Hot off the presses! May 13 Nature

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Latest Articles Include:

• Scientists' turn to win votes
  - Nature 465(7295):135 (2010)
  Nature | Editorial Scientists' turn to win votes Journal name:NatureVolume:465,Page:135Date published:(13 May 2010)DOI:doi:10.1038/465135aPublished online12 May 2010 Rather than bemoaning the loss of science-savvy politicians in last week's election, researchers in the UK should strive to make new friends. 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 As Nature goes to press, the United Kingdom's general election of 6 May has still not produced a clear outcome. Although the right-leaning Conservative Party won the most seats in the parliament, they failed to capture the majority needed to form a government. In the coming days, the Conservatives or the incumbent Labour Party are likely to form a coalition with the left-leaning Liberal Democrats to govern the country. What seems clear, however, is that the next parliament will contain fewer scientifically savvy members than the current one. An analysis by London's The Times newspaper shows that some 71 candidates with scientific backgrounds have been elected, down from 86 of the 650 members in the last parliament. Among the defeated is Evan Harris, a Member of Parliament (MP) for the Liberal Democrats, who was considered by many to be the most articulate voice during the election for science and its importance in policy-making. Harris's fate represents the dashed hopes of a small but vocal minority of scientists and policy-watchers who tried to shape research into a live campaign issue. In the aftermath of the election, The Times announced that it had been a "terrible night" for science, while New Scientist declared that "science is the loser". In truth, the UK's deep economic troubles meant that science was never likely to figure highly on the public's agenda. This parliament will be filled with fresh faces, and it now falls on the scientific community to begin the important and urgent work of educating these new MPs on scientific issues. The case must be made to members of all three major parties that science is an important driver of Britain's economy; that it can provide crucial solutions to major issues such as energy independence; and that it deserves strong support even during times of economic cutback. "Unless researchers act swiftly, science could end up at the front of the firing line." These arguments will hold more sway if they are cast in a non-partisan light. In the United States science has enjoyed strong support from the left and right for years, in part because academic societies such as the American Association for the Advancement of Science have scrupulously worked with both major parties to ensure a broad understanding of the benefits of science. In Britain, non-partisanship in the scientific establishment has been more fleeting. Since the then Prime Minister Margaret Thatcher imposed savage cuts to research in the 1980s, most academic scientists have shied away from the Conservative Party. Much of the effort to build an understanding of science has focused on Labour politicians, through groups such as Scientists for Labour, which was formed in the 1990s because of a perceived lack of scientific expertise within the party. Now is the time for such efforts to be extended to all parties. Unlike Thatcher's party, the Conservatives of today have made supportive noises about science — even if most members lack a strong understanding of scientific issues. Non-partisan organizations such as the Campaign for Science and Engineering in the UK (CaSE) and the Royal Society are well placed to make a broad appeal to the new parliament. In the run-up to the election, CaSE encouraged all parties to make their positions on science known, and in its aftermath the organization must work to inform a new government's science policies. The Royal Society, meanwhile, has a long-running programme matching scientists with MPs that could be particularly useful in educating new politicians. That programme should be put into high gear while the society considers other ways to engage parliament. Other scientific societies should rally their memberships to get the word out to new parliamentarians about the value of science. A well orchestrated, non-partisan appeal early in the life of the parliament could leave a lasting impression. And an early and enduring impression may be crucial to preserving Britain's scientific enterprise. Faced with a soaring budget deficit, whoever forms the new government will have to impose deep cuts on public spending. Unless researchers act swiftly, science could end up at the front of the firing line. Additional data
• Science subpoenaed
  - Nature 465(7295):135 (2010)
  Nature | Editorial Science subpoenaed Journal name:NatureVolume:465,Pages:135–136Date published:(13 May 2010)DOI:doi:10.1038/465135bPublished online12 May 2010 The University of Virginia should fight a witch-hunt by the state's attorney general. 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 Climate science is under scrutiny once again, this time over a modest half-a-million dollars — the collective sum of five federal and state grants being investigated by Kenneth Cuccinelli, a firebrand conservative who was elected late last year as attorney general of Virginia. The grants had multiple recipients, but the official target of the probe is Michael Mann, an internationally respected climate scientist who was an investigator on all five grants while working at the University of Virginia in Charlottesville between 1999 and 2005. On 23 April, Cuccinelli filed what amounts to a subpoena ordering the University of Virginia to hand over, by 26 July, all available documents, computer code and data relating to Mann's research on the five grants. He also demanded all correspondence, including e-mails — from 1999 to the present — between Mann, now at Pennsylvania State University in University Park, and dozens of climate scientists worldwide, as well as some climate sceptics. The order stated that Cuccinelli was investigating Mann's possible violation of the 2002 Virginia Fraud Against Taxpayers Act — although no evidence of wrongdoing was given to explain invoking the law, which is intended to prosecute individuals who make false claims in order to access government funds. Mann is the co-author of the famous 'hockey stick' graph, which shows estimated global temperatures over the last millennium to have been relatively constant until a drastic rise in the twentieth century. Mann has long been a target of climate-change deniers, and the scrutiny intensified last autumn when his e-mails were among those stolen from the Climatic Research Unit at the University of East Anglia, UK. But Mann's research has been upheld by the US National Academy of Sciences, and an investigation by Pennsylvania State University into the e-mails also cleared Mann of any misconduct. Given the lack of any evidence of wrongdoing, it's hard to see Cuccinelli's subpoena — and similar threats of legal action against climate scientists in a February report by climate-change denier Senator James Inhofe (Republican, Oklahoma) — as anything more than an idealogically motivated inquisition that harasses and intimidates climate scientists. Certainly Cuccinelli has lost no time in burnishing his credentials with far-right 'Tea Party' activists, many of whom hail him as a hero. In March, he instructed Virginia's state university presidents that they had no legal authority to protect homosexuals under their non-discrimination policies. He has also filed lawsuits challenging health-care reform and the Environmental Protection Agency's authority to issue greenhouse-gas regulations. Cuccinelli's actions against Mann hark back to an era when tobacco companies smeared researchers as part of a sophisticated public relations strategy to raise doubts over the science showing that tobacco caused cancer, and delayed the introduction of smoking curbs for decades. Researchers found themselves bogged down in responding to subpoenas and legal challenges, which deterred others from the field. Climate-change deniers have adopted similar strategies with alacrity and, unfortunately, considerable success. Cuccinelli has insisted that he is not "targeting scientific conclusions". But even several climate sceptics who count themselves among Mann's fiercest critics have publicly condemned the attorney general's move. Thankfully, so have many academic bodies. One of them was the University of Virginia's faculty senate, which on 5 May declared that Cuccinelli's "action and the potential threat of legal prosecution of scientific endeavor that has satisfied peer-review standards send a chilling message to scientists engaged in basic research involving Earth's climate and indeed to scholars in any discipline." Well said. Scientific organizations must respond quickly and forcefully any time political machinations threaten to undercut academic freedom. And, rather than complying, the University of Virginia should explore every avenue to challenge the subpoena. Additional data
• Advising the adviser
  - Nature 465(7295):136 (2010)
  Nature | Editorial Advising the adviser Journal name:NatureVolume:465,Page:136Date published:(13 May 2010)DOI:doi:10.1038/465136aPublished online12 May 2010 Europe's Joint Research Centre needs to find its place alongside the new chief scientific adviser. 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 Every night, under cover of darkness, boats set out into the Mediterranean Sea to fish illegally. But they can no longer do so invisibly, thanks to a detection system designed and operated by an institute of the European Commission's Brussels-based Joint Research Centre (JRC). The system collects location data signalled to coastal stations from transponders on approved fishing vessels, and combines them in real time with satellite images at a resolution as low as 8 metres that can identify the boats without transponder signals — among which are the illegal fishing ships. This is just one of several examples of how the JRC's seven institutes develop and apply technologies to support European Union (EU) policies. In the institute devoted to health and consumer protection, for instance, researchers validate methods to replace, reduce or refine the use of animals in safety testing of chemicals; develop tests to detect unauthorized genetically modified organisms in EU markets; and devise the kind of nanoscale measurement methods that will be needed to support future nanoparticle-safety studies. In some of its research areas, the JRC leads the world — which is all the more striking because the centre, with a budget close to 400 million (US$507 million) and a staff of 2,750, was once deemed to be a sluggish and inflexible body. But in the past 12 or so years it has transformed itself into a sharper and more effective organization that last year won unaccustomed approval from an evaluation panel led by the former UK science adviser David King. Since then, the JRC has been working to implement King's report recommendation that it should go further still, and have a more proactive role in advising the European Commission, Parliament and Council instead of just responding to their immediate needs. Last week the JRC unveiled its new — and still evolving — decadal strategy for achieving that goal. But because this comes at a time when commission president José Barroso is planning to create a new, high-level post of chief scientific adviser within his cabinet, a question arises. How should the new adviser and the JRC interact? It is still early days — the role of the chief scientific adviser has not been defined. But it is clear that the adviser's office will have a small staff, and will thus be dependent on information gathering from elsewhere. The JRC would like to be its major pillar of support, and its 10-year plan includes two activities that could help to achieve this. One is the provision of 'policy options', which would allow policy-makers to base their decisions on dispassionate technical analyses of the pros and cons of each course of action. The other is identifying new issues and technologies — synthetic biology, for example — that might require a policy response in the future. As valuable as those initiatives might be, however, and as admirable as the JRC's self-reform has been, the chief scientific adviser should not be constrained to listening to any single organization, least of all one that is part of the commission. He or she should be free to seek independent input from anywhere in the world. The JRC's prime mission must be to remain a world-class technical resource for the EU, helping to protect its people against everything from natural and man-made catastrophes to illegal fishing. If it develops its strategy in the same positive spirit that has animated its reinvention over the past dozen years, the long-awaited chief scientific adviser will no doubt be glad to give its technical input serious weight — without treating it like an absolute mandate. Additional data
• Neuroimaging: Stem cells on screen
  - Nature 465(7295):138 (2010)
  
• Synthetic biology: Search and destroy
  - Nature 465(7295):138 (2010)
  
• Neuroscience: Ageing on the brain
  - Nature 465(7295):138 (2010)
  
• Animal behaviour: Honeybee harmony
  - Nature 465(7295):138 (2010)
  
• Genetics: An eye for colour
  - Nature 465(7295):138 (2010)
  
• Quantum information: Leak-proof chips
  - Nature 465(7295):138 (2010)
  
• Neurodevelopment: Small brain roots
  - Nature 465(7295):139 (2010)
  
• Population genetics: Nautical niches
  - Nature 465(7295):139 (2010)
  
• Nanoelectronics: Protein transistor
  - Nature 465(7295):139 (2010)
  
• Journal club
  - Nature 465(7295):139 (2010)
  
• Correction
  - Nature 465(7295):139 (2010)
  Nature | Correction Correction Journal name:NatureVolume:465,Page:139Date published:(13 May 2010)DOI:doi:10.1038/465139ePublished online12 May 2010 Article tools * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg The Research Highlight 'Rat sequencing redux' (Nature465, 12–13; 2010) incorrectly stated that genome-sequence diversity between the spontaneously hypertensive rat and the reference brown Norway rat is less than that for currently sequenced mouse strains. In fact, the rat sequence diversity is about twice as high. Additional data
• News briefing: 13 May 2010
  - Nature 465(7295):140 (2010)
