stanford University, Stanford, California 94305, USA * Karl Deisseroth * Department of Psychiatry and Behavioral Sciences, Stanford, California 94305, USA * Karl Deisseroth Contributions J.H.L., F.Z., R.D., V.G. and K.D. designed the experiments. D.-S.K. provided information on the animal fMRI setup, J.H.L. developed the fMRI methods, and J.H.L. and R.D. conducted all ofMRI experiments. R.D., V.G. and F.Z. conducted animal surgery and preparation. J.H.L., L.E.F., R.D. and V.G. conducted optrode recordings. R.D., V.G. and I.G. acquired the confocal microscope images. J.H.L., R.D. and I.G. performed histology and confocal imaging for quantification. C.R. prepared the viral vectors. J.H.L., R.D., V.G., F.Z., D.-S.K. and K.D. prepared the figures and wrote the paper. K.D. supervised all aspects of the work. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Jin Hyung Lee (ljinhy@gmail.com) or * Karl Deisseroth (deissero@stanford.edu) Supplementary information * Change history * Author information * Supplementary information * Comments PDF files * Supplementary Information (4.9M) This file contains Supplementary Methods, References and Supplementary Figures S1-S5 with legends. Additional data
• Quiescent haematopoietic stem cells are activated by IFN-γ in response to chronic infection
  - Nature (London) 465(7299):793 (2010)
  Nature | Letter Quiescent haematopoietic stem cells are activated by IFN-γ in response to chronic infection * Megan T. Baldridge1, 4 Search for this author in: * NPG journals * PubMed * Google Scholar * Katherine Y. King2, 4 Search for this author in: * NPG journals * PubMed * Google Scholar * Nathan C. Boles3 Search for this author in: * NPG journals * PubMed * Google Scholar * David C. Weksberg1 Search for this author in: * NPG journals * PubMed * Google Scholar * Margaret A. Goodell1, 2, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:NatureVolume:465,Pages:793–797Date published:(10 June 2010)DOI:doi:10.1038/nature09135Received27 April 2009Accepted27 April 2010 Article tools * Full text * 日本語要約 * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Lymphocytes and neutrophils are rapidly depleted by systemic infection1. Progenitor cells of the haematopoietic system, such as common myeloid progenitors and common lymphoid progenitors, increase the production of immune cells to restore and maintain homeostasis during chronic infection, but the contribution of haematopoietic stem cells (HSCs) to this process is largely unknown2. Here we show, using an in vivo mouse model of Mycobacterium avium infection, that an increased proportion of long-term repopulating HSCs proliferate during M. avium infection, and that this response requires interferon-γ (IFN-γ) but not interferon-α (IFN-α) signalling. Thus, the haematopoietic response to chronic bacterial infection involves the activation not only of intermediate blood progenitors but of long-term repopulating HSCs as well. IFN-γ is sufficient to promote long-term repopulating HSC proliferation in vivo; furthermore, HSCs from IFN-γ-deficient mice have a lower proliferative r! ate, indicating that baseline IFN-γ tone regulates HSC activity. These findings implicate IFN-γ both as a regulator of HSCs during homeostasis and under conditions of infectious stress. Our studies contribute to a deeper understanding of haematological responses in patients with chronic infections such as HIV/AIDS or tuberculosis3, 4, 5. View full text Subject terms: * Stem cells * Immunology Figures at a glance * Figure 1: Infection with Mycobacterium avium induces changes in haematopoietic stem cells. , Absolute numbers of ST-HSCs, multipotent progenitors (MPPs) and LT-HSCs (KSL, Flk2−, CD34−) were determined after infection with M. avium. n = 3–7. , LT-HSCs (side population, lineage-negative, Sca-1+, c-Kit+ (SPKLS)) were not significantly (NS) changed after infection with M. avium. Plot is representative of three independent experiments, each with n = 3–5. , BrdU incorporation in SPKLS cells was determined at baseline and after infection. Data represent two independent experiments, each with n = 2–5. , Engraftment efficiency was determined after transplantation of 500 SPKLS from wild-type (WT) or M. avium-infected wild-type mice into lethally irradiated wild-type recipients. Data represent two independent experiments, each with n = 2–6. , The percentage of LT-HSCs (KSL, CD150+) in the spleen was determined at baseline and 4 weeks after M. avium infection. n = 4–5. Mean values ± s.e.m. are shown. *P < 0.05, **P < 0.01, ***P < 0.001. * Figure 2: The HSC response to M. avium infection is dependent upon intact IFN-γ signalling. , IFN-γ levels in bone marrow supernatant were quantified by cytokine bead array at baseline and 4 weeks after infection with M. avium. n = 3–6. , BrdU incorporation by HSCs (KSL, CD150+) of naive and M. avium-infected wild-type, Ifngr1-deficient, Stat1-deficient and Ifnar1-deficient mice was quantified. n = 3–6. , Absolute number of HSCs (KSL, CD150+) in the whole bone marrow of naive and infected wild-type, Ifngr1-deficient, Stat1-deficient and Ifnar1-deficient mice was determined. n = 3–7. Mean values ± s.e.m. are shown. *P < 0.05, **P < 0.01, ***P < 0.001. * Figure 3: IFN-γ is sufficient to induce HSC proliferation in vitro. , Relative quantities of Ifngr1 mRNA were determined in pooled samples of nucleated erythrocyte progenitors (Eryth), B cells, T cells, granulocytes (Gran), and LT-HSCs (SPKLS). n = 2–3 independent samples per cell type. , Irgm1 mRNA was quantified by real-time PCR in LT-HSCs (SPKLS) after IFN-γ treatment. , IRGM1 protein levels on LT-HSCs (SPKLS) were determined by immunofluorescence. Scale bars, 5 μm. , BrdU incorporation by primitive haematopoietic cells (KSL, CD150+) was assessed after 12-h in vitro treatment with PBS or IFN-γ. Data represent two independent experiments each performed in triplicate. Mean values ± s.e.m. are shown. *P < 0.05, **P < 0.01, ***P < 0.001. * Figure 4: IFN-γ is sufficient to induce HSC proliferation in vivo. , BrdU incorporation was measured in LT-HSCs (SPKLS) of wild-type mice 24 h after injection with IFN-γ or PBS and 12 h after injection with BrdU. n = 5. , Whole bone marrow was isolated from PBS or IFN-γ-injected wild-type mice 24 h after injection, and transplanted into lethally irradiated wild-type recipients in competitive transplant assays. Engraftment efficiency was determined 4, 8, 12 and 16 weeks after transplantation. n = 5–6. , The percentage of primitive haematopoietic cells (KSL, CD150+) in the spleen was determined 24 h after injection with either PBS or IFN-γ. n = 3. Mean values ± s.e.m. are shown. *P < 0.05, **P < 0.01, ***P < 0.001. * Figure 5: Basal IFN-γ tone affects HSC cycling and function. , BrdU incorporation by LT-HSCs (SPKLS) of wild-type and Ifng-deficient mice over 3 days was determined. n = 4–5. , Engraftment efficiency was determined 4, 8, 12 and 16 weeks after transplantation of whole bone marrow from wild-type or Ifng-deficient mice into lethally irradiated wild-type recipients in either noncompetitive or competitive transplant assays. n = 5. Mean values ± s.e.m. are shown. *P < 0.05, **P < 0.01, ***P < 0.001. Author information * Author information * Supplementary information * Comments Primary authors * These authors contributed equally to this work. * Megan T. Baldridge & * Katherine Y. King Affiliations * Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas 77030, USA * Megan T. Baldridge, * David C. Weksberg & * Margaret A. Goodell * Department of Pediatrics, Section of Infectious Diseases, Baylor College of Medicine, Houston, Texas 77030, USA * Katherine Y. King & * Margaret A. Goodell * Interdepartmental Program in Cell and Molecular Biology, Baylor College of Medicine, Houston, Texas 77030, USA * Nathan C. Boles & * Margaret A. Goodell Contributions M.T.B. and K.Y.K. designed experiments, performed flow cytometry, transplants, and quantitative real-time PCR analysis, and analysed data. K.Y.K. performed immunohistochemistry. N.C.B. performed flow cytometry and cytokine analysis. D.C.W. assisted with study design. M.T.B., K.Y.K. and M.A.G. wrote the manuscript. All authors discussed results and commented on the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Margaret A. Goodell (goodell@bcm.edu) Supplementary information * Author information * Supplementary information * Comments PDF files * Supplementary Figures (5.3M) This file contains Supplementary Figures 1-9 with legends. Additional data
• Control of mammary stem cell function by steroid hormone signalling
  Asselin-Labat ML Vaillant F Sheridan JM Pal B Wu D Simpson ER Yasuda H Smyth GK Martin TJ Lindeman GJ Visvader JE - Nature (London) 465(7299):798 (2010)
  Nature | Letter Control of mammary stem cell function by steroid hormone signalling * Marie-Liesse Asselin-Labat1 Search for this author in: * NPG journals * PubMed * Google Scholar * François Vaillant1 Search for this author in: * NPG journals * PubMed * Google Scholar * Julie M. Sheridan1 Search for this author in: * NPG journals * PubMed * Google Scholar * Bhupinder Pal1 Search for this author in: * NPG journals * PubMed * Google Scholar * Di Wu1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Evan R. Simpson3 Search for this author in: * NPG journals * PubMed * Google Scholar * Hisataka Yasuda4 Search for this author in: * NPG journals * PubMed * Google Scholar * Gordon K. Smyth1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * T. John Martin5 Search for this author in: * NPG journals * PubMed * Google Scholar * Geoffrey J. Lindeman1, 6, 7 Search for this author in: * NPG journals * PubMed * Google Scholar * Jane E. Visvader1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorsJournal name:NatureVolume:465,Pages:798–802Date published:(10 June 2010)DOI:doi:10.1038/nature09027Received10 October 2009Accepted22 March 2010Published online11 April 2010 Article tools * Full text * 日本語要約 * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg The ovarian hormones oestrogen and progesterone profoundly influence breast cancer risk1, 2, 3, underpinning the benefit of endocrine therapies in the treatment of breast cancer4. Modulation of their effects through ovarian ablation or chemoprevention strategies also significantly decreases breast cancer