  The week in science. This article is best viewed as a PDF. Policy|Events|People|Research|Business|Business watch|The week ahead|Number crunch|Sound bites A planned $434-million US environmental monitoring network was last week approved by the National Science Board, the oversight body of the National Science Foundation (NSF). The National Ecological Observatory Network aims to collect data on climate, ecology and biodiversity in 20 different ecosystems across the United States. The NSF has requested $20 million to begin construction in 2011; the entire network of distributed observatories would take five years to complete. Carbon emissions by the United States fell 7% in 2009, the US Energy Information Administration said in a report released on 5 May. The fall, a record since energy data collection began in 1949, was partly due to the recession, with gross domestic product falling 2.4%, and a particular decline in the energy-intensive industrial sector. But the agency, based in Washington DC, noted that the carbon intensity (emissions per unit of energy consumed) of the US energy supply also fell more than 2%, mainly because natural gas is replacing increasingly expensive coal. China faces an uphill struggle to meet its self-imposed target of reducing its energy intensity (energy consumption per unit of gross domestic product) by 20% from 2005 levels by the end of this year. Although energy intensity had improved by more than 14% between the start of 2006 and the end of 2009, the first quarter of this year has seen the figure fall back by 3.2%, according to a statement from Premier Wen Jiabao published by the country's Ministry of Industry and Information Technology on 6 May. Reiterating earlier findings, the third edition of Global Biodiversity Outlook — a United Nations (UN) report — declared that none of the 2002 targets to reduce the rate of biodiversity loss by 2010 has been achieved globally. "Humanity has fabricated the illusion that somehow we can get by without biodiversity," wrote Achim Steiner, the executive director of the UN Environment Programme. The report was released on 10 May at the opening of a two-week UN-led biodiversity meeting in Nairobi. USCG/S. LEHMANN Last week's attempt to contain oil leaking into the Gulf of Mexico with a giant dome failed after it became clogged with methane hydrates and had to be moved aside. BP had hoped that the dome could be used to collect oil flowing from a ruptured pipeline after the explosion and sinking of the Deepwater Horizon drilling rig. Dispersant chemicals are being deployed (pictured) in an attempt to manage the oil, but it has been coming ashore on the wildlife-rich Chandeleur Islands off the Louisiana coast. In the wake of the explosion, US interior secretary Ken Salazar declared that no new offshore drilling permits would be issued at least until a federal investigation into the accident is delivered at the end of May. Cancer researcher Suzanne Cory has been elected as president of the Australian Academy of Science, it was announced on 7 May. Cory — who takes over from geophysicist Kurt Lambeck — is the academy's first elected female president since its founding in 1954, although geologist Dorothy Hill led the organization for a short time in 1970. The US biomedical community will lose a powerful supporter with the departure of David Obey (Democrat, Wisconsin) from the House of Representatives after more than four decades of service. On 5 May, Obey announced that he would not seek re-election in November. He has long championed federal research funding, and last year helped to steer more than US$10 billion in stimulus money to the National Institutes of Health. Stringent US biosecurity regulations have increased the cost of experiments that use dangerous pathogens. But the red tape introduced by two acts passed after the 2001 anthrax and terrorist attacks — the 2002 Bioterrorism Preparedness and Response Act, and the 2001 USA PATRIOT Act — have not dissuaded scientists from performing research in these fields, contrary to some microbiologists' warnings. The conclusions come from a study published on 10 May (M. B. Dias et al. Proc. Natl Acad. Sci. USA doi:10.1073/pnas.0915002107). See go.nature.com/m5QO3J for more. AP/PA IMAGES Monju, Japan's prototype fast-breeder nuclear reactor (pictured), restarted operation on 6 May. Located on the coast at Tsuruga, the reactor was shut down more than 14 years ago following a coolant leak and a fire. Fast-breeder reactors, designed to produce more plutonium than they consume, are a key part of resource-poor Japan's nuclear ambitions (see Nature 464, 661; 2010). Monju will undergo a three-month test period, running at 1% of its 280,000 kilowatt capacity, and reach full capacity in 2012. The country hopes to commercialize fast-breeder reactors by 2050. The world's biggest free-electron laser is going to get bigger. The SLAC National Accelerator Laboratory in Menlo Park, California, announced on 5 May that it had received initial approval from the US Department of Energy for an upgrade to the Linac Coherent Light Source (LCLS). The $420-million LCLS, which began operation in April 2009, produces short pulses of coherent X-ray light for imaging single biomolecules and making movies of molecular events. The upgrade, capped at $380 million, would by 2017 widen the spectrum of available X-ray light, and increase the number of beams, allowing more experiment stations to work simultaneously. A one-stop-shop for DNA and RNA sequence data, the European Nucleotide Archive, was officially launched on 10 May. It provides access to three existing public databases through one web interface (ebi.ac.uk/ena). The backbone of the archive, hosted by the European Molecular Biology Laboratory's (EMBL's) European Bioinformatics Institute in Cambridge, UK, is the EMBL-Bank. It also holds raw sequence data from the European Trace Archive and the newer Sequence Read Archive, created in 2008 to house data from next-generation sequencing machines. The husband-and-wife owners of a pharmaceutical company shot to the top of China's rich list on 6 May, after their firm made its initial public offering on the Shenzhen Stock Exchange. Shenzhen Hepalink Pharmaceutical Company's share price rose by 18% on its first day of trading, putting the company's value at about 70 billion renminbi (US$10 billion). Co-founders Li Li and Tan Li own a 72% stake in the firm, which says it is the world's largest supplier of the blood thinner heparin. SOURCE: NYSE First-quarter earnings by large pharmaceutical companies exceeded analysts' expectations. But many companies have lowered their predictions for the rest of the year to incorporate the effects of US health-care reform. In the long term, the reform should provide drug manufacturers with more customers, but its measures this year include higher price rebates for patients in the government's Medicaid insurance plan. Overall, pharmaceutical stocks dipped below the Dow Jones Industrial Average in mid-February as the US health-care legislation took shape, and have remained there since (see chart). AstraZeneca, based in London, reported a 28% increase in earnings per share; revenue from sales in China alone soared 36%. Meanwhile, sales at New York-based Bristol-Myers Squibb rose 11% relative to the first quarter of 2009. The company expects changes in US health laws to reduce 2010 earnings per share by 12 cents. Sales by Swiss company Roche beat forecasts, helped by its cancer drug Avastin, and sales at London-based GlaxoSmithKline rose 9%, largely due to its pandemic flu vaccine. Two companies — Merck, in New Jersey, and Pfizer, headquartered in New York — reported lower profits than the same period last year, although better than expected. Both firms are dealing with the costs of large acquisitions. The InterAcademy Council, which is reviewing the procedures of the Intergovernmental Panel on Climate Change, hears its first presentations, in Amsterdam. They are open to the public. → go.nature.com/d9wy6A Giant earthquakes and their tsunamis are the subject of a one-off conference in Viña del Mar, Chile, convened by the American Geophysical Union on the 50th anniversary of the giant 1960 Chile earthquake (see Nature 465, 24–25; 2010). → go.nature.com/ZhmWap Japan's Venus Climate Orbiter 'Akatsuki' — its first probe to the planet — is scheduled to launch from the Tanegashima Space Center. → go.nature.com/zpRZYp The World Health Organization holds its 63rd annual assembly in Geneva, Switzerland; pandemic flu vaccines and a global strategy to reduce harmful use of alcohol are on the agenda. → go.nature.com/u7EdT2 Members of the UK House of Commons science and technology committee, before last week's elections. Number of committee members re-elected to parliament. See Editorial, page 135. Deepak Pental, the vice-chancellor of the University of Delhi, apologizes for a radioactive accident in which his institution sold off a γ-ray source for scrap metal. One worker has died and six have been ill since early April (see go.nature.com/KxRujd for more). 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.
• China drought highlights future climate threats
  - Nature 465(7295):142 (2010)
  Yunnan's worst drought for many years has been exacerbated by destruction of forest cover and a history of poor water management. Born into a farming family in south Yunnan province, China, Zhu Youyong's life has always been tied to the soil. At the age of 54, however, Zhu — now president of Yunnan Agricultural University in Kunming — says he "has never seen such severe drought in Yunnan". Since last September, the province has had 60% less rainfall than normal. According to the Ministry of Civil Affairs, 8.1 million people — 18% of Yunnan's population — are short of drinking water, and US$2.5-billion worth of crops are expected to fail. The drought in Yunnan province has left millions without water.AP PHOTO Scientists in China say that the crisis marks one of the strongest case studies so far of how climate change and poor environmental practice can combine to create a disaster. They are now scrambling to pin down exactly what caused the drought, and whether similar events are likely to hit the region more often in the future. Meanwhile, with most of the province's winter crops ruined, local farmers need immediate help. Zhu has been going from county to county to persuade farmers to grow different crops together in the same field, rather than as a monoculture. Intercropping can boost yields by up to 30%, and could help to avoid food shortages in the region later this year1. This summer, 80% of the farmland in Yunnan — a staggering 2.9 million hectares — will use the technique. But success will depend on a break in the weather. "If it still doesn't rain in late May, the consequences will be unthinkable," says Zhu. Dry spell It is not news that China is seriously short of water, but its southwestern region — including the Yunnan, Guizhou, Guangxi and Sichuan provinces and Chongqing municipality — usually sees ample precipitation. This year, however, the rains did not come, and people there want to know why. "Yunnan does experience droughts every few decades," says Xu Jianchu, an ecologist at the Kunming Institute of Botany, an institute of the Chinese Academy of Sciences (CAS). But the severity of this year's drought is unusual. Some say it is the worst in over a century. Xu is a contributor to a report on climate change in Yunnan and its myriad impacts2. Sponsored by CAS and the China Meteorological Administration, the report shows that Yunnan has got warmer and drier in the past half-century. Since 1960, the number of rainy days has decreased, whereas the number of extreme events, such as torrential rains and droughts, has increased. "If it still doesn't rain in late May, the consequences will be unthinkable." Some suggest that this year's drought in Yunnan might be caused by the El Niño/Southern Oscillation (ENSO), an atmospheric circulation system that originates in the western Pacific Ocean and brings rainfall to Southeast Asia. During El Niño years the wind from the Pacific weakens, leading to droughts in the region. "We've had a moderately strong El Niño event since October," says Dan Bebber, a climate researcher at the Earthwatch Institute in Oxford, UK, a non-profit environmental group. Although Yunnan is not directly under the influence of ENSO, "there is a statistical relationship between El Niño and the monsoon system in southwestern China through mechanisms that are unclear", he says. Indeed, the CAS report suggests that in previous strong El Niño years, the rainy season in Yunnan, which spans May to October, was delayed, with less rain in the summer and more rain in the autumn. But climate models are divided on how climate change will affect ENSO, with some showing increasing intensity and others decreasing intensity, says Bebber. Climate change is not the only factor affecting the drought. Deforestation in mountainous Yunnan is also being blamed. "Natural forests are a key regulator of climate and hydrological processes," says Xu, who is also China's representative at the World Agroforestry Centre, an international think tank headquartered in Nairobi, Kenya. The forest's thick litter layer of organic materials can absorb up to seven times its own weight in water, says Liu Wenyao, an ecologist at the Xishuangbanna Tropical Botanical Garden (XTBG), a research institute of CAS in Menglun in southwestern Yunnan. Natural forests also have an extensive network of roots that keep the ground moist, and the canopy can trap water vapour, creating a dense fog that keeps the myriad plant species alive during dry seasons. But in Xishuangbanna prefecture, renowned for the natural splendour of its tropical rainforests, forest clearance between 1976 and 2003 shrank the primary-forest cover to 3.6% of its 1976 value3. The rainforest has been replaced by rubber trees — known as 'water pumps' by locals because of their insatiable thirst — which now cover 20% of the prefecture's land. In the Ailao mountains north of Xishuangbanna, where it is too cold to grow rubber trees, plantations of fast-growing but thirsty eucalyptus are replacing primary forest to feed the paper industry. In other parts of Yunnan, logging, mining, quarrying and increasing human settlement have cleared huge areas of forest. The results are an increase in soil erosion, landslides and flash floods. "Such large-scale deforestation removes the valuable ecological services natural forests provide," says Liu. "The impact of deforestation on hydrological processes becomes particularly acute during prolonged droughts." The region could also be plagued by other natural hazards: with drought the risk of forest fire increases, whereas wetter monsoon seasons could see more floods wreaking havoc. Many scientists are now worried that severe droughts, such as Yunnan's, will become more common across southeast Asia. In addition to the effect on humans, "the impact on biodiversity could be huge," says Jennifer Baltzer, an ecologist at Mount Allison University in Sackville, New Brunswick, Canada. As existing plant species struggle to cope with the drought and die, they are replaced by hardier plants. Zhu Hua, an ecologist at XTBG, and his colleagues have already noted a 10% increase in the abundance of liana species over the past few decades in southwestern Yunnan's tropical forests4. Cao Kunfang, also an XTBG ecologist, says that lianas have a deep root system that allows them to absorb water deep in the soil5. They can also minimize evaporation by closing the minute stomatal pores in their leaves. But without a large trunk, lianas are poor at absorbing carbon dioxide — and even worse once their stomata close. "Having more lianas in tropical forests could compromise their function as a carbon sink," says Cao. Last-minute scramble As government officials scramble to deal with the emergency in Yunnan, the province's water management is being scrutinized. Most of its reservoirs were built more than 50 years ago, and half are either disused or do not function properly. Many of Yunnan's natural lakes are severely polluted and unusable, says Ma Jun, director of the Institute of Public and Environmental Affairs, a non-governmental organization in Beijing. Xu says that the region has not enough small-scale infrastructure — ponds, small reservoirs and canals — to distribute clean water to the hardest-hit areas. "There is an urgent need to develop an effective hydrological network in the province," he says. In recent years the region has instead focused on building huge reservoirs and hydropower stations, Xu says, because of the economic and political capital that such projects offer. Overall, the central government has been reactive, tackling droughts when they come rather than preparing for the worst, adds Yu Chaoqing, a hydrologist at the Beijing-based China Institute of Water Resources and Hydropower Research, part of the government's Ministry of Water Resources. Throughout southwestern China, where 2,000 drought-relief workers are drilling wells around the clock, the location of groundwater remains elusive because few geological surveys have been done. "It's a last-minute scramble because only 10% of the drought-ridden region has been surveyed," says Hao Aibing, a geologist at the China Geological Survey in Beijing, who is helping to locate groundwater in Yunnan, Guizhou and Guangxi provinces. "Even if we get live water wells, the water quality remains an issue," he says. "We just know so little about the groundwater in the region." Researchers are adamant that lessons must be learned from this year's drought in Yunnan. "Extreme weather events are likely to happen more frequently in the future," says Xu, referring to the findings of the CAS report. "I hope we will be better prepared when the next natural disaster strikes." * References * Li, C. et al. PLoS ONE4, e8049 (2009). | Article | PubMed | ChemPort | * Zhou, Y., Lu, X.-X., Xu, J.-C., Zhang, H. & Jiang, T. (eds) Systematic Assessment of Climate Change Impact in Yunnan Province (China Meteorological Press, in the press). * Li, H., Ma, Y., Aide, T. M. & Liu, W. Forest Ecol. Manag.255, 16-24 (2008). | Article * Zhu, H., Xu, Z.-F., Wang, H. & Li, B.-G. Biodivers. Conserv.13, 1355-1372 (2004). | Article * Zhu, S.-D. & Cao, K.-F. Plant Ecol.204, 295-304 (2009). | Article 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.