incidence5, 6. Conversely, there is an increased risk of breast cancer associated with pregnancy in the short term7. The cellular mechanisms underlying these observations, however, are poorly defined. Here we demonstrate that mouse mammary stem cells (MaSCs)8, 9 are highly responsive to steroid hormone signalling, despite lacking the oestrogen and progesterone receptors10. Ovariectomy markedly diminished MaSC number and outgrowth potential in vivo, whereas MaSC activity increased in mice treated with oestrogen plus progesterone. Notably, even three weeks of treatment with the aromatase inhibitor letrozole was sufficient to reduce the MaSC pool. In contrast, pregnancy led to ! a transient 11-fold increase in MaSC numbers, probably mediated through paracrine signalling from RANK ligand. The augmented MaSC pool indicates a cellular basis for the short-term increase in breast cancer incidence that accompanies pregnancy. These findings further indicate that breast cancer chemoprevention may be achieved, in part, through suppression of MaSC function. View full text Subject terms: * Cancer * Stem cells * Cell biology * Developmental biology Figures at a glance * Figure 1: Steroid hormone deprivation reduces MaSC activity. , LacZ+ outgrowths arising from transplantation of 75 double-sorted CD29hiCD24+ cells from glands of control or ovariectomized (Ovx) mice. The virgin recipient was collected at 8 weeks after transplantation (left panel) and the pregnant recipient at 18.5 days of pregnancy (right panel). Scale bars, 1 mm. , Bar chart showing percentage fat pad filling by outgrowths at 8 weeks after transplantation (n = 3). , Representative FACS dot plot showing the expression of CD29 and CD24 in the Lin– population (TER119–CD31–CD45–) of mammary glands from an 8-week-old virgin mouse implanted with slow-release pellets (placebo or oestrogen plus progesterone, E2+Pg) for 3 weeks. , Histogram showing the fold-increase in the absolute number of repopulating cells in placebo, oestrogen (E2), progesterone (Pg) or oestrogen plus progesterone (E2+Pg) treated mice. Data represent the mean ± s.e.m. of three experiments. *P < 0.05. , LacZ+ outgrowths arising from transplantation of! 75 double-sorted CD29hiCD24+ cells isolated from vehicle- or letrozole-treated animals (left panel; scale bars, 1 mm). Right panel: smooth muscle actin (SMA) immunostaining of sections of LacZ+ outgrowths harvested from letrozole- or vehicle-treated donor mice. Insert represents control isotype antibody. Scale bars, 50 μm. , Bar chart showing percentage fat pad filling by outgrowths 8 weeks after transplantation (n = 3). * Figure 2: Marked increase in the number of MaSCs in mid-pregnancy. , Histogram showing the absolute number of CD29hiCD24+ cells per mouse (four glands) in virgin, pregnant (day of pregnancy) and involuting (days involution) glands. Data represent the mean ± s.e.m. of 3–5 animals per group. , Representative FACS dot plot showing the expression of CD29 and CD24 in the Lin– population (TER119–CD31–CD45–) of mammary glands from an 8-week-old virgin or 12.5 day pregnant mouse. , Histogram showing the fold-increase in repopulating cells in virgin, 12.5 day pregnant and 18.5 day pregnant mice. Data represent the mean ± s.e.m. of three experiments. *P < 0.05. , LacZ+ outgrowths arising from transplantation of 150 CD29hiCD24+ cells isolated from virgin or 12.5 day pregnant donor mice (top panel). Glands were harvested from 18.5 day pregnant recipients. Scale bars, 1 mm. Bottom panel: sections of LacZ+ outgrowths counterstained with nuclear fast red. Scale bars, 50 μm. , Bar chart showing percentage fat pad filling by! outgrowths collected 8 weeks after transplantation of CD29hiCD24+ cells from either virgin or 12.5 day pregnant mice (n = 3 experiments). * Figure 3: RANKL is involved in MaSC activation observed during pregnancy. , mRNA levels of RANKL, RANK and Id2 in the MaSC-enriched (CD29hiCD24+) and luminal cell-enriched (CD29loCD24+) subpopulations from virgin or pregnant mammary glands (days 12.5 to 18.5) by quantitative RT–PCR relative to 18S rRNA. Data represent the mean ± s.e.m. of two to five experiments. , Clonogenic capacity of CD29hiCD24+ and CD29loCD24+ cells isolated from control versus anti-RANKL-treated virgin mice, plated on fibroblast feeder layers. Data represent the mean ± s.e.m. (n = 2 experiments). Bottom panel shows Giemsa-stained colonies of a representative experiment. *P < 0.05. . Clonogenic capacity of CD29hiCD24+ and CD29loCD24+ cells isolated from control versus anti-RANKL-treated mice at 12.5 days of pregnancy, plated on fibroblast feeders. CD29loCD24+ cells isolated from pregnant glands have a higher clonogenic capacity than from virgin glands, suggesting expansion of an alveolar progenitor. Data represent the mean ± s.e.m. (n = 4 experiments). *P!  < 0.05. Accession codes * Accession codes * Author information * Supplementary information * Comments Primary accessions Gene Expression Omnibus * GSE20401 * GSE20402 Author information * Accession codes * Author information * Supplementary information * Comments Affiliations * The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia * Marie-Liesse Asselin-Labat, * François Vaillant, * Julie M. Sheridan, * Bhupinder Pal, * Di Wu, * Gordon K. Smyth, * Geoffrey J. Lindeman & * Jane E. Visvader * Department of Medical Biology, The University of Melbourne, Parkville, Victoria 3010, Australia * Di Wu, * Gordon K. Smyth & * Jane E. Visvader * Prince Henry's Institute of Medical Research, Monash Medical Centre, Clayton, Victoria 3168, Australia * Evan R. Simpson * Nagahama Institute for Biochemical Science, Oriental Yeast Company, Nagahama, Shiga 526-0804, Japan * Hisataka Yasuda * St Vincent's Institute, Fitzroy, Victoria 3065, Australia * T. John Martin * Department of Medical Oncology, The Royal Melbourne Hospital, Parkville, Victoria 3050, Australia * Geoffrey J. Lindeman * Department of Medicine, The University of Melbourne, Parkville, Victoria 3010, Australia * Geoffrey J. Lindeman Contributions M.-L.A.-L. conceptualized and designed the experiments and performed most of the experiments and data analysis; F.V. performed transplantation experiments and analysis; J.S. performed in vitro inhibitor experiments; B.P. performed quantitative RT–PCR; D.W. and G.K.S. performed bioinformatics analyses; E.R.S. provided ArKO mice and advice; H.Y. provided anti-RANKL inhibitor and advice; T.J.M. provided advice and helped design RANKL experiments; J.E.V. and G.J.L. conceived and directed the study, and J.E.V., G.J.L. and M.-L.A.-L. wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Geoffrey J. Lindeman (lindeman@wehi.edu.au) or * Jane E. Visvader (visvader@wehi.edu.au) All microarray data are available from the Gene Expression Omnibus database (http://www.ncbi.nlm.nih.gov/geo) under accession codes GSE20401 and GSE20402. Supplementary information * Accession codes * Author information * Supplementary information * Comments Excel files * Supplementary Table 1 (623K) This file contains the gene profiling data (Log2-intensity and the log2-fold-change) for mammary populations from control versus ovariectomized (Ovx) and 12.5 day pregnant mice. PDF files * Supplementary Figures (2M) This file contains Supplementary Figures S1-S7 with legends. The Supplementary Tables were added on 14 April 2010. A small correction was made to Supplementary Table 5 on 19 May 2010. Additional data
• Progesterone induces adult mammary stem cell expansion
  Joshi PA Jackson HW Beristain AG Di Grappa MA Mote P Clarke C Stingl J Waterhouse PD Khokha R - Nature (London) 465(7299):803 (2010)
  Nature | Letter Progesterone induces adult mammary stem cell expansion * Purna A. Joshi1 Search for this author in: * NPG journals * PubMed * Google Scholar * Hartland W. Jackson1 Search for this author in: * NPG journals * PubMed * Google Scholar * Alexander G. Beristain1 Search for this author in: * NPG journals * PubMed * Google Scholar * Marco A. Di Grappa1 Search for this author in: * NPG journals * PubMed * Google Scholar * Patricia A. Mote2 Search for this author in: * NPG journals * PubMed * Google Scholar * Christine L. Clarke2 Search for this author in: * NPG journals * PubMed * Google Scholar * John Stingl3 Search for this author in: * NPG journals * PubMed * Google Scholar * Paul D. Waterhouse1 Search for this author in: * NPG journals * PubMed * Google Scholar * Rama Khokha1 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:NatureVolume:465,Pages:803–807Date published:(10 June 2010)DOI:doi:10.1038/nature09091Received19 March 2010Accepted19 April 2010Published online05 May 2010Corrected online10 June 2010 Article tools * Full text * 日本語要約 * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Reproductive history is the strongest risk factor for breast cancer after age, genetics and breast density1, 2. Increased breast cancer risk is entwined with a greater number of ovarian hormone-dependent reproductive cycles, yet the basis for this predisposition is unknown3, 4, 5. Mammary stem cells (MaSCs) are located within a specialized niche in the basal epithelial compartment that is under local and systemic regulation6. The emerging role of MaSCs in cancer initiation warrants the study of ovarian hormones in MaSC homeostasis. Here we show that the MaSC pool increases 14-fold during maximal progesterone levels at the luteal dioestrus phase of the mouse. Stem-cell-enriched CD49fhi cells amplify at dioestrus, or with exogenous progesterone, demonstrating a key role for progesterone in propelling this expansion. In aged mice, CD49fhi cells display stasis upon cessation of the reproductive cycle. Progesterone drives a series of events where luminal cells probably provide Wn! t4 and RANKL signals to basal cells which in turn respond by upregulating their cognate receptors, transcriptional targets and cell cycle markers. Our findings uncover a dynamic role for progesterone in activating adult MaSCs within the mammary stem cell niche during the reproductive cycle, where MaSCs are putative targets for cell transformation events leading to breast cancer. View full text Subject terms: * Stem cells * Cell biology * Cancer Figures at a glance * Figure 1: MaSCs fluctuate during the reproductive cycle. , Serum progesterone levels during the oestrous cycle (mean ± s.e.m.). The four phases of the cycle are denoted as: P, pro-oestrus; E, oestrus; M, metoestrus; D, dioestrus; n = 5 (P), n = 8 (E), n = 7 (M), n = 6 (D) mice. , Total cell numbers generated from oestrous-staged inguinal glands (mean ± s.e.m.); n = 5 (P), n = 4 (E), n = 7 (M), n = 5 (D) mice. , Mammary whole mounts from mice at the indicated oestrous phases show pronounced lobuloalveolar morphology at dioestrus. Scale bars, 500 μm. , Two examples of positive ductal outgrowths from limiting dilution analyses into cleared fat pads, reconstituted with 500 (top) or 5,000 (bottom) dioestrus cells. Scale bars, 500 μm. , Comparison of take rates of dioestrus and oestrus mammary cells at limiting dilutions show a 7.6-fold increase in the mammary repopulating unit frequency of dioestrus cells. Take rate indicates positive outgrowths as detailed in Supplementary Table 1. Data are consistent with a single-h! it process (P = 0.9998, dioestrus cells; P = 0.7534, oestrus cells). * Figure 2: Progesterone drives expansion of the MaSC-enriched subpopulation in vivo. , Representative FACS plots of CD24+CD49flo (luminal) and CD24+CD49fhi (basal) populations generated from oestrous-staged glands. , Histogram showing total numbers of CD24+CD49flo and CD24+CD49fhi cells at stages of the oestrous cycle (mean ± s.e.m.); n = 5 (P), n = 4 (E), n = 7 (M), n = 5 (D) cycling mice. (See Fig. 1 legend for definitions.) , CD24+CD49flo and CD24+CD49fhi populations after 17β-oestradiol treatment alone or with progesterone of ovariectomized mice. , Further segregation of stem (CD24+CD61+CD49fhi), progenitor (CD24+CD61+CD49flo) and differentiated (CD24+CD61−CD49flo) cells using CD49f and CD61 markers within the CD24+ subset. , , Histograms showing quantification of the indicated cell populations after hormone treatments. E, 17β-oestradiol; EP, 17β-oestradiol plus progesterone; P, progesterone; S, sham; V, vehicle (sesame oil). Data represent mean ± s.e.m. of n = 4 (S), n = 3 (V), n = 3 (E), n = 3 (P), n = 4 (EP). , The colony-forming capaci! ty of 5,000 total mammary cells in Matrigel from dioestrus or oestrus glands (mean ± s.e.m., n = 3 per group). , Representative gross morphology of acinar and solid colonies arising in Matrigel cultures. Scale bars, 500 μm. , , Histogram showing colonies formed from 17β-oestradiol versus 17β-oestradiol plus progesterone cells () and representative tiled image composites spanning a Matrigel droplet of 1.2-cm diameter, with insets showing a 3.5× magnification (). Data represent mean ± s.e.m., n = 3 mice per group. * Figure 3: Dynamic mammary cell turnover in cycling females whereas stem-cell-enriched basal cells show stasis in aged mice. , , The percentage of PR+ cells in oestrous-staged and hormone-treated mice as quantified after immunostaining. , , Histograms showing the percentage of Ki67+ cells at dioestrus and after 17β-oestradiol plus progesterone treatment. In –, data represent mean ± s.e.m.; n = 2 (, ), n = 3 (, ); E, oestrus and D, dioestrus in , ; E, 17β-oestradiol and EP, 17β-oestradiol plus progesterone in , . , Diminished serum progesterone levels in non-cycling, 20-month-old aged (A) mice compared with those in 10-week-old oestrus (E) and dioestrus mice (D). Data are mean ± s.e.m.; n = 6 (D), n = 8 (E), n = 3 (A). , Mammary whole mount from a 20-month-old non-cycling female mouse. , Histogram showing the clonogenic capacity of cells derived from 10-week-old oestrus (E) versus 20-month-old (A) glands. Results are mean ± s.e.m. of n = 3 mice each. , FACS profiles of luminal CD24+CD49flo and basal CD24+CD49fhi cell populations in 20-month-old mice are comparable to 10-week-old! oestrus mice. , Histogram showing the number of CD24+CD49flo and CD24+CD49fhi cells in 20-month-old (A) mice compared to those at oestrus (E). Data are mean ± s.e.m.; n = 4 (E) and n = 3 (A). * Figure 4: RANKL and Wnt4 as paracrine effectors of progesterone-induced MaSC expansion. –, Luminal (L, CD24+CD49flo) and MaSC-enriched basal (B, CD24+CD49fhi) populations were sorted by flow cytometry from dioestrus, oestrus, 17β-oestradiol (E) and 17β-oestradiol plus progesterone (EP) mammary glands and analysed by qRT–PCR for gene expression relative to β-actin. Expression is relative to that of CD24+CD49fhi basal cells (B) from oestrus mice or ovariectomized mice with 17β-estradiol treatment, set at 1, except where it was undetectable (PR-B, Wnt4). In this case, expression is relative to CD24+CD49fhi (B) cells from 17β-oestradiol plus progesterone glands. Data represent mean ± s.e.m.; n = 3 each for oestrus and dioestrus; n = 4 each for E and EP. Statistically significant differences (P < 0.05) are denoted with an asterisk. Change history * Change history * Author information * Supplementary information * CommentsCorrected online 10 June 2010Minor corrections were made to affiliation 2 and Fig. 2a, c, d. Author information * Change history * Author information * Supplementary information * Comments Affiliations * Ontario Cancer Institute, Department of Medical Biophysics and Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto M5G 2M9, Ontario, Canada * Purna A. Joshi, * Hartland W. Jackson, * Alexander G. Beristain, * Marco A. Di Grappa, * Paul D. Waterhouse & * Rama Khokha * Westmead Institute for Cancer Research, University of Sydney at Westmead Millennium Institute, Westmead, New South Wales 2145, Australia * Patricia A. Mote & * Christine L. Clarke * Cancer Research UK, Cambridge Research Institute, Li Ka Shing Centre, Cambridge CB2 0RE, UK * John Stingl Contributions P.A.J. designed and performed majority of the experiments and data analysis; H.W.J. conducted CFC assays and contributed to transplantation experiments; A.G.B. extracted RNA and performed quantitative RT–PCR; M.A.D.G. administered hormones and designed graphics; P.M. and C.C. provided PR antibody and advice; J.S. advised on multiple aspects of stem cell analyses; P.D.W. conceptualized the importance of the reproductive cycle; and R.K. directed the study. P.A.J. and R.K. wrote the paper. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Rama Khokha (rkhokha@uhnres.utoronto.ca) Supplementary information * Change history * Author information * Supplementary information * Comments PDF files * Supplementary Information (9.7M) This file contains Supplementary Methods and References, Supplementary Table 1 and Supplementary Figures S1-S5 with legends. Additional data
• Patient-specific induced pluripotent stem-cell-derived models of LEOPARD syndrome
  - Nature (London) 465(7299):808 (2010)
  Nature | Letter Patient-specific induced pluripotent stem-cell-derived models of LEOPARD syndrome * Xonia Carvajal-Vergara1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Ana Sevilla1, 10 Search for this author in: * NPG journals * PubMed * Google Scholar * Sunita L. D'Souza1, 10 Search for this author in: * NPG journals * PubMed * Google Scholar * Yen-Sin Ang1 Search for this author in: * NPG journals * PubMed * Google Scholar * Christoph Schaniel1 Search for this author in: * NPG journals * PubMed * Google Scholar * Dung-Fang Lee1 Search for this author in: * NPG journals * PubMed * Google Scholar * Lei Yang1 Search for this author in: * NPG journals * PubMed * Google Scholar * Aaron D. Kaplan3 Search for this author in: * NPG journals * PubMed * Google Scholar * Eric D. Adler3 Search for this author in: * NPG journals * PubMed * Google Scholar * Roye Rozov1 Search for this author in: * NPG journals * PubMed * Google Scholar * YongChao Ge4 Search for this author in: * NPG journals * PubMed * Google Scholar * Ninette Cohen5 Search for this author in: * NPG journals * PubMed * Google Scholar * Lisa J. Edelmann5 Search for this author in: * NPG journals * PubMed * Google Scholar * Betty Chang1 Search for this author in: * NPG journals * PubMed * Google Scholar * Avinash Waghray1 Search for this author in: * NPG journals * PubMed * Google Scholar * Jie Su1 Search for this author in: * NPG journals * PubMed * Google Scholar * Sherly Pardo5, 6 Search for this author in: * NPG journals * PubMed * Google Scholar * Klaske D. Lichtenbelt7 Search for this author in: * NPG journals * PubMed * Google Scholar * Marco Tartaglia8 Search for this author in: * NPG journals * PubMed * Google Scholar * Bruce D. Gelb5, 6, 9, 10 Search for this author in: * NPG journals * PubMed * Google Scholar * Ihor R. Lemischka1, 10 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorsJournal name:NatureVolume:465,Pages:808–812Date published:(10 June 2010)DOI:doi:10.1038/nature09005Received16 December 2009Accepted08 March 2010 Article tools * Full text * 日本語要約 * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg The generation of reprogrammed induced pluripotent stem cells (iPSCs) from patients with defined genetic disorders holds the promise of increased understanding of the aetiologies of complex diseases and may also facilitate the development of novel therapeutic interventions. We have generated iPSCs from patients with LEOPARD syndrome (an acronym formed from its main features; that is, lentigines, electrocardiographic abnormalities, ocular hypertelorism, pulmonary valve stenosis, abnormal genitalia, retardation of growth and deafness), an autosomal-dominant developmental disorder belonging to a relatively prevalent class of inherited RAS–mitogen-activated protein kinase signalling diseases, which also includes Noonan syndrome, with pleomorphic effects on several tissues and organ systems1, 2. The patient-derived cells have a mutation in the PTPN11 gene, which encodes the SHP2 phosphatase. The iPSCs have been extensively characterized and produce multiple differentiated cell ! lineages. A major disease phenotype in patients with LEOPARD syndrome is hypertrophic cardiomyopathy. We show that in vitro-derived cardiomyocytes from LEOPARD syndrome iPSCs are larger, have a higher degree of sarcomeric organization and preferential localization of NFATC4 in the nucleus when compared with cardiomyocytes derived from human embryonic stem cells or wild-type iPSCs derived from a healthy brother of one of the LEOPARD syndrome patients. These features correlate with a potential hypertrophic state. We also provide molecular insights into signalling pathways that may promote the