• Shake-up for fusion team
  - Nature 465(7295):143 (2010)
  Appointment of new chief may trigger wider changes. The international fusion experiment ITER may get a new director-general after suffering delays and cost overruns. Osamu Motojima, a distinguished Japanese physicist, is being floated as the project's new chief, Nature has learned. The appointment, if made, may trigger further changes for the project. "I wouldn't be surprised if there's a huge shake-up in ITER management under him," said one fusion scientist familiar with the project. Motojima would replace Kaname Ikeda, who has led the programme since its inception in 2007. Ikeda was originally appointed for a five-year term, and his departure would be the second high-level management change in recent months. In February, Europe's project head, Didier Gambier, was replaced by British physicist Frank Briscoe (see Nature 463, 721; 2010). ITER spokesman Neil Calder said that the organization would consider management changes at the next council meeting in June. However, he would not confirm whether Motojima is a candidate for the directorship. ADVERTISEMENT Researchers hope that ITER, based in the south of France, will prove the viability of nuclear fusion as a power source. The doughnut-shaped reactor will heat and squeeze hydrogen isotopes until they fuse together, forming helium. The process is expected to release ten times the power it consumes. ITER's seven members — Europe, Japan, the United States, South Korea, China, Russia and India — hoped to build the project by 2016 at a cost of €5 billion (US$6.3 billion). But that cost is expected to double, and first experiments are now set for late 2019. On 5 May, the European Commission announced that it faces a €1.4-billion funding gap for construction between 2012 and 2013. Motojima has a long career in fusion research. From 1999 to 2002, he oversaw construction of a fusion machine called the Large Helical Device (LHD) at the National Institute for Fusion Science in Toki City, Japan. The LHD uses a twisted loop of magnets to wrangle hot gas, a more complex set-up than the doughnut-shaped ITER device. Despite this, Motojima saw the LHD completed on schedule, says Hutch Nielson of Princeton Plasma Physics Laboratory in New Jersey. "I think Motojima's record there was a complete success," he says. 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.
• Genomics goes beyond DNA sequence
  - Nature 465(7295):145 (2010)
  A technology that simultaneously reads a DNA sequence and its crucial modifications makes its debut. What makes two individuals different? Biologists now know that the genome sequence holds only a small part of the answer, and that key elements of development and disease are controlled by the epigenome — a set of chemical modifications, not encoded in DNA, that orchestrate how and when genes are expressed. But whereas faster, cheaper and more accurate sequencing technologies have developed rapidly, techniques to map the epigenome have lagged behind. DNA polymerase (shown flanking the double helix) can reveal genomic and epigenomic detail.LAGUNA DESIGN/SPL Sequencing company Pacific Biosciences, based in Menlo Park, California, has now developed an integrated system that simultaneously reads a genome sequence and detects an important epigenetic marker called DNA methylation. "I think it's an important step forward, although I think it is a baby step," says Joseph Ecker, a plant geneticist at the Salk Institute for Biological Studies in La Jolla, California, who was not involved in the work. DNA methylation — the addition of methyl groups to individual bases — is just one of many epigenetic markers of DNA and its associated proteins. Others include modification of the histone proteins that DNA winds around to form chromatin — the tightly packed cluster that makes up chromosomes — and the activation of small non-coding RNA molecules. DNA methylation, which reduces gene expression, is linked to key developmental events, as well as many types of cancer. It is the best-studied epigenetic modification, mainly because tools have existed to study it, says Susan Clark, an epigeneticist at the Garvan Institute of Medical Research in Sydney, Australia. The gold-standard method for detecting DNA methylation, which Clark's group developed more than 15 years ago, is bisulphite sequencing, in which unmethylated versions of the base cytosine are chemically converted into another base, uracil. Sequencing the converted DNA allows scientists to reconstruct a genome-wide methylation map. But the technique has several drawbacks. Not only is it expensive and time consuming, it also damages DNA, reducing the map's accuracy. And it doesn't detect methylation at adenine bases, which are very prevalent in organisms such as bacteria. Pacific Biosciences' approach for detecting DNA methylation, published this month in Nature Methods1, builds on the company's sequencing technology. The system uses an enzyme called DNA polymerase to read a strand of DNA and build a complementary strand out of nucleo­tides labelled with fluorescent mol­ecules. As each component is added to the growing strand, it produces a flash of light — the colour of the light corresponds to the identity of the base, and thus reveals the sequence of the template DNA. Analysing the pulses of light, and the time between them, can also show whether methylation is affecting polymerase activity. This has now been exploited to detect methyl­adenine, methylcytosine and a poorly understood modification called 5-hydroxymethylcytosine. "We foresee with this technology that in the future there will be a unification of the fields of epigenomics and genomics," says Stephen Turner, the company's founder and chief technology officer. Game changer? Although the data are promising, obstacles remain. "There are distinct advantages, but we're not rushing out tomorrow to apply this because it's not prime time for human methyl­ome mapping," says Ecker. One problem is that although the technique is great at distinguishing adenine from methyl­adenine, it doesn't quite reach single-base resolution for cytosine and methylcytosine. It also lacks one of the key promised benefits of Pacific Biosciences' sequencing technology: its ability to read long sequences of DNA, up to 8,000–10,000 base pairs, which makes it easier to assemble the data into complete genomes. Instead, the reported methylation read-length is only about 1,000 base pairs. "This is exactly the technology you could use to look for epigenomic changes in specific cell types." Turner says that the company is working to solve these problems. It will ship the first sequencers that use fluorescent labelling this year, and plans to add the methylation mapping capability next year. "What needs to be done now is to make it robust and accurate," says Clark, a steering-committee member of the Inter­national Human Epigenome Consortium, a bid launched in January to map the epigenome in multiple cell types2. "There's a lot of trouble­shooting that needs to be done to get it to be accurate enough to be able to compete with bisulphite sequencing." Several companies are working on similar technologies. UK-based Oxford Nanopore Technologies published a report last year showing that it could detect methylated DNA at a single-molecule level3. But that system and others are still at an earlier stage of development. Some say that the promise for such a technique is huge. Bisulphite sequencing for a single human genome can cost up to US$100,000, says Robert Martienssen, a geneticist at Cold Spring Harbor Laboratory in New York. With the latest technique, the cost of a full-genome methylation map would drop to $100–1,000, he says. "That will change everything." There is no shortage of epigenetic questions ripe for probing. One example is in tumour biology, where different cancer cells are likely to have different methylation patterns. Another is how cells in a single organism take on different functions despite having identical genomes. "This is exactly the technology you could use to look for epigenomic changes in specific cell types," says Martienssen, who is also on the International Human Epigenome Consortium's steering committee. Ecker says researchers still haven't pinned down the significance of, say, having a methylation mark in one position and not another, and what's really needed is more studies that unify genomic and epigenomic information. "As you get more genomes to compare, then of course the differences take on some meaning," he says. "We're just lacking numbers at this point." * References * Flusberg, B. A. et al. Nature Meth.doi:10.1038/nmeth.1459 (2010). * Abbott, A. Nature463, 596-597 (2010). | Article | PubMed | ChemPort | * Clark, J. et al. Nature Nanotechnol.4, 265-270 (2009). | Article | ChemPort | 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.
• Hot science from a volcanic crisis
  - Nature 465(7295):146 (2010)
  The eruption of Mount St Helens in 1980 left an indelible mark on the field of volcanology. Janet Fang reports. Since the avalanche that uncorked Mount St Helens, eruptions have rebuilt part of the volcano.USGS/J. PALLISTER Thirty years ago this week, Mount St Helens in Washington state was swollen to bursting point. The northern flank of the mountain was bulging outward at a rate of more than one metre per day as magma built up inside. By 18 May 1980, the volcano could withstand the pressure no longer. The side of St Helens collapsed in an immense landslide, unleashing the largest explosive eruption in US recorded history. An avalanche of rock raced 22 kilometres downhill while a plume of debris shot 25 kilometres skyward, punching into the stratosphere. The eruption killed 57 people near the volcano and blanketed 10 states with a layer of ash. Amid all the destruction, however, the blast stimulated unheralded interest in eruptions and sparked many careers in volcanology. Not since the annihilation of Pompeii by Mount Vesuvius in Italy had a volcanic event garnered so much attention from scientists and public officials. After St Helens blew, the US government boosted funding for research in this area by more than a factor of 10, opened up new volcano observatories, and within a few years developed an international programme to respond to volcanic crises around the world (see 'A blast from the past'). "A whole generation of volcanologists came out of that period." "St Helens blew up in a very photogenic way near a major American city and caused dozens of fatalities, millions of dollars of destruction, and incredibly violent stories and pictures," says geologist Jonathan Fink from Arizona State University in Tempe. "It was impossible to ignore." With increased funding and new research opportunities, "a whole generation of volcanologists came out of that period", he says. So did a new understanding of explosive volcanism and how to forecast it — tools that have helped save thousands of lives over the intervening decades. One of those who joined the field then was John Pallister, now chief of the Volcano Disaster Assistance Program, which is part of the US Geological Survey (USGS). At the time of the explosion, he was a graduate student at the University of California, Santa Barbara, working on a project in Oman looking at the ocean crust and mantle. "Everybody in the geological world turned their heads and listened to the news every day and thought, 'I sure would like to understand what happened'." Click for a larger version.B. MARQUEZ/AP PHOTO Mount St Helens provided "unprecedented access to an erupting volcano", says Cynthia Gardner, scientist-in-charge at the Cascades Volcano Observatory in Vancouver, Washington. In 1980, Gardner had just started with the USGS in Denver, Colorado, in an office that also housed the volcano hazards group. "You could see what the inside of a volcano looked like — it was revolutionary," says Gardner. One of the biggest insights for volcanologists was the lateral blast that occurred when the landslide unroofed the volcano's highly pressurized magma dome. Before that eruption, scientists had never witnessed the violence of a sideways eruption, which blew down enough trees to build 300,000 two-bedroom homes. Mount St Helens showed in dramatic fashion how volcanoes can grow unstable and fall apart. As volcanologists picked through the debris, they identified distinctive rounded knolls, or hummocks, left behind by the avalanche that started the eruption. Researchers realized that similar hummocky deposits elsewhere must have formed in the same way, when the flank of a volcano collapsed. Since 1980, volcanologists have found at least 200 similar deposits worldwide, such as those at Mount Galunggung in Indonesia and at Unzen and Bandai in Japan. "It was the 'aha' heard around the world," Gardner says. Furthermore, the lateral blast left a thin but characteristic type of deposit that geologists have also spotted elsewhere. Such deposits serve as a warning sign that a volcano has previously directed its energy sideways: at Lokon in Indonesia, for example, a series of thin surge deposits clued scientists in to the hazard for nearby villages. The St Helens eruption advanced knowledge about volcanoes in many disciplines, but petrology and seismology benefited most. It allowed petrologists to decipher 'volcano plumbing', by using the erupted rock to determine the depth and temperature of the magma reservoir and how quickly magma rose to the surface. And from the 10,000 or more earthquakes that shook St Helens before the eruption, seismologists identified specific seismic patterns that can help to predict eruptions. Explosion in funding In 1979, funding for the USGS Volcano Hazards Program was less than US$1 million, nearly all of which went towards studies of Hawaiian volcanoes. In the federal budget for 1981, support for the programme jumped to $12.6 million. The USGS established the Cascades Volcano Observatory to monitor St Helens and nearby mountains. Later, the agency and local institutions set up observatories in Alaska and at Long Valley in California, as well as one to monitor activity in the Yellowstone National Park region. Funding remained fairly steady until the Mount Redoubt eruption in Alaska in 1989, which brought total appropriations up to about $16 million. Currently, the programme receives around $24 million. But, adjusted for inflation, overall funding has remained flat since 1991. Fink, who chaired a National Research Council review of the programme in 2000, observes that support for volcano research increases steeply right after a calamity, then declines to a steady, lower level until the next event. "The amount of the temporary increase tends to scale with the number of deaths or the total dollars of destruction or the media coverage," he adds. Five years after the St Helens event, mudflows from a small eruption of Nevado del Ruiz in Colombia killed 23,000 people. The fatalities and economic losses from the two eruptions prompted the USGS and the US Agency for International Development to create the Volcano Disaster Assistance Program in 1986 to help foreign countries forecast eruptions. Work by that programme and others during the 1991 eruption of Mount Pinatubo in the Philippines led to an early warning that allowed crisis-response teams to evacuate tens of thousands of people. The 1980 eruption was not the first time Mount St Helens had lost its top. About 2,500 years ago, the volcano collapsed in another flank failure, and then rebuilt itself over the following 150 years. A similar pattern may be occurring today. In September 2004, magma that had lingered underground since the 1980s began erupting and forming a new dome inside the mountain's crater. The series of dome-building eruptions continued until January 2008, reconstructing about 7% of what was lost in 1980. It is likely that more small eruptions will continue to rebuild the dome over the coming decades, according to Gardner, who adds that there will probably not be another flank collapse or lateral blast until the summit has re-formed. A 2005 USGS report ranked the 169 US volcanoes by their threat levels and monitoring capabilities. St Helens, which was erupting at the time, is second, after Kilauea in Hawaii. One of the other mountains in the top five is Mount Hood in Oregon, which has had more than 50 small earthquakes in the past year. ADVERTISEMENT Jay Wilson, a hazard-mitigation coordinator for Clackamas County where Mount Hood sleeps, was a high-school student in Birmingham, Alabama, in 1980, who caught the volcano bug from the St Helens event. Three weeks ago, Wilson joined USGS geologists to brief members of Congress on volcano risks. They were also garnering support for a recently introduced bill, which asks for $15 million annually for a National Volcano Early Warning and Monitoring System. Such a project would expand existing efforts that monitor only a few volcanoes to keep tabs on all active US volcanoes that pose a risk to people. That kind of work will require new blood in the volcanology field. As researchers mark the thirtieth anniversary of the St Helens eruption, many who studied the volcano are nearing the end of their careers. Frederick Swanson, a geologist with the US Forest Service's Pacific Northwest Research Station in Corvallis, Oregon, rushed to Washington in 1980 and toured St Helens by helicopter to study the landslide's effects. Now he's contemplating retirement. "We have a rich legacy of information from 30 years of volcano research," he says. "We're keen to continue studies, as well as ignite new ones." 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.