disease phenotype. View full text Subject terms: * Cell biology * Stem cells * Molecular biology Figures at a glance * Figure 1: Gene expression profile in LEOPARD syndrome iPSCs is similar to human ES cells. , Quantitative real-time PCR assay for the expression of endogenous human OCT4, NANOG and SOX2 in iPSCs and parental fibroblasts (Fib). PCR reactions were normalized against β-actin and plotted relative to expression levels in HES2. Error bars indicate ±s.d. of triplicates. , Bisulphite sequencing analyses of the OCT4 and NANOG promoters. The cell line and the percentage of methylation are indicated to the left of each cluster. , Heat map showing hierarchical clustering of 3,657 genes with at least twofold expression change between the average of the three fibroblast cell lines versus all the iPSC lines/human ES cell samples. Expression levels are represented by colour; red indicates lower and yellow higher expression. * Figure 2: LEOPARD syndrome iPSCs differentiate in vitro and in vivo into all three germ layers. , L2-iPS6 cells were differentiated as floating EBs for 8 days and then plated onto gelatin-coated dishes and allowed to differentiate for another 8 days. Immunocytochemistry showed cell types positively stained for differentiation markers including desmin/α-SMA (mesoderm), AFP (endoderm), vimentin (mesoderm) and GFAP/βIII-tubulin (ectoderm). The arrow indicates a βIII-tubulin-positive cell. Scale bar, 100 μm. , HES2, L1-iPSC and L2-iPSC were injected subcutaneously into the right hind leg of immuno-compromised NOD-SCID mice. The resulting teratomas were stained with haematoxylin and eosin and tissues representative of all three germ layers were observed. Scale bar, 100 μm. * Figure 3: Cardiomyocytes derived from LEOPARD syndrome iPSCs show hypertrophic features. , HES2, H1, wild-type S3-iPS4 and three LEOPARD syndrome iPSC clones were differentiated into cardiac lineage. Cell areas of 50 random cTNT-positive cardiomyocytes of each cell line were measured using ImageJ. Boxes show the span from the median (50th percentile) to the first and third quartiles. The lines represent the largest/smallest sizes that are no more than 1.5 times the median to quartile distance. Additional points drawn represent extreme values, ≥1.5 times (open circles) and ≥3 times (filled circle) the median to quartile distance. , Sarcomeric organization was assessed in 50 cTNT-positive (red) cardiomyocytes. Data are presented as mean ± s.d. n = 3; **P < 0.01 (Student's t-test). , S3-iPS4 and L2-iPS10 cell-derived cardiomyocytes were re-stained with NFATC4 antibody, and the nuclear versus cytosolic expression was analysed. n = 3; **P < 0.01 (Student's t-test). , Nuclear localization of NFATC4 protein in a cTNT-positive cell from L2-iPS10 is ! shown. * Figure 4: Phosphoproteomic and MAPK activation analyses. , Protein extracts of two iPSCs from each LEOPARD syndrome patient (L1 and L2), wild-type iPSCs (BJ-iPSB5) and HES2 cells were hybridized to an antibody microarray. The heat map represents the most significant protein changes preserved in all the comparison groups. , p-MEK1 and p-EGFR expression was confirmed by western blot using phospho-specific antibodies. Band density was measured (ImageJ software) and normalized to β-actin. , HES2, wild-type S3-iPS4 and LEOPARD syndrome iPSCs were serum- and bFGF-starved for 6 h and then treated with bFGF (20 ng ml−1) for the indicated time. Phosphorylated ERK1/2 (p-ERK1/2) and total ERK were assessed by immunoblotting and quantified. p-ERK1/2 levels were compared to the untreated p-ERK1/2 level in each sample, normalized to the total ERK1/2 and represented graphically at the right of each panel. Accession codes * Accession codes * Author information * Supplementary information * Comments Primary accessions Gene Expression Omnibus * GSE20473 Author information * Accession codes * Author information * Supplementary information * Comments Primary authors * These authors contributed equally to this work. * Ana Sevilla, * Sunita L. D'Souza, * Bruce D. Gelb & * Ihor R. Lemischka Affiliations * Department of Gene and Cell Medicine, Department of Developmental and Regenerative Biology, Black Family Stem Cell Institute, Mount Sinai School of Medicine, New York, New York 10029, USA * Xonia Carvajal-Vergara, * Ana Sevilla, * Sunita L. D'Souza, * Yen-Sin Ang, * Christoph Schaniel, * Dung-Fang Lee, * Lei Yang, * Roye Rozov, * Betty Chang, * Avinash Waghray, * Jie Su & * Ihor R. Lemischka * Department of Regenerative Cardiology, Centro Nacional de Investigaciones Cardiovasculares, 28029 Madrid, Spain * Xonia Carvajal-Vergara * Department of Medicine, Cardiovascular Institute, Mount Sinai School of Medicine, New York, New York 10029, USA * Aaron D. Kaplan & * Eric D. Adler * Department of Neurology, Mount Sinai School of Medicine, New York, New York 10029, USA * YongChao Ge * Department of Genetics and Genomic Sciences, Mount Sinai School of Medicine, New York, New York 10029, USA * Ninette Cohen, * Lisa J. Edelmann, * Sherly Pardo & * Bruce D. Gelb * Child Health and Development Institute, Mount Sinai School of Medicine, New York, New York 10029, USA * Sherly Pardo & * Bruce D. Gelb * Department of Medical Genetics, University Medical Center Utrecht, 3584 EA Utrecht, the Netherlands * Klaske D. Lichtenbelt * Dipartimento di Ematologia, Oncologia e Medicina Molecolare, Istituto Superiore di Sanità, 00161 Rome, Italy * Marco Tartaglia * Department of Pediatrics, Mount Sinai School of Medicine, New York, New York 10029, USA * Bruce D. Gelb Contributions X.C.-V. (iPSC establishment, project planning, experimental work and preparation of manuscript); A.S., S.L.D., Y.-S.A., L.Y., A.D.K., E.D.A., D.-F.L., A.W., B.C., J.S. and S.P. (experimental work); R.R. and Y.G. (microarray analysis); N.C. and L.J.E. (karyotype analysis); K.D.L. and M.T. (obtaining of fibroblast samples from patients); C.S. (project planning, experimental work); B.D.G. and I.R.L. (project planning, preparation of manuscript). Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Ihor R. Lemischka (ihor.lemischka@mssm.edu) or * Xonia Carvajal-Vergara (xcarvajal@gmail.com) Microarray data have been deposited in NCBI-GEO under the accession number GSE20473. Supplementary information * Accession codes * Author information * Supplementary information * Comments Movies * Supplementary Movie 1 (5.5M) This movie shows beating embryoid bodies at day 18 of cardiac differentiation: cell line HES2. * Supplementary Movie 2 (5.5M) This movie shows beating embryoid bodies at day 18 of cardiac differentiation: cell line L1-iPS6. * Supplementary Movie 3 (2.5M) This movie shows beating embryoid bodies at day 18 of cardiac differentiation: cell line L1-iPS13. * Supplementary Movie 4 (5.6M) This movie shows beating embryoid bodies at day 18 of cardiac differentiation: cell line L2-iPS6. * Supplementary Movie 5 (6.3M) This movie shows beating embryoid bodies at day 18 of cardiac differentiation: cell line L2-iPS10. * Supplementary Movie 6 (14.8M) This movie shows beating embryoid bodies at day 18 of cardiac differentiation: cell line L2iPS16. * Supplementary Movie 7 (6.2M) This movie shows beating embryoid bodies at day 18 of cardiac differentiation: cell line S3-iPS4. PDF files * Supplementary Information (2M) This file contains Supplementary Figures 1-12 with legends and Supplementary Table 1. Additional data
• Tumour angiogenesis is reduced in the Tc1 mouse model of Down's syndrome
  - Nature (London) 465(7299):813 (2010)
  Nature | Letter Tumour angiogenesis is reduced in the Tc1 mouse model of Down's syndrome * Louise E. Reynolds1 Search for this author in: * NPG journals * PubMed * Google Scholar * Alan R. Watson1, 14 Search for this author in: * NPG journals * PubMed * Google Scholar * Marianne Baker1, 14 Search for this author in: * NPG journals * PubMed * Google Scholar * Tania A. Jones3 Search for this author in: * NPG journals * PubMed * Google Scholar * Gabriela D'Amico1 Search for this author in: * NPG journals * PubMed * Google Scholar * Stephen D. Robinson1 Search for this author in: * NPG journals * PubMed * Google Scholar * Carine Joffre2 Search for this author in: * NPG journals * PubMed * Google Scholar * Sarah Garrido-Urbani5 Search for this author in: * NPG journals * PubMed * Google Scholar * Juan Carlos Rodriguez-Manzaneque6 Search for this author in: * NPG journals * PubMed * Google Scholar * Estefanía Martino-Echarri6 Search for this author in: * NPG journals * PubMed * Google Scholar * Michel Aurrand-Lions7 Search for this author in: * NPG journals * PubMed * Google Scholar * Denise Sheer3 Search for this author in: * NPG journals * PubMed * Google Scholar * Franca Dagna-Bricarelli8 Search for this author in: * NPG journals * PubMed * Google Scholar * Dean Nizetic4 Search for this author in: * NPG journals * PubMed * Google Scholar * Christopher J. McCabe9 Search for this author in: * NPG journals * PubMed * Google Scholar * Andrew S. Turnell10 Search for this author in: * NPG journals * PubMed * Google Scholar * Stephanie Kermorgant2 Search for this author in: * NPG journals * PubMed * Google Scholar * Beat A. Imhof5 Search for this author in: * NPG journals * PubMed * Google Scholar * Ralf Adams11 Search for this author in: * NPG journals * PubMed * Google Scholar * Elizabeth M. C. Fisher12 Search for this author in: * NPG journals * PubMed * Google Scholar * Victor L. J. Tybulewicz13 Search for this author in: * NPG journals * PubMed * Google Scholar * Ian R. Hart2 Search for this author in: * NPG journals * PubMed * Google Scholar * Kairbaan M. Hodivala-Dilke1 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:NatureVolume:465,Pages:813–817Date published:(10 June 2010)DOI:doi:10.1038/nature09106Received08 December 2009Accepted14 April 2010 Article tools * Full text * 日本語要約 * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Down's syndrome (DS) is a genetic disorder caused by full or partial trisomy of human chromosome 21 and presents with many clinical phenotypes including a reduced incidence of solid tumours1, 2. Recent work with the Ts65Dn model of DS, which has orthologues of about 50% of the genes on chromosome 21 (Hsa21), has indicated that three copies of the ETS2 (ref. 3) or DS candidate region 1 (DSCR1) genes4 (a previously known suppressor of angiogenesis5, 6) is sufficient to inhibit tumour growth. Here we use the Tc1 transchromosomic mouse model of DS7 to dissect the contribution of extra copies of genes on Hsa21 to tumour angiogenesis. This mouse expresses roughly 81% of Hsa21 genes but not the human DSCR1 region. We transplanted B16F0 and Lewis lung carcinoma tumour cells into Tc1 mice and showed that growth of these tumours was substantially reduced compared with wild-type littermate controls. Furthermore, tumour angiogenesis was significantly repressed in Tc1 mice. In particul! ar, in vitro and in vivo angiogenic responses to vascular endothelial growth factor (VEGF) were inhibited. Examination of the genes on the segment of Hsa21 in Tc1 mice identified putative anti-angiogenic genes (ADAMTS18, 9and ERG10) and novel endothelial cell-specific genes11, never previously shown to be involved in angiogenesis (JAM-B12 and PTTG1IP), that, when overexpressed, are responsible for inhibiting angiogenic responses to VEGF. Three copies of these genes within the stromal compartment reduced tumour angiogenesis, explaining the reduced tumour growth in DS. Furthermore, we expect that, in addition to the candidate genes that we show to be involved in the repression of angiogenesis, the Tc1 mouse model of DS will permit the identification of other endothelium-specific anti-angiogenic targets relevant to a broad spectrum of cancer patients. View full text Subject terms: * Cancer * Cell biology Figures at a glance * Figure 1: Tumour angiogenesis is restricted in Tc1 mice. , B16F0 and LLC tumour size was reduced significantly in the Tc1 mice. Representative tumour images are given. n = 20 per group. , Blood vessel density was reduced significantly in tumours from Tc1 mice. Representative staining for endomucin in tumour sections is given. n = 5–10 per group. , Perfusion of tumour blood vessels in Tc1 mice was reduced in comparison with wild-type (WT) controls. Graph shows the mean number of dextran-FITC-perfused blood vessels. Arrowheads, perfused vessels; arrows, non-perfused vessels. , Blood vessel density was similar in unchallenged skin of WT and Tc1 mice. n = 5 per group. Two asterisks, P < 0.05; asterisk, P < 0.01, n.s., not statistically significant. Scale bars, 10 mm () and 100 μm (). All values are means and s.e.m. * Figure 2: VEGF-mediated angiogenic responses are inhibited in Tc1 mice. , VEGF-stimulated neovascularization into subcutaneously implanted sponges, quantified by numbers of endomucin-positive blood vessels, was decreased in Tc1 mice in comparison with wild-type (WT) mice. n = 20 per group. , VEGF-induced vessel sprouting ex vivo from Tc1 aortic rings was inhibited. Representative images of aortic ring sprouts are given. n = 6–12 aortic rings per test. Asterisk, aortic ring; arrows, microvessel sprouts. , Phospho-ERK1 (pp44) was increased in WT but not in Tc1 primary endothelial cells stimulated with VEGF. , Phospho-ERK1 (pp44) was increased in normal human cells stimulated with VEGF but not in DS cells. Asterisk, P < 0.01; two asterisks, P < 0.05; n.s., not statistically significant. Scale bars, 100 μm () and 500 μm (). All values are means and s.e.m. * Figure 3: Reduction of copy number of candidate genes from three to two can rescue the angiogenic defect in Tc1 mice. , siRNA depletion of the candidate human genes inhibited human (Hu) transcript expression levels (left panel) but not mouse transcript expression levels (right panel). , Knockdown of human candidate genes in wild-type aortic tissue has no effect. Non-transfected (NT) wild-type (WT) aortic rings treated with VEGF (+) enhanced microvessel sprouting compared with untreated aortic rings (−). Scr-siRNA or human-specific siRNAs did not alter VEGF-mediated microvessel sprouting. , Knockdown of human candidate genes in Tc1 aortic tissue restores disomy. Non-transfected (NT) and Scr-siRNA-transfected Tc1 aortic rings did not respond to VEGF (+). Transfection with human-specific siRNAs, except ETS2, effectively restoring expression of two copies of the genes, increased VEGF-mediated vessel sprouting. n = 20-40 aortic rings per test. Filled bars, WT; open bars, Tc1. Two asterisks, P < 0.005; n.s., not statistically significant. All values are means and s.e.m. * Figure 4: Reduction of copy number from three to one rescues the angiogenic defect in Tc1 mice. , siRNA depletion of the mouse (Ms) candidate genes inhibited mouse endothelial transcript expression levels. , Knockdown of mouse candidate genes in Tc1 aortic tissue induces monosomy. Non-transfected (NT) and Scr-siRNA-transfected Tc1 aortic rings did not sprout in response to VEGF (+). Transfection with Adamts1, Jam-b and Pttg1ip siRNA, effectively generating one copy of the genes, increased VEGF-mediated vessel sprouting. Transfection with Erg siRNA had no effect on VEGF-mediated microvessel sprouting. n = 20–40 aortic rings per test. , Administration of the anti-mouse JAM-B antibody (JB4.2) to tumour-burdened Tc1 mice enhanced tumour growth and blood vessel density. Asterisk, P < 0.005; two asterisks, P < 0.001; n.s., not statistically significant. , Tumour volume was increased in JAM-B+/− mice. Asterisk, P < 0.05. , Tumour blood vessel density was increased in JAM-B+/− mice. Asterisk, P < 0.05. , VEGF-mediated microvessel sprouting was increased i! n JAM-B+/− aortic rings. n = 24–68 aortic rings per genotype per test. Scale bars, 100 μm. Asterisk, P < 0.05; two asterisks, P < 0.005. All values are means and s.e.m. Author information * Author information * Supplementary information * Comments Primary authors * These authors contributed equally to this work. * Alan R. Watson & * Marianne Baker Affiliations * Adhesion and Angiogenesis Laboratory, Barts Institute of Cancer, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, John Vane Science Centre, Charterhouse Square, London EC1M 6BQ, UK * Louise E. Reynolds, * Alan R. Watson, * Marianne Baker, * Gabriela D'Amico, * Stephen D. Robinson & * Kairbaan M. Hodivala-Dilke * Centre for Tumour Biology, Institute of Cancer, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, John Vane Science Centre, Charterhouse Square, London EC1M 6BQ, UK * Carine Joffre, * Stephanie Kermorgant & * Ian R. Hart * Neuroscience Centre, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, Institute of Cell and Molecular Sciences, 4 Newark Street, London E1 2AD, UK * Tania A. Jones & * Denise Sheer * Paediatrics Centre, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, Institute of Cell and Molecular Sciences, 4 Newark Street, London E1 2AD, UK * Dean Nizetic * Department of Pathology and Immunology, Centre Medical Universitaire, University of Geneva Medical School (CMU), rue Michel Servet 1, CH-1211 Geneva, Switzerland * Sarah Garrido-Urbani & * Beat A. Imhof * GENYO, Avenida Del Conocimiento, s/n Armilla 18100, Granada, Spain * Juan Carlos Rodriguez-Manzaneque & * Estefanía Martino-Echarri * INSERM, 27, Boulevard Lei Roure, 13009 Marseille, France * Michel Aurrand-Lions * Human Genetics Institute, Galliere Hospital, Via Volta 10, 16128 Genoa, Italy * Franca Dagna-Bricarelli * School of Clinical and Experimental Medicine, University of Birmingham, Birmingham B15 2TT, UK * Christopher J. McCabe * School of Cancer Sciences, University of Birmingham, Birmingham B15 2TT, UK * Andrew S. Turnell * Max Planck Institute for Molecular Biomedicine, Röntgenstrasse 20, D-48149 Münster, Germany * Ralf Adams * Department of Neurodegenerative Disease, UCL Institute of Neurology, Queen Square, London WC1N 3BG, UK * Elizabeth M. C. Fisher * Division of Immune Cell Biology, MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK * Victor L. J. Tybulewicz Contributions L.E.R. and K.M.H-D. designed the experiments. L.E.R. performed the experiments. A.R.W. performed the bone marrow transplant experiments and stained for Y chromosome and conducted RT–PCR. G.D'A. performed the aortic ring assay. S.D.R. performed the phospho-VEGFR2 western blot analysis. T.A.J. and D.S. performed the tumour cell karyotyping. M.B. assisted with the immunostaining, tumour and sponge harvesting and flow cytometric analysis. C.J. and S.K. conducted flow cytometry and immunofluorescence of cells. B.A.I., R.A. and S.G.-U. supplied the JAM-B antibodies for western blot analysis and JAM-B wild-type and heterozygous mice for in vivo and ex vivo studies and JAM-B biochemistry in JAM-B heterozygotes. J.C.R.-M. and E.M.-E. provided the ADAMTS1 heterozygous aortae and ADAMTS1 PCR analysis. C.J.M. and A.T. provided the PTTG1IP antibody for western blot analysis. F.D.-B. and D.N. provided the human DS and normal control cells. V.J.T. and E.M.C.F. designed , developed an! d provided the Tc1 mice. L.E.R., K.M.H-D. and I.R.H. wrote the paper with substantial input from the other authors. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Louise E. Reynolds (l.reynolds@qmul.ac.uk) Supplementary information * Author information * Supplementary information * Comments PDF files * Supplementary Information (2.3M) This file contains Supplementary Methods and Supplementary Figures 1-13 with legends. Additional data
• Structural basis for 5′-nucleotide base-specific recognition of guide RNA by human AGO2
  Frank F Sonenberg N Nagar B - Nature (London) 465(7299):818 (2010)