• Ancient DNA set to rewrite human history
  - Nature 465(7295):148 (2010)
  Discovery that some humans are part-Neanderthal reveals the promise of comparing genomes old and new. The worlds of ancient and modern DNA exploration have collided in spectacular fashion in the past few months. Last week saw the publication of a long-awaited draft genome of the Neanderthal, an archaic hominin from about 40,000 years ago1. Just three months earlier, researchers in Denmark reported the genome of a 4,000-year-old Saqqaq Palaeo-Eskimo2 that was plucked from the Greenland permafrost and sequenced in China using the latest technology. Neanderthals once bred with Homo sapiens.PHOTOLIBRARY As researchers compare these ancient genomes with the ever-expanding number from today's humans, they expect to gain insights into human evolution and migration — with more discoveries to come as they decipher DNA from other branches of the human evolutionary tree. "For the first time, ancient and modern genetic research is going hand in hand," says Eske Willerslev, whose team at the University of Copenhagen led the Palaeo-Eskimo sequencing project. "It is really a fantastic time." Already, analysis of the Neanderthal genome has helped to resolve a debate about whether there was interbreeding between Neanderthals and Homo sapiens: genome comparisons suggest that the two groups mated an estimated 45,000–80,000 years ago in the eastern Mediterranean area. The sequencing study, from a consortium led by Svante Pääbo of the Max Planck Institute for Evolutionary Anthropology in Leipzig, Germany, found that the genomes of non- African H. sapiens today contain around 1–4% of sequence inherited from Neanderthals. The breakthroughs have been driven by the plummeting cost of sequencing, together with new strategies for reducing or detecting contamination by near-identical modern human DNA. These days, labs such as Pääbo's and Willerslev's might piece together a complete genome from the degraded scraps of DNA present in ancient bone, hair or teeth in as little as a month. Researchers from geneticists to fossil specialists can't wait for more. "For the first time, ancient and modern genetic research is going hand in hand." Some hope to use ancient–modern genome comparisons to chart splits in human populations and how they might have correlated with climatic changes. "I call this molecular stratigraphy," says Jeffrey Long, a genetic anthropologist at the University of New Mexico in Albuquerque. "I then want to use this relative chronology of genetic events to compare to the palaeoclimate of Earth's biomes." For Willerslev, ancient genomes offer the opportunity to trace prehistoric migration routes. By comparing the ancient Saqqaq genome with those of modern human populations, Willerslev and his team linked it to the present-day Chukchi people of Siberia, revealing that ancestors of this group trekked from eastern Siberia to Greenland about 5,500 years ago. "The genomes will allow us to test theories about peoples and migrations debated for a century," says Willerslev. "In the next five years, we will see a whole spectrum of discoveries." For example, the work could reveal whether the first Native Americans included migrants from Europe who crossed the ice-age Atlantic Ocean. Pääbo and his team had nearly completed the Neanderthal genome by early 2009, about four years after the sequencing effort began. But, to carry out their analysis, the researchers raced to sequence five genomes of people from diverse modern populations in Europe, Asia and Africa. By comparing these to the Neanderthal genome, they found 78 protein-altering sequence changes that seem to have arisen since the divergence from Neanderthals several hundred thousand years ago, plus a handful of other genomic regions that show signs of positive selection in modern humans. These are linked to sperm motility, wound healing, skin function, genetic transcription control and cognitive development. The team also found that only the modern African genomes lacked segments of Neanderthal ancestry, indicating that interbreeding between the two groups probably occurred after humans migrated out of Africa. That revelation is likely to revive the debate about whether or not the two groups are separate species, says anthropologist Fred Smith of Illinois State University in Normal, who has studied Neanderthals in Europe. Smith thinks that they are a subspecies of H. sapiens. Now that the genomes can be compared, it will be possible to investigate the genetic roots of some shared features. For instance, he points to the development of the occipital bun, a bulge at the back of the skull that is found in Neanderthals and in some modern humans. "We need to look and clarify certain characteristics in Neanderthal morphology with genetics," he says. ADVERTISEMENT Most researchers in the field anticipate that the next ancient human genome will be completed by Pääbo's group, from a tiny finger bone found in a cave in the Altai Mountains in southern Siberia. In March, the group reported the mitochondrial DNA sequence from this individual3, an unknown hominin that, so far, does not genetically match either Neanderthals or H. sapiens and may represent a new species. The team dated the bone to about 40,000 years ago, but others say that the sediments around the bone may be as old as 100,000 years. There is speculation that the bone could be the remains of an older species of Homo, perhaps even of a remnant population of Homo heidelbergensis, known in Europe from 300,000 to 500,000 years ago, or of Homo erectus, found as early as 1.8 million years ago from Africa to Indonesia. A full sequence may help to resolve this. Obtaining the genome of a human ancestor this old was previously unimaginable. "I honestly believe this new era will change our view of human evolution," Willerslev says. * References * Green, R. E.et al. Science328, 710-722 (2010). * Rasmussen, M.et al. Nature463, 757-762 (2010). * Krause, J.et al. Nature464, 894-897 (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.
• China and Taiwan strengthen academic ties
  - Nature 465(7295):148 (2010)
  Legislators opt for pragmatism over nationalism. After a year of debate that on several occasions descended into fisticuffs, Taiwanese legislators this week opted for pragmatism over nationalism and put the finishing touches to amendments that will allow about 2,000 mainland Chinese students to enter Taiwan's graduate and undergraduate university programmes every year. The change, to be finalized this month, will open the door to a large and much-needed pool of young minds for Taiwan's universities as early as this autumn. Taiwan has strong research universities but a shortage of students. Many mainlanders already head to Taiwan for postdoctoral studies, and some go as short-term graduate or undergraduate exchange students. But, thus far, Taiwanese law has barred them from graduate or undergraduate degree programmes. Taiwan's universities are set to take in Chinese students.J.-M. HU/INST. ECOl. AND EVOL. BIOL., NATL TAIWAN UNIV. Taiwan's Chinese Nationalist Party government proposed relaxing these rules more than a year ago, as part of a warming relationship that has seen increased business and transportation links with mainland China. But, fearing that both the universities and the workforce could be overrun by mainlanders, opposition party members resisted, with some resorting to grappling a speaker to the floor of the legislature last month in an attempt to block the motion. On 10 May, violence broke out again before a subcommittee finalized the amendments. Many scientists see the plan as an opportunity to keep the island internationally competitive. "The talent and the hard-working attitude in general of the mainland students will be a boost to research," says Ben Chao, dean of the Earth sciences college at Taiwan's National Central University in Jhongli City. Kuo-Fong Ma, a seismologist at the university, says that it will also benefit science on the mainland. Taiwan has a strong record in earthquake early warning systems, for example, and educating mainland students could help to establish more research collaborations and transfer knowledge that could benefit the whole of China. An explosion in the number of universities in Taiwan — from 40 to 175 during the past 20 years — along with a declining birth rate means that there are plenty of places available for mainlanders: about 55,000 by 2015, according to census figures. And there will be no shortage of applicants, says Cong Cao at the Levin Institute in New York, who studies China's science and technology manpower issues. He points out that students will not suffer the language problems or culture shock that study abroad could bring. "Moreover, professors at Taiwan's universities are more serious about advising students," Cao argues. But the amendments still bar the major national universities from accepting Chinese undergraduates — a big caveat — and withhold Taiwanese government scholarships from mainlanders. This is a potential deal breaker, says Xiao-fan Wang, a cancer biologist at Duke University in Durham, North Carolina, and the first mainland-born scientist to be president of the Society of Chinese Bioscientists in America, a formerly Taiwanese-dominated academic networking group (see Nature 459, 1044; 2009). Wang says that when Hong Kong opened up to mainland students, scholarships were a big draw. "I am not sure there will be a flood of students who want to go to Taiwan," he says. Yi Rao, dean of the life-sciences school at Peking University in Beijing, adds that many mainland students prefer to do graduate work at US universities. Andrew Wang, vice-president of Taiwan's premier scientific institute, Academia Sinica, says that mainlanders may be wary of returning from Taiwan with a freshly minted diploma bearing the phrase 'National Taiwan University' — a rubric that challenges Beijing's assertion that Taiwan is part of China, and not an independent nation. There is also concern that if places at top universities are held for mainland graduate students, Taiwanese people might feel slighted. "These issues have to be worked out before we move ahead," says Andrew Wang. 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.