  Nature | Letter Structural basis for 5′-nucleotide base-specific recognition of guide RNA by human AGO2 * Filipp Frank1, 2, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Nahum Sonenberg1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Bhushan Nagar1, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:NatureVolume:465,Pages:818–822Date published:(10 June 2010)DOI:doi:10.1038/nature09039Received16 December 2009Accepted19 March 2010Published online26 May 2010 Article tools * Full text * 日本語要約 * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg MicroRNAs (miRNAs) mediate post-transcriptional gene regulation through association with Argonaute proteins (AGOs)1. Crystal structures of archaeal and bacterial homologues of AGOs have shown that the MID (middle) domain mediates the interaction with the phosphorylated 5′ end of the miRNA guide strand and this interaction is thought to be independent of the identity of the 5′ nucleotide in these systems2, 3. However, analysis of the known sequences of eukaryotic miRNAs and co-immunoprecipitation experiments indicate that there is a clear bias for U or A at the 5′ position4, 5, 6, 7. Here we report the crystal structure of a MID domain from a eukaryotic AGO protein, human AGO2. The structure, in complex with nucleoside monophosphates (AMP, CMP, GMP, and UMP) mimicking the 5′ end of miRNAs, shows that there are specific contacts made between the base of UMP or AMP and a rigid loop in the MID domain. Notably, the structure of the loop discriminates against CMP and GMP a! nd dissociation constants calculated from NMR titration experiments confirm these results, showing that AMP (0.26 mM) and UMP (0.12 mM) bind with up to 30-fold higher affinity than either CMP (3.6 mM) or GMP (3.3 mM). This study provides structural evidence for nucleotide-specific interactions in the MID domain of eukaryotic AGO proteins and explains the observed preference for U or A at the 5′ end of miRNAs. View full text Subject terms: * Structural biology * Biophysics * Biochemistry * Molecular biology Figures at a glance * Figure 1: Overall structure of the hAGO2 MID domain. , Ribbon representation of the MID domain, with UMP depicted in stick representation. Highlighted in yellow is the nucleotide specificity loop. The phenylalanine residues, F470 and F505 (shown as sticks in red), proposed to be involved in cap binding24 are both buried within the hydrophobic core of the protein (Supplementary Fig. 3). , Mapping of surface conservation of selected eukaryotic AGOs (see Supplementary Fig. 2). , Electrostatic potential surface representation25. All molecular figures were generated using PyMol (http://www.pymol.org). * Figure 2: Crystal structures of hAGO2 MID domain in complex with UMP, AMP, CMP and GMP. –, Human AGO2 MID domain in complex with UMP (), AMP (), CMP () and GMP (). UMP and AMP are modelled and shown in stick representation. Only the phosphate groups of GMP and CMP were modelled (not shown). The nucleotide specificity loop is shown without side chains and highlighted in yellow. Relevant backbone atoms in the loop are indicated with blue spheres (nitrogen) and red sticks (oxygen). Difference electron density contoured at 2.5σ is shown before inclusion of any nucleotide in the model. Dotted black lines indicate hydrogen bonds. Adjacent to each structure are two-dimensional representations of the contacts between NMPs and the MID domain. The 5′-nucleotide specificity loop is highlighted in yellow and orange. In green are hydrogen bonds with distances in Å. 'Eyelashes' represent van der Waals contacts. Atoms of UMP and AMP that interact with the nucleotide specificity loop are labelled and circled. Curved red lines mark repulsive interactions between the b! ase of GMP or CMP and the nucleotide specificity loop. Clashing hydrogen atoms for GMP are shown to highlight the repulsive interactions. * Figure 3: Determination of dissociation constants using NMR titration experiments. , Representative 15N HSQC NMR spectra from the MID domain of hAGO2 with increasing amounts of UMP added. Significantly shifting peaks used for the determination of the dissociation constant of UMP are boxed and marked by arrows. , Chemical shift differences were calculated as Δδ = [(ΔδH)2 + (0.2*ΔδN)2]1/2, where ΔδH and ΔδN are the observed chemical shift changes for 1H and 15N, respectively. For determination of dissociation constants, Δδ was plotted as a function of the molar ratio (nucleotide:protein) and the data for multiple peaks were fitted using the maximum shift and dissociation constant as adjustable parameters. Accession codes * Accession codes * Author information * Supplementary information * Comments Primary accessions Protein Data Bank * 3LUC * 3LUD * 3LUG * 3LUH * 3LUJ * 3LUK * 3LUC * 3LUD * 3LUG * 3LUH * 3LUJ * 3LUK Author information * Accession codes * Author information * Supplementary information * Comments Affiliations * Department of Biochemistry, McGill University, Montréal, Québec H3G 0B1, Canada * Filipp Frank, * Nahum Sonenberg & * Bhushan Nagar * Goodman Cancer Center, McGill University, Montréal, Québec H3G 0B1, Canada * Filipp Frank & * Nahum Sonenberg * Groupe de Recherche Axe sur la Structure des Proteines, Montréal, Québec H3G 0B1, Canada * Filipp Frank & * Bhushan Nagar Contributions B.N., N.S. and F.F. designed the project. B.N. and F.F. wrote the manuscript. F.F. performed all of the NMR and crystallographic work. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Bhushan Nagar (bhushan.nagar@mcgill.ca) Coordinates and structure factors have been deposited in the Protein Data Bank under accession codes 3LUC, 3LUD, 3LUG, 3LUH, 3LUJ and 3LUK. Supplementary information * Accession codes * Author information * Supplementary information * Comments PDF files * Supplementary Information (10.1M) This file contains Supplementary Table 1, Supplementary Figures S1-S15 with legends and References. Additional data
• Structural biology: The gatekeepers revealed
  - Nature (London) 465(7299):823 (2010)
  Nature | Technology Feature Structural biology: The gatekeepers revealed * Monya Baker1 Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:NatureVolume:465,Pages:823–826Date published:(10 June 2010)DOI:doi:10.1038/465823aPublished online09 June 2010 Proteins in cell membranes are notoriously hard to crystallize, but new techniques give scientists the means to map them. Monya Baker scouts out the tools for cracking the structure of membrane proteins. Introduction * Introduction * References * Author information * Comments Article tools * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg If a cell is a house and the cell membrane its walls, then proteins serve as the doors, windows and electricity and telephone lines. Membrane-bound proteins, anchored within the cell's lipid bilayer, regulate the influx and efflux of molecules and information. How and when these membrane proteins change shape determines essential processes, including whether a drug slows a racing heart, an eye detects light or a virus invades a cell. Yet scientists studying these proteins often know only the rough outlines of their shapes. Structures have been solved for fewer than 250 membrane proteins, and almost all of these are from microbes such as bacteria and yeast. Of the 7,000 human membrane proteins, researchers have found high-resolution structures for fewer than 12, and each structure captures only one of the protein's many possible forms. Functional protein assays are important, but are better suited to determining whether a protein performs a certain task than to explaining how or why it does so. Mapped-out structures can give researchers ideas about why a mutation changes a protein's behaviour or help researchers to design drugs. But without a structure, researchers can only speculate about the reasons for drug or mutation effects, says Brian Kobilka, a biochemist at Stanford University in Palo Alto, California. "When you have a structure," he says, "you can begin to understand." B. KOBILKA LAB. The floppy helices of a G-protein-coupled receptor can be stabilized with an antibody fragment (left, orange) or by the insertion of another protein (right, green). Researchers hoping to solve membrane-protein structures face a cruel paradox: to work on a protein's shape, they must remove it from the cell membrane, destabilizing it and disrupting the conformation. "When you solubilize the protein, you are taking away the belt that holds it together," says Raymond Stevens, a biochemist at the Scripps Research Institute in La Jolla, California. Researchers have tended to avoid this added hassle by focusing on proteins that float free in water. According to So Iwata, head of the Human Receptor Crystallography Project at the Japan Science and Technology Agency in Kyoto, membrane structural biology is lagging 20–30 years behind the study of soluble proteins. But the field is catching up fast as researchers learn better ways to make, purify and crystallize membrane proteins. The first atomic-resolution crystal structure of a membrane protein, the reaction centre of a photosynthetic bacterium, was published in 1985 (ref. 1). It was important not just for the structure, but as proof that membrane proteins could be crystallized. Still, it was 13 years before crystal structures had been solved for 20 membrane proteins. Techniques have improved: in 2006 alone, 21 proteins were reported, mostly from Escherichia coli and other bacteria; this year, that number was surpassed by mid-May (see 'Protein progress'). But there's a long way to go. James Bowie, a structural biologist at the University of California, Los Angeles, estimates that representing 90% of structural families would need structures from around 1,700 membrane proteins2. His work indicates that most disease-causing mutations are likely to perturb the structure3, so the number of clinically relevant structures could be much greater. One sign that crystal structures are easier to solve is that pharmaceutical companies are looking for the structures of membrane proteins to target with drugs. "Before, they wouldn't spend money on it because it was too risky," says Iwata. Added stability The largest family of membrane proteins is among the most challenging for biologists. The G-protein-coupled receptors (GPCRs) consist of seven helices that twist and turn through the membrane. Flexible loops extending beyond the lipid bilayer interact with water and the inner helices are normally surrounded by lipids. Ligands that bind these receptors on the outside of the cell membrane cause conformational shifts on the inside that can trigger the cell to respond. B. KOBILKA Brian Kobilka solves high resolution crystal structures. The roughly 800 different GPCRs control pretty much everything that happens in the body — smell, sight, even response to neuro-transmitters and immune signals. Many frequently prescribed drugs, from antihistamines to β-blockers, target this class of receptor, and researchers think that crystal structures can help to find more and better drugs more quickly. The first known GPCR structure, published in low resolution in 1993 (ref. 4) and in high resolution in 2000 (ref. 5), was of the bovine version of rhodopsin, the photoreceptor that enables vision in low light conditions. Rhodopsin, however, is an unusual GPCR because it is particularly stable and is expressed in high enough concentrations to be collected from natural sources — two characteristics not usually associated with this protein family. As such, "rhodopsin didn't tell us how to get structures for other GPCRs", says Kobilka. It wasn't until 2007 that researchers solved the structure of a second GPCR6 — the human β2-adrenergic receptor, which is involved in cardiovascular and pulmonary function. Kobilka led a team that designed an antibody to bind two of the protein's helices, stabilizing the receptor and providing a polar surface that helped crystals to form7. In other work, Kobilka and the Scripps Research Institute's Stevens solved a high-resolution structu! re from another crystal, in which the protein was stabilized by replacing an intracellular loop with another protein6, 8. SOURCE: S. WHITE, UNIV. CALIFORNIA, IRVINE Researchers are also hunting for compounds that can boost proteins' stability without adding another protein, a strategy that has allowed Stevens to obtain a portrait of another important GPCR, the A2A adenosine receptor9, which is involved in many physiological processes and is blocked by caffeine. Receptos, a drug development company co-founded by Stevens and based in San Diego, California, used the same method to solve the structure for sphingosine-1-phosphate receptor subtype 1, a drug target for multiple sclerosis. This year, the company announced a clinical drug candidate designed with reference to this structure. R. STEVENS LAB. a, adrenoreceptor β2AR; b, adenosine receptor A2AAR; c, chemokine receptor CXCR4; d, dopamine receptor D3. Another tactic is to stabilize GPCRs through targeted mutagenesis rather than through third-party agents such as antibodies and extra proteins. Researchers led by Chris Tate and Gebhard Schertler at the Medical Research Council's Laboratory of Molecular Biology in Cambridge, UK, for example, identified a handful of mutations in GPCRs that boost stability with no apparent effect on function10. In 2007, Tate co-founded Heptares Therapeutics in Welwyn Garden City, UK, which uses the stabilized GPCRs, known as StaRs, to inform drug design and has solved several crystal structures of GPCRs with bound ligands. Sticking with a membrane Techniques for purifying membrane proteins without denaturing them go beyond tool compounds and engineering. Anatrace in Maumee, Ohio, part of Affymetrix, based in Santa Clara, California, sells detergents and lipids used for solubilizing and stabilizing proteins, including Chobimalt, a water-soluble cholesterol derivative; A8-35, a polymer that wraps itself around the membrane protein; and tripod amphipiles, which limit protein mobility and interactions. In 2007, the company launched three or four new products for membrane proteins; last year, it rolled out twenty. The growing number of publications and tools has brought in scientists who previously restricted themselves to soluble proteins, says Ben Travis, the company's research and development manager. "They're finding the membrane-protein field more accessible," he says. Perhaps the biggest shift in the field is the ability to accommodate membrane proteins' structural need for fat. "Before, people tried to purify membrane proteins so they didn't have lipids associated with them, but now we know that it probably isn't a good idea," says Stephen White, a biophysicist at the University of California, Irvine. To address this, many structural biologists have turned to a technique called lipidic cubic phase (LCP) crystallization, also known as in meso crystallization. In this technique, proteins are dissolved in lipids to form membrane-like bilayers around water-filled cavities. This mixture feeds lipids and proteins into crystals as they grow. The idea that one could obtain crystals from a protein embedded in a bilayer was "pretty radical", says Bowie, who has developed a variant of the technique using bicelles of lipid and detergent. "A number of protein structures would remain unsolved without the LCP method." The LCP mixture, however, is incredibly difficult to work with. "You end up with something that looks and feels like very sticky toothpaste," says Martin Caffrey, a biochemist from Trinity College Dublin, Ireland, who is widely credited with popularizing the technique by inventing a way for researchers to homogenize the solutions. His method involves two syringes, one filled with protein, detergent and water, and the other with lipid. The syringes are coupled together so that each injects into the other; researchers mix the contents by pushing the plungers back and forth. Finally, the mixture is placed onto a crystallization plate along with a solution that promotes precipitation. "And then," he says, "you pray for crystals." Although Caffrey says that researchers with access to a good machine shop should be able to build syringe-coupling devices themselves, Emerald Biosystems, a protein reagents and services firm in Bainbridge Island, Washington, makes a kit consisting of plates, a variety of precipitant solutions formulated for cubic phase and a syringe device prefilled with lipids. Formulatrix in Waltham, Massachusetts, sells crystal-imaging and other technologies that work with the LCP technique. Late last year, QIAGEN in Hilden, Germany, began offering a product that allows researchers to grow crystals using the LCP method while following many of the protocols for soluble proteins, such as vapour diffusion. The hardest part, says Frank Schäfer, associate director of protein sciences at QIAGEN, was outfitting fluid-handling robots so that they could deal with such viscous material and dispense it into standard crystallization plates. Their success meant that researchers who buy the product don't have to work with the lipids at all. "This has the potential to be used by everybody. There is no special equipment," he says. Stevens, who switched to using LCP technology about five years ago, after Vadim Cherezov, who had previously worked with Caffrey, joined his lab, welcomes the development of commercial products for the technique, but believes that they need further refinement. "Those kits have not advanced to the stage where it's routine," he says. Researchers familiar with LCP methods don't use the kits, and less-experienced labs have trouble using them. Time and communication should solve that problem, though. The National Institutes of Health Roadmap meeting on membrane-protein technologies this November includes a workshop, organized by Stevens and Cherezov, on LCP crystallization technologies. F. SIGWORTH Large membrane complexes can be embedded in liposomes, and then imaged with cryo-electron microscopy to yield a structure. Scale bar, 25 nm. Computational boosts Laboratory techniques will continue to improve, but some of the most important advances will happen in silico. Computer programs are getting better at filling in gaps from incomplete or ambiguous data sets. In April, for example, Axel Brunger, professor of molecular and cellular physiology at Stanford University, described a technique for improving the accuracy of low-resolution structures11. The added precision provided by his software means that researchers can pinpoint specific amino acids, which should help with protein and drug engineering. So far, Brunger has reported using the algorithm only with soluble protein structures, but it should work for membrane proteins too, he says. But what if you don't have a structure to start with, even a crude one at poor resolution? David Baker at the University of Washington in Seattle is pursuing an approach that builds models of proteins using data that are too sparse to solve any structure at all. His computer program, Rosetta, evaluates possible protein conformations to find the most stable — and hence most likely — shape. Although the number of possibilities makes such calculations impractical for all but the smallest proteins, even sparse structural data can be used to refine the search by excluding improbable conformations. Baker compares his software to a vast team of explorers scouting Earth for the lowest possible point. If data showed that it was not in North America, say, then the explorers could search more effectively by focusing their efforts only on other continents, he says. Another program, MODELLER, by Andrej Sali at the University of California, San Francisco, is also very popular among structural biologists and modellers. Sali's software uses sequence homology to create three-dimensional best guesses for proteins of unknown structure. These and other computer programs will improve as more protein structures are solved — and will in turn allow more structures to be found. They could pave the way for researchers to get high-resolution structures using assorted data sources, including X-ray diffraction (see 'Crystal-clear images'), cryoelectron microscopy (cryoEM) and nuclear magnetic resonance (NMR). Structure without crystals The prospect of refining coarse structures through computer modelling entices scientists such as Fred Sigworth at Yale University, New Haven, Connecticut, who studies ion channels using cryoEM, a type of electron microscopy performed at very low temperatures. CryoEM can't match the resolution of classic methods, but it addresses a nagging question: is this how the protein looks in the membrane or does the lack of the lipid bilayer distort it? Instead of extracting proteins and putting them through a crystallization matrix, cryo-EM involves embedding membrane proteins in artificial liposomes, which are then frozen and can yield thousands of pictures. In recent years, robots from companies including FEI in Hillsboro, Oregon, which sells the Vitrobot, and Gatan in Pleasanton, California, which sells the Cryoplunge, have helped to automate liposome preparation and freezing, preserving researchers' time and samples. With the cryoEM photo library, researchers can apply a technique! called single-particle reconstruction to sort through all the two-dimensional images and calculate what kind of three-dimensional proteins could have generated them. Besides falling short of the resolution of most crystal structures, the