• Life after death
  - Nature 465(7295):150 (2010)
  In February, a biologist gunned down three colleagues at the University of Alabama, Huntsville. Meredith Wadman reports how their department is trying to move past the tragedy. Download a PDF of this story. Last month, Joseph Ng, a biologist at the University of Alabama, Huntsville (UAH), sat down with very mixed feelings to write a job advertisement for a new chair of the biology department. The provisional draft said that the department was seeking "an energetic and visionary leader" who could preside over the hiring of several junior faculty members. What the ad didn't talk about, and couldn't possibly describe, were the events that left so many holes to fill. On a Friday afternoon in early February, Amy Bishop, an assistant professor in the department, pulled out a black 9-millimetre pistol during a biology faculty meeting. "She just went down the line", wearing a look that was "cold, very cold", says Ng. At point-blank range, Bishop shot five of her colleagues in the head, killing three of them and critically wounding two others. Ng, seated at the opposite end of the table, thought she would murder them all. In the space of seconds, Bishop cut the 14-strong faculty by more than a third. Ever since, the survivors have been struggling with the enormous task of repairing the shattered department even as they try to heal their own emotional wounds. With 473 undergraduate majors, the biology department is the second largest on the Huntsville campus, and the shooting left nine courses without teachers. Twenty-one master's and doctoral students suddenly had no mentors. And seven research grants lost their principal investigators (see 'Keeping research grants alive'). Administrators and faculty members have rallied to keep the department running: they found substitute teachers, temporarily recalled the department's previous chairman and his assistant from retirement, and sought new mentors for graduate students. They took over the orphaned grants (except for Bishop's own project) and added dozens of student advisees to their lists of responsibilities — all on top of carrying their own teaching and research loads. "Right now, it's a sort of managed chaos. As each thing comes up, we deal with it," says Debra Moriarty, a biologist who is also dean of graduate studies and was in the conference room that day in February. Amy Bishop opened fire in a faculty meeting.R. CONN/THE HUNTSVILLE TIMES/LANDOV/PA IMAGES The surviving faculty members know that their success or failure will have effects well beyond the campus. The city of Huntsville has been building itself into a regional biotechnology research hub and has relied on the biology department to supply many of the ideas and scientists that feed that enterprise. Such ambitions were dealt a blow by Bishop's attack — the latest of the mass shootings that have become all too common on US campuses. This weekend, the remaining faculty members in the Huntsville biology department will take a moment to celebrate the graduation of 40 biology undergraduates, four master's students and a PhD candidate. But it will be only a temporary respite. All but two of the surviving faculty members witnessed the shooting and must cope with having seen friends gunned down before their eyes and thinking that their own turn was seconds away. In the aftermath of the shooting, the biologists attended counselling sessions, but the true scale of the loss is still sinking in (see 'The department that was'). "There are times when you feel very, very empty," says Ng, who has carried out research in structural biology in the department for 12 years. On 12 February at 3 p.m., members of the biology faculty gathered for a meeting in Room 369R — a small, windowless conference room tucked away in a corner of the Shelby Center for Science and Technology, where the biology department occupies the whole of the third floor. Budgeting issues were on the agenda and attendance was good: 12 of the 14 faculty members squeezed around the table. Stephanie Monticciolo, the department's staff assistant, joined them to take notes. Bishop arrived shortly before the meeting started and positioned herself at the corner of the table nearest the door. Roughly 50 minutes into the meeting, she produced what Ng remembers as "a big, black pistol". Aiming for the head in each case, Bishop fired systematically, getting off at least five rounds in what survivors say was probably 20 to 30 seconds. Starting on her immediate right, Bishop aimed at the face of Gopi Podila, killing the department's affable chairman, who was a nationally recognized plant biochemist. She then shot Monticciolo in the left temple, gravely injuring her. Bishop turned to her left and killed Adriel Johnson, a gastrointestinal physiology expert, who had made a career of encouraging minority students into science. The next lethal shot hit plant scientist Maria Ragland Davis, who had come to the university from Research Genetics, a local biotechnology firm. A ricocheting bullet or bone fragment hit Luis Cruz Vera, a molecular biologist and the department's newest faculty member, who sustained a minor chest wound. Bishop then fired at Joseph Leahy, sending a bullet slicing through his skull and critically wounding him. She pointed the gun next at Moriarty and pulled the trigger, but the gun jammed and wouldn't fire. After Bishop stepped into the hall, the survivors locked and barricaded the door with a coffee table and called the police. Bishop was arrested a few minutes later outside the building and has been charged with capital murder and attempted murder. She is being held without bail while prosecutors prepare to bring her case before a grand jury. Bishop's husband, James Anderson, whom she called to pick her up after the shooting, was briefly detained by police and released. The couple has four children, aged 8 to 18 at the time of the shootings. Huntsville, nicknamed 'Rocket City', has been a space- and engineering-science Mecca ever since 1950, when German rocket scientist Wernher von Braun was brought to the tiny cotton town to develop ballistic missiles for the US Army. Today, Huntsville and its suburbs boast a population of 387,000, with a higher percentage of engineers than any other US city. A decade ago, as human-genome sequencers raced to the finish line, biologists in Huntsville decided it was time to put their city on the map for a different reason. They set themselves the ambitious goal of building Huntsville into a biotechnology hub; the region's answer, in spirit if not in size, to the San Francisco Bay Area (see Nature 453, 818–820; 2008). They were led by Jim Hudson, a local entrepreneur whose biotech reagents company, Research Genetics, had been bought in 2000 by Invitrogen of Carlsbad, California, for more than US$200 million. Hudson, working with an anonymous donor, eventually launched the non-profit HudsonAlpha Institute for Biotechnology to conduct genomics research, educate the public and provide a home to fledgling biotech firms. The institute opened its doors in 2008, in a huge research park three miles from UAH. Today, it houses 13 of the city's 20 biotechnology companies. Ties between HudsonAlpha and the UAH biology department are tight. Many graduates from the department have gone on to work at the biotech start-ups housed at HudsonAlpha, and half of the institute's academic faculty members are adjunct professors at UAH. One of the many tenants at the institute with links to the biology department is iXpressGenes, which Ng launched to synthesize genes and enzymes for researchers. Within days of the attack, HudsonAlpha's scientists were stepping in to help. They teamed up to take over Bishop's neuroscience class. And the institute has taken in half of the now-mentorless doctoral students, who have been doing rotations in HudsonAlpha labs to find a new PhD adviser. Rick Myers, who directs the institute, is also helping in the search for a new UAH biology chairman. That person will be expected to work closely and collegially, as the late Podila did, with HudsonAlpha, says Ng. There is particular concern about supporting the university's interdisciplinary doctoral programme in biotechnology, which started in 2001 and is headed by Ng."For biotech to succeed here and for HudsonAlpha to succeed, we need people coming through that [PhD] programme," says Troy Moore, an entrepreneur at HudsonAlpha who studied biology at UAH. He went on to co-found Huntsville-based Open Biosystems, which was acquired in 2008 by Thermo Fisher Scientific of Waltham, Massachusetts. One of his first thoughts on learning of the shootings, Moore says, was: "Oh no, who's going to want to come into that programme?" But UAH administrators are confident it will survive. "The faculty members who put the PhD programme together were actually very astute in making sure that it wasn't built on just one or two faculty members," says Vistasp Karbhari, the UAH provost. "Normally I would worry about a programme [in this situation]. In this case, I am not worried." One of the longest-serving professors in the biology department is Moriarty, an outgoing scientist who studies growth-factor signalling. A department stalwart since 1982, Moriarty had become in recent years one of Bishop's closest colleagues. The two were even talking about submitting a grant proposal together to the National Institutes of Health to study an enzyme that is thought to inhibit breast cancer. Moriarty was glad to make the faculty meeting that Friday in February because her commitments as a dean often prevented her attending. As the meeting neared its end, Moriarty was jotting something on her notepad when the first shots rang out. By the time Moriarty looked up, Bishop had already hit two people and was rapidly selecting new targets. "There was only a second or two seconds between" each shot, Moriarty says. "At that point I realized what was happening. And all I thought was: 'This has to stop'." Moriarty crawled under the table and grabbed one of Bishop's legs, pleading with her to end the attack. Bishop freed herself, chased Moriarty to the room's doorway and pulled the gun's trigger. It just clicked. "She pointed the gun at me again and it clicked." Moriarty rolled into the corridor outside and Bishop followed her. Moriarty saw "a very, very evil-looking stare" in the face of her colleague. "She pointed the gun at me again and it clicked," Moriarty recalls. "At that point, I kind of threw myself back into the room and shut the door. As I was doing that I heard 'click, click, click'." Bishop had been quirky and outspoken ever since she arrived at the university in 2003. She came to Huntsville from Harvard University in Cambridge, Massachusetts, where she had earned her PhD a decade earlier. During a postdoc position in Bruce Demple's lab at the Harvard School of Public Health in Boston, she had studied the role of nitric oxide in central nervous system injury and diseases. At UAH, she continued that research, along with teaching introductory neuroscience, anatomy and physiology. Survivors: Debra Moriarty and Joseph Ng.B. DILL/ She also spent what some colleagues considered an inordinate amount of time developing a portable cell incubator with her husband, a biologist and computer engineer. They helped found a company, Prodigy Biosystems, to license the incubator from the university and commercialize it. The device has drawn $2.25 million in funding from local investors — $1 million of that since early March — making Bishop and Anderson a local biotech success story. Dick Reeves, the chairman of Prodigy Biosystems, says that the company formally parted ways with Bishop's husband in April, although Anderson says he has not yet accepted the severance agreement offered by Prodigy. All that outside activity did not help Bishop — who turned 45 just before the shooting — when it came to her tenure decision. She was denied tenure in March 2009 and the university rejected her appeal in November. After the tenure denial, "she started to get a lot more agitated about things", says Moriarty. Bishop found someone who told her how each of her colleagues had voted on her tenure application. According to Moriarty, the decision "was not super close". Moriarty has no doubt that the right call was made on Bishop, who had a history of causing graduate students to leave her lab in search of more congenial work environments. At the time of the shootings, Bishop was not supervising any PhD students. "Her teaching was weak," says Moriarty. "And her research output was very weak." She had received repeated warnings in her annual reviews to publish more papers, says Moriarty. In the past three years, Bishop published two papers in the Journal of Neurochemistry, and one in the Inter-national Journal of General Medicine, which includes three of her children as authors. Yet Moriarty is hard pressed, in hindsight, to find red flags. Bishop was vocal, and highly strung, but so are lots of academics. She had a big ego, but so do plenty of other scientists. "As long as she worked here, she was just sort of odd in her reaction to things. But not violent," Moriarty says. Within days of the murders, however, Bishop's history of violence emerged. In 1986, when she was 21, Bishop shot and killed her 18-year-old brother with her father's shotgun, which was ruled to be an accident. Last month, an inquest into that shooting was held, and the District Attorney in Norfolk County, Massachusetts, may decide to seek an indictment against Bishop in her brother's death. Bishop was also charged with assault in 2002 after she punched a woman in the head in a Massachusetts restaurant; the woman had taken the last child's booster seat there. Bishop was sentenced to probation in that case. Nearly a decade earlier, Bishop and her husband were both questioned by law-enforcement authorities in connection with a mail bomb sent to Paul Rosenberg, a neurologist and neuroscientist at Children's Hospital Boston and Harvard Medical School. Bishop had worked for Rosenberg as a postdoc for eight months, until the end of November 1993. He received the bomb in December of that year after a discussion with her about whether he would be able to write her a strong letter of recommendation. That case has not been solved, but the US Attorney's office in Boston issued a statement after the February shooting, saying, "We have commenced a thorough review of the information related to this incident to confirm that all appropriate steps were taken in that matter." Anderson says the case was closed in 2001 and he told The New York Times in February that he and his wife had nothing to do with the bomb. On 11 February, the day before the Huntsville shootings, Kimberly Hobbs, a third-year PhD student in biology, was wrestling with a problem: she had to take blood from rats for her studies of pancreatic function in diabetes, but she always seemed to miss the tiny veins in their tails. She sought out her mentor and thesis adviser, Adriel Johnson, for help. "Watch what I'm doing and you'll be able to do it too," said Johnson, showing Hobbs how to infuse the tail vein with blood by stroking it from the base to the tip, and how to insert the needle directly on top of the vein. Johnson had been the first faculty member Hobbs had met when, as a high-school senior from Chicago, she toured the Huntsville campus in 1998 with her mother, trying to decide between UAH and closer-to-home competitors such as the University of Minnesota. Meeting Johnson clinched her decision. It wasn't only that he was African-American, like herself. The man's demeanour reassured Hobbs and her mother. "We felt that he genuinely cared about students and wanted you to do well." But Johnson hardly pampered his recruits. On the contrary, he was known for his tough love, which included requiring them to work extra hours designing lab experiments. Without him, Hobbs is preparing for biology and biochemistry cumulative exams this summer as well as searching for a