technique has other drawbacks. For one, it works best on very large, rigid structures, such as ribosomes. Last year, Sigworth used the single-particle technique to solve the structure of a membrane protein — in this case, the human large-conductance calcium- and voltage-activated potassium channel — which, with a molecular mass of 0.5 megadaltons, is small for cryoEM12. Other important groups of proteins, such as the GPCRs, are flexible and just a fraction of that size, and thus are not amenable to cryo-EM studies. That hasn't dimmed Sigworth's enthusiasm, particularly for large membrane complexes. "X-ray crystallography is very powerful," he says. "CryoEM has the same kind of potential, it's just 30 years behind. That makes it really fun to be in because new methods are being invented every day." M. CAFFREY LAB. Martin Caffrey mixes lipids, detergents and proteins to stabilize crystallizing membrane proteins. Perhaps the most surprising technique to be applied to membrane structural biology is mass spectrometry. Proteins analysed by mass spectrometry are usually broken down and studied as fragments, but Carol Robinson, a chemist at the University of Oxford, UK, applies the technique to large protein complexes, under controlled conditions that cause subunits to separate from the main complex. In 2008, she and her colleagues showed that the technique could be used to study a membrane-protein transporter complex called BtuC2D2 (ref. 13), which imports vitamin B12 into the cell. Since then, Robinson has applied mass spectrometry to four more membrane transport complexes, each with differing subunits. She is now applying the technique to larger complexes, with up to 20 subunits. QIAGEN QIAGEN offers a crystallization plate for growing crystals in lipidic cubic phase. The red dye is added for visualization. But mass spectrometry, cryoEM and X-ray crystallography share a problem: they cannot show the dynamics of protein movement. "Drugs probably don't work by stabilizing a single state but an ensemble of states, and we need to understand what those are," says Kobilka. To gain this understanding, many researchers are turning to technologies borrowed, with a lot of tweaking, from studies on soluble proteins. Stevens subjects membrane proteins to a short burst of deuterium, then fragments the proteins and uses mass spectrometry to identify which peptides are most heavily deuterated, and thus are most mobile in solution. Kobilka is using fluorescence quenching to try to learn how far apart bits of the protein are in various drug-induced conformations. Researchers are also resorting to NMR, in solid state or in solution. The advantage of solid-state NMR is that it can be performed with proteins of any size, in conditions very similar to those of the cell membrane. However, solid-state NMR is technically challenging and expensive. It is often carried out at cold temperatures and so may not show how proteins move in living cells. Solution NMR is more commonly used, but it can follow only small proteins or parts of proteins. These proteins must be encased in protective groups called micelles, which add to the protein's weight and dampen the NMR signal. It does, however, offer the ability to look at different parts of the protein at once and show how they move in response to drugs. "NMR can really show us the dynamics. We're beginning to appreciate the value of that kind of study," says Kobilka. "It was such a difficult task for so many years that many people were unwilling to undertake it. It isn't such a high-risk prospect any more." In fact, it's possible that the study of membrane-protein movement could follow a similar course to the one that membrane-protein structures are currently on: a series of tiny steps from impossible-to-get to essential-to-have. References * Introduction * References * Author information * Comments * Deisenhofter, J., Epp, O., Miki, K., Huber, R. & Michel, H.Nature318, 618–624 (1985). * ISI * Article * Oberai, A., Ihm, Y., Kim, S. & Bowie, J. U.Protein Sci.15, 1723–1734 (2006). * ChemPort * PubMed * Article * Oberai, A., Joh, N. H., Pettit, F. K. & Bowie, J. U.Proc. Natl Acad. Sci. USA106, 17747–17750 (2009). * PubMed * Article * Schertler, G. F., Villa, C. & Henderson, R.Nature362, 770–772 (1993). * ChemPort * ISI * PubMed * Article * Palczewski, K.et al. Science289, 739–745 (2000). * ChemPort * ISI * PubMed * Article * Cherezov, V.et al. Science318, 1258–1265 (2007). * ChemPort * PubMed * Article * Rasmussen, S. G.et al. Nature450, 383–387 (2007). * ChemPort * PubMed * Article * Rosenbaum, D. M.et al. Science318, 1266–1273 (2007). * ChemPort * PubMed * Article * Jaakola, V.-P.et al. Science322, 1211–1217 (2008). * ChemPort * PubMed * Article * Warne, T.et al. Nature454, 486–491 (2008). * ChemPort * PubMed * Article * Schroeder, G. F., Levitt, M. & Brunger, A. T.Nature464, 1218–1222 (2010). * ChemPort * PubMed * Article * Wang, L. & Sigworth, F. J.Nature461, 292–295 (2009). * ChemPort * PubMed * Article * Barrera, N. P.et al. Science321, 243–246 (2008). * ChemPort * PubMed * Article Download references Author information * Introduction * References * Author information * Comments Affiliations * Monya Baker is technology editor of Nature and Nature Methods. Additional data
• Structural biology: Crystal-clear images
  - Nature (London) 465(7299):824 (2010)
  Nature | Technology Feature Structural biology: Crystal-clear images Journal name:NatureVolume:465,Pages:824–825Date published:(10 June 2010)DOI:doi:10.1038/465824aPublished online09 June 2010 Article tools * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Getting proteins to form crystals is only one step for the structural biologist. The next step to sleuthing out a protein's structure involves placing the crystals in an intense beam of X-rays. This radiation bears little resemblance to the broad, diffuse X-rays used in medicine: the powerful X-rays that work best for protein crystallography are produced at giant facilities called synchrotrons, of which only a few dozen exist. At the Advanced Photon Source synchrotron at Argonne National Laboratory, Illinois, for example, electrons race around a 1.1-kilometre track at close to the speed of light. Radiation generated from the electrons is collected into a 70-metre beamline, which focuses X-rays into a 25-square-micrometre area where crystals can be positioned for analysis. ARGONNE NATL LAB. Robert Fischetti matches crystals with X-rays. Proteins in the crystal scatter the X-rays as they pass through, and researchers can decipher a protein's structure from the resulting diffraction pattern. To generate a complete pattern, the crystal must be rotated within the beam so that X-rays pass through in different orientations. The process requires precision: researchers have to collect enough data to solve a structure, while limiting radiation damage to the crystal. Technologies for manipulating crystals and keeping them at temperatures below 0 °C to decrease radiation damage have got better, but the most dramatic improvement is that experiments can now be done using very small crystals or crystals with many poorly diffracting regions, says So Iwata, who heads the Human Receptor Crystallography Project at the Japan Science and Technology Agency in Kyoto. "Crystals that would have been turned away ten years ago are welcome now," he says. Still, a crystal must be as wide as or wider than the beam passing through it to generate a reliable diffraction pattern. That's a problem, because crystals of membrane proteins tend to be small, says Robert Fischetti, a senior scientist at Argonne National Laboratory, which has produced data for crystal structures of several membrane proteins, including the β2-adrenergic receptor (S. G. Rasmussenet al. Nature450, 383–387; 2007). Technologies such as lipidic cubic phase crystallization have helped researchers to grow crystals, he says, but these are often only 5–10 micrometres across, a tenth the size of most crystals submitted for analysis and much smaller than the X-ray beam used in crystallography studies. Researchers led by Fischetti have developed a new version of a collimator, a device that blocks most of the X-rays to produce a 'minibeam' of 5 micrometres or less. Collimators, essentially engineered strips of platinum, are placed about 3 centimetres from the sample and are much more than simple pinhole apertures, says Fischetti. "We started out with a single collimator with three parts — the beam-defining pinhole aperture, the capsule around the pinhole and a forward scatter guard tube." The first versions of the device caused X-rays to scatter in a way that interfered with the diffraction pattern, but his team has since engineered features, such as a layer of molybdenum, to overcome these problems. They also created double and triple collimators to let users pick the beam size. After one user damaged a collimator by spilling liquid nitrogen on it, they modified the design, eventually making a more robust version with four aperture settings but fewer parts. What has made the collimator most practical, says Fischetti, is automating the process of selecting the aperture to match the crystal. In the past, technicians had to refocus the beam manually to shrink its size, which took hours. Now, he says, "we can do it in seconds with just clicking". Besides allowing the study of smaller crystals, smaller beams let researchers identify the parts of the crystal that diffract better. "In the past, if you had a large crystal that was not homogeneous, you'd look at the crystal and say it was bad," says Fischetti. "Now, people can find the region that is the best to look at." ARGONNE NATL LAB. A collimator produces a 5-micrometre minibeam that allows the study of a tiny crystal, mounted on a copper post. Additional data
• Structural biology: Table of suppliers
  - Nature (London) 465(7299):827 (2010)
  Nature | Technology Feature Structural biology: Table of suppliers Journal name:NatureVolume:465,Pages:827–828Date published:(10 June 2010)DOI:doi:10.1038/465827aPublished online09 June 2010 Article tools * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Table 1 Table 1 Full table Additional data
• Penumbra
  - Nature (London) 465(7299):836 (2010)
  Welcome to the twilight zone.