new thesis adviser. No remaining UAH biologist works in her area, so Hobbs has been observing in several labs at the HudsonAlpha Institute, and is likely to move her doctoral work there this summer. Transferring to HudsonAlpha, with its supercharged intellectual atmosphere, could be a great career move, but she has mixed feelings about taking advantage of an opportunity created by a tragedy. With Johnson gone, the department has a large hole to fill in terms of recruiting and mentoring minority students. Davis also supported disadvantaged minority students. These efforts were particularly important in Alabama, where African Americans make up more than a quarter of the population but only 13% of science graduate students. Kimberly Hobbs lost her mentor in the shooting.B. DILL/ The president of UAH, David Williams, calls this dimension of the shootings "the saddest thing in so many respects". The biology faculty "was in so many ways a poster child for the diverse scientific department," he says. "That's something that we need to work to preserve and build on." The last time Hobbs saw Johnson was when he showed her how to draw blood. The next afternoon, just after 3 p.m. on Friday 12 February, Hobbs confronted the rats again, by herself. She quickly succeeded, with all the animals. "I was so happy," she laughs, recalling how she resolved to find Johnson and tell him immediately. But she eventually decided to wait until Monday. A while later, Hobbs walked out of Johnson's third-floor lab, heading for her office down the corridor, past Conference Room 369R. She ran straight into two black-clad policemen bearing rifles. "I just put my hands up and said 'I work here.' I didn't know what was going on." Hobbs was sent outside, where she waited in the chill air as paramedics emerged with stretchers carrying the wounded. She didn't recognize Monticciolo or Leahy as they were borne past her, beyond the fact that they were people, and that they were grievously wounded. "That's when I knew it was real," Hobbs says. At the time of the shootings, Leland Cseke was not in the conference room with the other faculty members. As a research assistant professor who is not tenure track and does not normally teach classes, Cseke was not required to attend and Podila had given him the option of bowing out. Cseke had spent much of his adult life working with Podila in one way or another. They met in 1990, when Cseke was an undergraduate at Michigan Technological University in Houghton, and Podila was his adviser. Cseke was drawn by Podila's commitment to using plant molecular biology to tackle environmental issues. In 2002, when Podila was recruited to chair the biology department at UAH, he brought along Cseke, who by then had earned a doctorate and had become Podila's chief lab lieutenant. Leland Cseke (left) has stepped in to teach and to oversee research grants.B. DILL/ Now he is on his own. "I lost my closest colleague," says Cseke, who since the shootings has been trying to do the work of two men. He routinely puts in 13-hour days running the Podila lab, aided by a cadre of students without whom he would be "screwed", he says. He has become faculty adviser for four doctoral students and also oversees two master's students and five undergraduates. In Podila's stead, he is teaching a course in advanced molecular techniques. In addition to his own research, he is working to keep up with the demands of Podila's grants from the Argonne National Laboratory in Illinois and the Energy and Resources Institute in New Delhi, India. When Cseke gets overwhelmed, he looks at a smiling picture of Podila he has propped up on top of a filing cabinet in his office. He is not sure what will happen in the long run — he knows he can't keep up his current pace indefinitely. Cseke may be considered for one of the tenure-track jobs that has opened up, but that is not a certainty. Shortly before 4 p.m. on the day of the faculty meeting, Cseke remembers pulling his office door closed and heading towards the lifts to the fourth-floor greenhouse. As he walked down the hall, he saw Amy Bishop coming towards him from the direction of the conference room, "running fast — faster than I have ever seen her". "She said: 'Hey, Leland,' like nothing had ever happened. She clearly was panicked and I was tempted to ask her if everything was okay. I figured she must be really late to something. So I just let her go." Cseke went up to the greenhouse and was pondering how to tackle a white-fly infestation when a SWAT team burst through the greenhouse door. They threw him on the floor and frisked him before sending him down the stairs and out of the building. "If only I could have been there, maybe to have done something." Over the past few months Cseke has disciplined himself to stop replaying that day in his head. Among the 'what ifs' that tormented him was the fact that he is a seventh-degree black belt in bujinkan budo taijutsu, a traditional Japanese martial art. He wondered if he could have disarmed Bishop, if he had attended the meeting. "If only I could have been there, maybe to have done something," he says. "But eventually I figured out that I just wasn't given that opportunity." Instead, Cseke continued on his way as Bishop fled from the floor. She later borrowed a phone and called her husband to pick her up. While waiting at the building's loading dock, she was arrested by police. On 23 March, Bishop appeared in a Huntsville courtroom, handcuffed and dressed in orange prison clothes. During a preliminary hearing, police investigator Charlie Gray testified that police searching the building on 12 February had found a bloodstained woman's jacket and a 9-milli-metre pistol in a rubbish bin in the second-floor toilets of the Shelby Center. Gray also testified that Bishop had told police that the shooting hadn't happened, that she had not been present at the meeting and that "it wasn't her". The presiding judge at the preliminary hearing, Ruth Ann Hall, ruled that Bishop should be kept in custody without bail, and that her case should be turned over to a grand jury, which will examine the possibility of indicting her. That process will take months. Hall also imposed a gagging order on attorneys in the case. Before that, Bishop's lawyer, Roy Miller, had told the media that he will be mounting an insanity defence, but she could plead in a different way if this case moves to trial. In Alabama, capital murder is punishable with life in prison without parole, or with the death penalty. In late April, a crowd of minority students gathered at a research conference in the Westin Hotel in Huntsville. Normally, the conference would have been overseen by Johnson. Instead, the registration material included a copy of a resolution by the Board of Trustees of the University of Alabama, expressing their deep sorrow at Johnson's loss. Many of the students are supported by 'Bridge to the Doctorate' awards from the National Science Foundation, the same type of grant that Johnson had helped Hobbs to land six years earlier. During a break from her duties at the conference, Hobbs says that she has been coping by "keeping myself busy". She has been visiting labs at HudsonAlpha and continuing to work with the diabetic rats in Johnson's lab. Now that the initial shock has worn off, a new species of desolation has set in. The once-collegial third floor of the Shelby Center, where she used to enjoy hanging out, has become a lonely place that she leaves as soon as she can. "Every time you are in the building you are thinking about it," she says. "On Fridays, when the clock strikes three or four, you are thinking about it." Ng is also at the Westin Hotel on this April morning, judging the poster competition and energetically quizzing a freshman nursing student about her work. Ng has been working with his colleagues to develop strategies for moving ahead. They have already posted job vacancies for three visiting professors to start in time for the new academic year in September, and they will soon send out the job announcement for a permanent chairman. "You go into the building and you are really missing these people." The events of the past few months have refocused him. "Your biggest problems all became minuscule. The things you worry about: getting a manuscript done, the grant proposal that didn't make it. All that stuff just became low priority." After months of avoiding the lab at night, he is starting to work late there again, "accepting that if something happens, it will happen". He has also lost his obsession with collecting news articles about the shootings. "The adrenaline is gone," he says. But the sadness has moved in. "You go into the building and you are really missing these people." Moriarty feels much the same. "I told somebody a week ago that I felt worse than I have the whole time," she says. She also sees similar signs in her students. "I have had a number of good students who are not doing well at all now. They come in to me and say, 'I just can't get my mind on it'. I send them all to counsellors." Moriarty talks to a lot of students. Like her colleagues, she has doubled her advising load and is now mentoring an additional 30 or so undergraduates who had been the charges of Podila, Johnson or Davis. She has also taken on Johnson's mantle as the special adviser for undergraduates bound for the health professions. Moriarty tries to stay focused on the positives. She and colleagues were buoyed when Monticciolo, the staff assistant, was discharged from a local hospital in March. And they all rejoiced when Joe Leahy returned to the department for a visit in late April. ADVERTISEMENT Leahy, who had spent more than two months in hospital, was wearing a helmet covering a spot where his skull had been shattered. Eventually a metal plate will be inserted over the hole. He also had an external jaw brace that was scheduled to be removed three days later. Bishop's last bullet had severed his right optic nerve, blinding his right eye. He had also had lost peripheral vision in his left eye. Still, says Moriarty, he was "the same old Joe". Students and faculty members clustered around him and he was eagerly planning his return to work. It was uplifting "just to know that even though he had such a terrible injury, that it hasn't taken him away", says Moriarty. "I was so, so glad to see the amazing progress he has made." 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.
• Asian education must change to promote innovative thinking
  - Nature 465(7295):157 (2010)
  As Asian economies ramp up R&D, and high-tech companies relocate to China and India, Asian science looks unstoppable — apart from one last hurdle: a shortage of local talent. A radical change in Asia's education culture is needed to foster the human capital necessary for innovation-led economies.
• Volcanic ash should not be presumed harmless in long term
  - Nature 465(7295):157 (2010)
  The scientific community and public authorities need to thoroughly investigate the potential effects of the potential effects of the volcanic ash cloud originating in Iceland and now covering large areas of Europe (Nature464, 1253; 2010).Although the associated climate and meteorological phenomena are being well documented, questions arise as to what the environmental and public-health effects might be in the longer term.
• Making the case for low-template DNA analysis
  - Nature 465(7295):157 (2010)
  Your News Feature 'DNA's identity crisis' (Nature464, 347–348; 2010) implies that the UK Court of Appeal in the Reed brothers' case cast doubt on the validity of analysing small amounts of DNA. This is misleading and not borne out by the judgement of the Court (see http://go.nature.com/msMgum
• How best to log local temperatures?
  - Nature 465(7295):158 (2010)
  The climate community must work together to create a single, clean, comprehensive and open repository of detailed temperature data, say Peter A. Stott and Peter W. Thorne.
• Vaccinate before the next pandemic?
  - Nature 465(7295):161 (2010)
  Klaus Stöhr of Novartis argues that pre-pandemic immunization with a cocktail of likely strains could be a cheap, practical and equitable way to protect people against influenza.
• The beauty of ant antics
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  Deborah M. Gordon enjoys a photographic paean to individual ants and their rarely glimpsed exploits on behalf of the collective.
• Back to basics by way of evolution
  - Nature 465(7295):164 (2010)
  The public misunderstands and mistrusts the scientific explanation of evolution more than any other branch of research, particularly in the United States. Molecular biologist John Ellis's student primer, How Science Works: Evolution, seeks to teach the nature of science using evolution as an exemplar, in the hope that a public with greater scientific literacy will be able to tackle "looming world problems" more effectively.
• Books in brief
  - Nature 465(7295):164 (2010)
  Why do so many people reject scientific evidence in favour of anecdote? In Nonsense on Stilts (Univ.
• Q&A: Sylvia Earle on protecting our seas
  - Nature 465(7295):165 (2010)
  Oceanographer and underwater explorer Sylvia Earle served as chief scientist of the US National Oceanic and Atmospheric Administration during 1990–92, and is a US national committee member of the Census of Marine Life, due to conclude this October. Earle advised on Disney's newly released cut of the documentary film Oceans; here she explains why films are important for raising awareness of the state of our seas.
• Nanotechnology: Molecular robots on the move
  - Nature 465(7295):167 (2010)
  Robots have to store lots of information in order to coordinate their actions, but how can this be done for nanometre-scale robots? One answer is to program data into the robots' environment instead.
• Origins of life: Common ancestry put to the test
  - Nature 465(7295):168 (2010)
  The question of whether or not all life on Earth has an ultimate common origin is a subtle one, complicated by the phenomenon of lateral gene transfer. It has now been tackled with a formal statistical analysis.
• Earth science: Mountains without erosion
  - Nature 465(7295):169 (2010)
  Increased erosion associated with the rise of the world's great mountain ranges has been held to be the cause of a prolonged episode of past climate cooling. That connection is now brought into doubt.
• 50 & 100 years ago
  - Nature 465(7295):170 (2010)
  To meet the great world-wide interest in nuclear science and its applications, it has often been suggested that regional centres should be created ... The first of such centres to be established is the CENTO ... Institute of Nuclear Science, formerly the Baghdad Pact Nuclear Centre, which now serves the three countries of the CENTO region, Iran, Pakistan and Turkey. The Institute is concerned exclusively with the peaceful uses of nuclear science ... The original Nuclear Centre in Baghdad was formally opened on March 31, 1957 ... and trained about sixty Middle East scientists in nuclear techniques, before the Iraqi revolution brought operations to a close.
• Biophysics: Transporter in the spotlight
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  Membrane transporter proteins switch between conformational states to move substrates across membranes. The transition between these states can now be studied using single-molecule experiments.
• Materials chemistry: Controlled nanotube reactions
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  For many potential applications, carbon nanotubes must be chemically modified, but the reactions involved aren't easily controlled. The discovery of a reversible modification process is a step towards such control.
• Transcription: Enhancers make non-coding RNA
  - Nature 465(7295):173 (2010)
  Genomes don't just encode protein-coding RNAs. They also give rise to various groups of RNAs that can regulate gene expression. Short RNAs that form from enhancer sequences might be one such class of regulatory RNA.
• Aberrant silencing of imprinted genes on chromosome 12qF1 in mouse induced pluripotent stem cells
  Stadtfeld M Apostolou E Akutsu H Fukuda A Follett P Natesan S Kono T Shioda T Hochedlinger K - Nature 465(7295):175 (2010)
  Nature | Article Aberrant silencing of imprinted genes on chromosome 12qF1 in mouse induced pluripotent stem cells * Matthias Stadtfeld1, 2, 3, 7 Search for this author in: * NPG journals * PubMed * Google Scholar * Effie Apostolou1, 2, 3, 7 Search for this author in: * NPG journals * PubMed * Google Scholar * Hidenori Akutsu4 Search for this author in: * NPG journals * PubMed * Google Scholar * Atsushi Fukuda5 Search for this author in: * NPG journals * PubMed * Google Scholar * Patricia Follett1 Search for this author in: * NPG journals * PubMed * Google Scholar * Sridaran Natesan6 Search for this author in: * NPG journals * PubMed * Google Scholar * Tomohiro Kono5 Search for this author in: * NPG journals * PubMed * Google Scholar * Toshi Shioda2 Search for this author in: * NPG journals * PubMed * Google Scholar * Konrad Hochedlinger1, 2, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:NatureVolume:465,Pages:175–181Date published:(13 May 2010)DOI:doi:10.1038/nature09017Received06 September 2009Accepted17 March 2010Published online25 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 Induced pluripotent stem cells (iPSCs) have been generated by enforced expression of defined sets of transcription factors in somatic cells. It remains controversial whether iPSCs are molecularly and functionally equivalent to blastocyst-derived embryonic stem (ES) cells. By comparing genetically identical mouse ES cells and iPSCs, we show here that their overall messenger RNA and microRNA expression patterns are indistinguishable with the exception of a few transcripts encoded within the imprinted Dlk1–Dio3 gene cluster on chromosome 12qF1, which were aberrantly silenced in most of the iPSC clones. Consistent with a developmental role of the Dlk1–Dio3 gene cluster, these iPSC clones contributed poorly to chimaeras and failed to support the development of entirely iPSC-derived animals ('all-iPSC mice'). In contrast, iPSC clones with normal expression of the Dlk1–Dio3 cluster contributed to high-grade chimaeras and generated viable all-iPSC mice. Notably, treatment ! of an iPSC clone that had silenced Dlk1–Dio3 with a histone deacetylase inhibitor reactivated the locus and rescued its ability to support full-term development of all-iPSC mice. Thus, the expression state of a single imprinted gene cluster seems to distinguish most murine iPSCs from ES cells and allows for the prospective identification of iPSC clones that have the full development potential of ES cells. View full text Subject terms: * Stem cells * Molecular biology * Genetics * Genomics * Developmental biology Figures at a glance * Figure 1: Aberrant silencing of the Dlk1–Dio3 gene cluster in mouse iPSCs. , Strategy for comparing genetically matched ES cells and iPSCs generated with the doxycycline-controllable collagen-OKSM system. , Morphology of collagen-OKSM ES cells and iPSCs. , Unsupervised clustering of four ES cell and six derivative iPSC lines based on microarray expression data. , Scatter plot of microarray data comparing iPSCs and ES cells with differentially expressed genes highlighted in green (twofold, P < 0.05, t-test with Benjamini–Hochberg correction). , Heat map showing relative expression levels of selected mRNAs in ES cells and iPSCs. , Schematic representation of the Dlk1–Dio3 gene cluster with maternally and paternally expressed transcripts shown in red and blue, respectively. , Heat map showing miRNAs that are differentially expressed between ES cells and iPSCs (twofold, P < 0.01, t-test). * Figure 2: Developmental consequences of Dlk1–Dio3 silencing. , Heat map showing relative expression levels of Gtl2, Rian and other select genes in ES cells and iPSCs derived from haematopoietic stem cells (HSC), granulocyte-macrophage progenitors (GMP), granulocytes (Gran), peritoneal fibroblasts (PF) and tail-tip fibroblasts (TTF). Four iPSC clones expressing ES-cell-like levels of Gtl2 and Rian were identified (highlighted by asterisks) (iPSC clone number 18 was analysed only by qPCR; see Supplementary Fig. 1b). , Strategy for assessing the developmental potential of iPSC clones by injection into diploid (2n) and tetraploid (4n) blastocysts to produce chimaeric or all-iPSC mice, respectively. , Images of representative chimaeras with agouti coat colour indicating iPSC origin. , Quantification of coat colour chimaerism in mice derived from indicated Gtl2off clones (green diamonds), Gtl2on iPSC clones (red diamonds) and ES cells (open diamonds). , Statistical analysis of coat colour chimaerism in mice derived form Gtl2off and Gtl2on i! PSC clones. Error bars indicate standard deviations, n = 38 for Gtl2off clones and n = 11 for Gtl2on clones, P < 0.001. , Images of two GFP+ all-iPSC neonates (left) and two agouti all-iPSC mice (right). , Scatter plot showing intensity levels of all probe sets covered by microarray analysis; highlighted in green are those probe sets that were significantly different between 4n complementation-competent iPSCs (clones 19, 44, 47 and 49) and non-4n complementation-competent iPSCs (clones 18, 20, 45 and 48) (twofold, P < 0.05, t-test with Benjamini–Hochberg correction). * Figure 3: Epigenetic silencing of the Gtl2 locus in iPSCs. , Structure of the Dlk1–Dio3 locus with the position of the genomic regions (I–VII) analysed by pyrosequencing indicated by black bars. , Degree of DNA methylation at IG-DMR and Gtl2 DMR in three Gtl2off iPSC clones (green bars), three Gtl2on iPSC clones (red bars), three ES cell clones (red open bars), as well as the parental tail-tip fibroblasts (TTF, grey bars). Analysis of the other regions is shown in Supplementary Fig. 5. , Prevalence of activation-associated (acH3, acH4 and H3K4me) and repression-associated (H3K27me) chromatin marks at the Gtl2 promoter in two Gtl2off iPSC clones, two Gtl2on iPSC clones and ES cells. , Gtl2 expression levels as measured by qPCR in subclones derived from Gtl2off clone 45 and Gtl2on clone 49 in the absence (-) or presence (+) of doxycycline (Dox). , Bright-field images of iPSC culture in the absence or presence of all-trans retinoic acid (RA). , Expression levels of Gtl2, other imprinted genes (Igf2, Igf2r), and the pluripotency mar! ker Pou5f1 in cells cultured with (+) or without (-) RA. All error bars indicate standard deviations with n = number of CpGs within the corresponding region in and n = 3 in and . * Figure 4: Developmental defects in embryos derived from Gtl2off iPSCs. , Images of 'all-iPSC' E11.5 embryos obtained with Gtl2on clone 47 and Gtl2off clone 48, both of which express EGFP ubiquitously from the ROSA26 locus. , Frequency of dead and live E11.5 all-iPSC embryos obtained with two Gtl2on (red bars) and two Gtl2off (green bars) iPSC clones upon 4n blastocyst injection. Number of blastocysts transferred per clone is indicated in brackets. , Expression of Gtl2, Rian, Mirg and the paternally expressed gene Dlk1 in Gtl2off MEFs relative to Gtl2on MEFs (upper panel) as well as in Gtl2mKO MEFs relative to MEFs isolated from wild-type embryos (lower panel). , In situ hybridization for Gtl2 mRNA in MEFs derived from all-iPSC embryos generated with either Gtl2on clone 44 or Gtl2off clone 48. , Expression levels of Gtl2, Rian, Mirg and Dlk1 in the indicated tissues isolated from all-iPSC embryos produced with Gtl2off iPSCs relative to the levels seen in tissues derived from Gtl2on iPSCs. , Degree of DNA methylation at the indicated Dlk1–D! io3 regions in Gtl2off, Gtl2on, Gtl2mKO and wild-type MEFs. , Gtl2 expression levels in iPSC lines derived by subcloning Gtl2off clone 45 in the presence of valproic acid (VPA). , Images of a neonatal stillborn pup (left) and a uterus filled with resorptions (right) derived after 4n blastocyst injections with either VPA-10 or the parental iPSC clone 45, respectively. All error bars indicate standard deviations with n = 3 in , n = 5 in and n = number of CpGs in . Accession codes * Abstract * Accession codes * Author information * Supplementary information * Comments Primary accessions Gene Expression Omnibus * GSE20576 Author information * Abstract * Accession codes * Author information * Supplementary information * Comments Primary authors * These authors contributed equally to this work. * Matthias Stadtfeld & * Effie Apostolou Affiliations * Howard Hughes Medical Institute at Massachusetts General Hospital, Center for Regenerative Medicine; Harvard Stem Cell Institute, 185 Cambridge Street, Boston, Massachusetts 02114, USA * Matthias Stadtfeld, * Effie Apostolou, * Patricia Follett & * Konrad Hochedlinger * Massachusetts General Hospital Cancer Center and Harvard Medical School, 149 13th Street, Charlestown, Massachusetts 02129, USA * Matthias Stadtfeld, * Effie Apostolou, * Toshi Shioda & * Konrad Hochedlinger * Department of Stem Cell and Regenerative Biology, Harvard University and Harvard Medical School, 42 Church Street, Cambridge, Massachusetts 02138, USA * Matthias Stadtfeld, * Effie Apostolou & * Konrad Hochedlinger * Department of Reproductive Biology, National Institute for Child Health and Development, Tokyo 157-8535, Japan * Hidenori Akutsu * Department of BioScience, Tokyo University of Agriculture, Tokyo 156-8502, Japan * Atsushi Fukuda & * Tomohiro Kono * Sanofi-Aventis, 270 Albany Street, Cambridge, Massachusetts 02139, USA * Sridaran Natesan Contributions M.S., E.A. and K.H. conceived the ideas for this study, designed and analysed experiments and wrote the manuscript. M.S. derived iPSC lines, conducted in vitro differentiation assays and performed expression array analysis. E.A. conducted qPCR analyses, in situ hybridizations and chromatin immunoprecipitations. H.A. and A.F. performed nuclear transfer experiments. P.F. did blastocyst injections. T.S. performed microarray experiments and analyses. S.N. and T.K. provided important study materials. Competing financial interests K.H. is on the advisory board of iPierian. Corresponding author Correspondence to: * Konrad Hochedlinger (khochedlinger@helix.mgh.harvard.edu) The mRNA profiling data discussed in this paper have been deposited in NCBI's Gene Expression Omnibus and are accessible through GEO series accession number GSE20576. Supplementary information * Abstract * Accession codes * Author information * Supplementary information * Comments Excel files * Supplementary Table 2 (41K) This table shows expression levels of imprinted genes in ES cells and iPSCs. * Supplementary Table 3 (224K) This table shows the global miRNA expression in ES cells and iPSCs. * Supplementary Table 5 (188K) This table shows the global miRNA expression of 4n complementation-competent and non-competent iPSCs. Pease note that the descriptions for Tables 2, 3 and 5 were updated on 2 May 2010 PDF files * Supplementary Information (4.9M) This file contains Supplementary Figures 1-10 with legends and Supplementary Tables 1, 4, 6 and 7. Additional data
• Widespread transcription at neuronal activity-regulated enhancers
  Kim TK Hemberg M Gray JM Costa AM Bear DM Wu J Harmin DA Laptewicz M Barbara-Haley K Kuersten S Markenscoff-Papadimitriou E Kuhl D Bito H Worley PF Kreiman G Greenberg ME - Nature 465(7295):182 (2010)
  Nature | Article Widespread transcription at neuronal activity-regulated enhancers * Tae-Kyung Kim1, 9, 10 Search for this author in: * NPG journals * PubMed * Google Scholar * Martin Hemberg2, 9 Search for this author in: * NPG journals * PubMed * Google Scholar * Jesse M. Gray1, 9 Search for this author in: * NPG journals * PubMed * Google Scholar * Allen M. Costa1 Search for this author in: * NPG journals * PubMed * Google Scholar * Daniel M. Bear1 Search for this author in: * NPG journals * PubMed * Google Scholar * Jing Wu3 Search for this author in: * NPG journals * PubMed * Google Scholar * David A. Harmin1, 4 Search for this author in: * NPG journals * PubMed * Google Scholar * Mike Laptewicz1 Search for this author in: * NPG journals * PubMed * Google Scholar * Kellie Barbara-Haley5 Search for this author in: * NPG journals * PubMed * Google Scholar * Scott Kuersten6 Search for this author in: * NPG journals * PubMed * Google Scholar * Eirene Markenscoff-Papadimitriou1, 10 Search for this author in: * NPG journals * PubMed * Google Scholar * Dietmar Kuhl7 Search for this author in: * NPG journals * PubMed * Google Scholar * Haruhiko Bito8 Search for this author in: * NPG journals * PubMed * Google Scholar * Paul F. Worley3 Search for this author in: * NPG journals * PubMed * Google Scholar * Gabriel Kreiman2 Search for this author in: * NPG journals * PubMed * Google Scholar * Michael E. Greenberg1 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:NatureVolume:465,Pages:182–187Date published:(13 May 2010)DOI:doi:10.1038/nature09033Received21 October 2009Accepted25 March 2010Published online14 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 We used genome-wide sequencing methods to study stimulus-dependent enhancer function in mouse cortical neurons. We identified ~12,000 neuronal activity-regulated enhancers that are bound by the general transcriptional co-activator CBP in an activity-dependent manner. A function of CBP at enhancers may be to recruit RNA polymerase II (RNAPII), as we also observed activity-regulated RNAPII binding to thousands of enhancers. Notably, RNAPII at enhancers transcribes bi-directionally a novel class of enhancer RNAs (eRNAs) within enhancer domains defined by the presence of histone H3 monomethylated at lysine 4. The level of eRNA expression at neuronal enhancers positively correlates with the level of messenger RNA synthesis at nearby genes, suggesting that eRNA synthesis occurs specifically at enhancers that are actively engaged in promoting mRNA synthesis. These findings reveal that a widespread mechanism of enhancer activation involves RNAPII binding and eRNA synthesis. View full text Subject terms: * Neuroscience * Genomics * Molecular biology * Genetics Figures at a glance * Figure 1: Enhancers near the c-fos gene with increased CBP/RNAPII/NPAS4 binding and eRNA production upon membrane depolarization. ChIP-Seq: for each histone modification or transcription factor, two horizontal rows display the numbers of input-normalized ChIP-Seq reads across the locus, with '+' and '–' denoting the membrane-depolarized (2 h KCl) and unstimulated conditions, respectively. RNA-Seq: for each of 0, 1, or 6 h of membrane depolarization, the numbers of reads aligning to forward (F) and reverse (R) genomic strands are separately displayed. Enhancers identified in this study are highlighted by light-blue vertical bars (e1–e5), and the promoter region of c-fos gene is shown by a vertical light-red bar. * Figure 2: Comparison of binding profiles between promoters and neuronal activity-regulated enhancers. , , Binding profiles of methylated histones and transcription factors at the promoter transcription start sites (TSSs) of 25,562 annotated genes () versus 5,117 extragenic enhancers (). In each panel, binding profiles of methylated histones (top), CBP and NPAS4 (middle), and CREB and SRF (bottom) from unstimulated and membrane-depolarized (2 h KCl) neurons are shown. The y axes denote the degree of binding averaged across all promoters or enhancers, expressed as the mean number of input-normalized ChIP-Seq reads. Promoters are aligned at their annotated TSSs and enhancers are aligned at their CBP binding sites, with the x axes indicating the distance (kb) to either the TSS or the CBP peak. * Figure 3: Activity-induced luciferase expression mediated by neuronal enhancers. The Arc enhancer was replaced by six randomly chosen neuronal enhancers and one of the c-fos enhancers (e2; see Fig. 1) in the context of the ~7-kb region upstream of the Arc gene. The resulting fragments were placed upstream of a luciferase reporter gene, and activity-dependent expression of luciferase was measured in the presence or absence of the Arc proximal promoter after 6 h KCl treatment in rat cortical neurons. In additional control experiments, the Arc enhancer was removed, or three randomly chosen extragenic loci that do not show enhancer features were inserted. The red dotted line indicates the mean induction value of the three negative regions tested. Error bars indicate s.e.m. (n = 3 biological replicates); P-value from t-test. * Figure 4: Enhancers bind RNA polymerase II (RNAPII) and produce eRNAs. , Binding profile of RNAPII at 25,562 TSSs of annotated genes using two different anti-RNAPII antibodies (8WG16 or 4H8). , Binding profile of RNAPII at 5,117 extragenic enhancers. , , Profile of RNA expression at 5,117 extragenic enhancers () and at 6,718 intragenic enhancers () based on RNA sequencing of the total RNA and poly(A)+ RNA fractions. The y axes report RNA expression as the normalized number of RNA-Seq reads per bp (Methods). In , F and R denote forward (+) and reverse (-) genomic strands. In , enhancers are aligned oriented relative to the gene in which they reside to allow for sense and antisense RNA-Seq reads to be shown separately. Although sense eRNAs cannot be detected due to overlapping mRNA transcription, the red arrow indicates a local increase in antisense RNA expression attributable to eRNAs (statistics in Methods). Note different scales on the y axis in and . * Figure 5: eRNAs are transcribed bi-directionally, and their activity-dependent induction correlates with induction of nearby genes. , RNA expression at 315 representative extragenic enhancers (see Methods for the enhancer selection and clustering). The enhancers are grouped into six categories using k-means clustering based on eRNA, RNAPII, CBP, NPAS4, CREB, SRF and H3K4me1 levels, with categories separated by horizontal black lines3. , Directional bias of transcription initiated from enhancers and promoters, where f and r represent the numbers of reads (forward and reverse, respectively) aligning to the regions indicated (see Methods). , The distribution of the number of RNA-Seq reads found within 1.5 kb of the extragenic enhancer loci, adjacent regions and random regions (see Methods). , Changes in RNAPII binding and eRNA levels at extragenic enhancers versus changes in mRNA expression levels of nearby genes upon membrane depolarization. Each dot represents a set of genes that have similar mRNA induction indices and a corresponding set of enhancers nearby those genes (see Methods). The lines are the ! best linear fits to the points, and ρ is the Spearman correlation coefficient. * Figure 6: eRNA synthesis but not RNAPII binding at the Arc enhancer requires the presence of the Arc promoter. , The mouse Arc genomic locus with ChIP-Seq and RNA-Seq data as in Fig. 1. Also shown are the region deleted in the Arc knockout (Arc KO) mouse and a non-polyadenylated eRNA transcript defined by the RNA circularization method (Methods). , Binding profiles of RNAPII and SRF at various loci determined by ChIP-qPCR from both wild-type and Arc knockout neurons. Error bars indicate s.e.m. (n = 2 biological replicates) , RT–qPCR detection of the presence of eRNAs from wild-type and Arc knockout neurons. 'No RT' represents the qPCR signal from cDNA samples generated from reactions in which reverse transcriptase was omitted. Error bars are s.e.m. (n = 3 biological replicates); P-values are from t-test. NS, not significant. Accession codes * Abstract * Accession codes * Author information * Supplementary information * Comments Primary accessions Gene Expression Omnibus * GSE21161 Author information * Abstract * Accession codes * Author information * Supplementary information * Comments Primary authors * These authors contributed equally to this work. * Tae-Kyung Kim, * Martin Hemberg & * Jesse M. Gray Affiliations * Department of Neurobiology, Harvard Medical School, 220 Longwood Avenue, Boston, Massachusetts 02115, USA * Tae-Kyung Kim, * Jesse M. Gray, * Allen M. Costa, * Daniel M. Bear, * David A. Harmin, * Mike Laptewicz, * Eirene Markenscoff-Papadimitriou & * Michael E. Greenberg * Department of Ophthalmology, Children's Hospital Boston, Center for Brain Science and Swartz Center for Theoretical Neuroscience, Harvard University, 300 Longwood Avenue, Boston, Massachusetts 02115, USA * Martin Hemberg & * Gabriel Kreiman * The Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, Maryland 21205, USA * Jing Wu & * Paul F. Worley * Children's Hospital Informatics Program at the Harvard-MIT Division of Health Sciences and Technology, 300 Longwood Avenue, Boston, Massachusetts 02115, USA * David A. Harmin * Molecular Genetics Core facility, Children's Hospital Boston, 300 Longwood Avenue, Boston, Massachusetts 02115, USA * Kellie Barbara-Haley * Epicentre Biotechnologies, 726 Post Road, Madison, Wisconsin 53713, USA * Scott Kuersten * Institute for Molecular and Cellular Cognition (IMCC), Center for Molecular Neurobiology (ZMNH), University Medical Center Hamburg-Eppendorf (UKE), Falkenried 94, 20251 Hamburg, Germany * Dietmar Kuhl * Department of Neurochemistry, Graduate School of Medicine, University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan * Haruhiko Bito * Present addresses: University of Texas Southwestern Medical Center, Department of Neuroscience, 5323 Harry Hines Blvd, Dallas, Texas 75390-9111, USA (T.-K.K.); Graduate Program in Neuroscience, University of California San Francisco, 1550 4th Street, San Francisco, California 94158, USA (E.M.-P.). * Tae-Kyung Kim & * Eirene Markenscoff-Papadimitriou Contributions T-K.K., J.M.G. and M.E.G. conceived and designed experiments. T-K.K., J.M.G., M.H., G.K. and M.E.G. wrote the manuscript. T-K.K. optimized the protocol for ChIP-Seq library preparation to be suitable for the SOLiD sequencer and made all ChIP-Seq libraries used in this study. S.K. invented the library construction methodology used for all RNA sequencing reported here. J.M.G., A.M.C. and E.M.-P. made all RNA-Seq libraries. M.H., J.M.G. and D.A.H. performed bioinformatic analyses. K.B.-H. carried out the SOLiD bead preparation and sequencing. T-K.K., J.W., P.F.W. and A.M.C. performed the Arc knockout experiment. D.M.B. performed the luciferase experiments. M.L. performed the RNA circularization experiment. H.B. provided the pArc7000 plasmid. D.K. provided the Arc knockout mouse. All authors reviewed the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Michael E. Greenberg (michael_greenberg@hms.harvard.edu) Sequencing data have been submitted to the GEO repository under accession numbers GSE21161 (for all ChIP-Seq and RNA-Seq data) and HM047267 (for circularized Arc enhancer RNA). The bigWig files for genome browser visualization are posted online (see Supplementary Table 6). Supplementary information * Abstract * Accession codes * Author information * Supplementary information * Comments Zip files * This file contains Supplementary Figures 1-11 with legends. (14.3M) This zipped file comprises Supplementary Tables as follows: Supplementary Table S1 shows the number of CBP sites that were removed at each step of filtering in order to produce a list of high-confidence enhancers. Supplementary Table S2 shows ChIP-Seq, RNA-Seq, and other data associated with each CBP peak, with ~41,000 CBP peaks as rows and nearly 200 columns of information about each peak. Supplementary Tables S3a and S3b show lists of TF peaks found in the 2hour KCl stimulated and unstimulated conditions, with positive values for any given factor indicating the presence of a peak. Supplementary Table S4 shows the number of extragenic enhancers that have TFs, RNAPII, or eRNAs detected, as well as the number of enhancers with any two of these features detected. Supplementary Table S5 shows the number of ChIP-Seq/RNA-Seq reads for each experiment and the antibody used for each ChIP-Seq experiment. Supplementary Table S6 contains text that can be pasted into the UCSC Genome Br! owser to display the raw ChIP-Seq/RNA-Seq sequencing data using the mm9 mouse genome. Supplementary Table S7 shows DAVID analysis of the genes whose promoters bind CREB. Supplementary Table S8 shows a list of genes with expression changes that were significant following 6 hours KCl stimulation, based on RNA-Seq biological replicate 1. Supplementary Tables S9a and 9b show DAVID analysis of strongly up-and down-regulated genes. Supplementary Table S10 shows primers used for RT-qPCR validation. PDF files * Supplementary Figures (5.8M) DESCRIPTION Additional data
• Single-molecule dynamics of gating in a neurotransmitter transporter homologue
  - Nature 465(7295):188 (2010)
  Nature | Article Single-molecule dynamics of gating in a neurotransmitter transporter homologue * Yongfang Zhao1, 2, 3, 7 Search for this author in: * NPG journals * PubMed * Google Scholar * Daniel Terry4, 7 Search for this author in: * NPG journals * PubMed * Google Scholar * Lei Shi4, 5 Search for this author in: * NPG journals * PubMed * Google Scholar * Harel Weinstein4, 5 Search for this author in: * NPG journals * PubMed * Google Scholar * Scott C. Blanchard4 Search for this author in: * NPG journals * PubMed * Google Scholar * Jonathan A. Javitch1, 2, 3, 6 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorsJournal name:NatureVolume:465,Pages:188–193Date published:(13 May 2010)DOI:doi:10.1038/nature09057Received27 November 2009Accepted23 March 2010 Abstract * Abstract * 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 Neurotransmitter:Na+ symporters (NSS) remove neurotransmitters from the synapse in a reuptake process that is driven by the Na+ gradient. Drugs that interfere with this reuptake mechanism, such as cocaine and antidepressants, profoundly influence behaviour and mood. To probe the nature of the conformational changes that are associated with substrate binding and transport, we have developed a single-molecule fluorescence imaging assay and combined it with functional and computational studies of the prokaryotic NSS homologue LeuT. Here we show molecular details of the modulation of intracellular gating of LeuT by substrates and inhibitors, as well as by mutations that alter binding, transport or both. Our direct observations of single-molecule transitions, reflecting structural dynamics of the intracellular region of the transporter that might be masked by ensemble averaging or suppressed under crystallographic conditions, are interpreted in the context of an allosteric mechan! ism that couples ion and substrate binding to transport. View full text Subject terms: * Biophysics * Biochemistry * Structural biology * Chemical biology Figures at a glance * Figure 1: Structural landmarks and the disposition of the engineered Cys pairs in the crystal structure of LeuT. , Side view of the LeuT crystal structure equilibrated in a POPC lipid bilayer, showing Leu in the S1 site, CMI in the S2 site, sodium ions as yellow spheres and the surrounding lipid molecules in thin stick rendering. The intracellular surface is at the bottom. Residues involved in conserved ionic:cation-π interactions in both the putative extracellular and intracellular gates are shown in volume rendering. , , The Cys pairs used to monitor rearrangements at the extracellular () and intracellular () ends of the transporter, under designated conditions. * Figure 2: Single-molecule imaging of LeuT. , His-tagged, dye-labelled LeuT-H7C/R86C was immobilized by biotin-NTA-Ni2+ to the streptavidin-treated surface. , Representative fluorescence (Cy3 donor in green, Cy5 acceptor in red) and FRET (grey) time traces from experiments in 200 mM KCl. , FRET traces were summed into histograms in 200 mM KCl (left), 200 mM NaCl (centre), and 200 mM NaCl with 20 µM leucine (right). Each two-dimensional histogram was summed over time (grey bars, on side), filtered to remove fluorophore dark states (see Methods), and fit to the sum (red) of two Gaussian distributions (blue) to estimate the mean value and relative occupancies of each FRET state. , Histograms are shown for experiments performed in 5 mM NaCl with no substrate (left), 200 nM leucine (centre) and 20 μM leucine (right). Scale on right indicates the relative population. * Figure 3: The structural context of the observed dynamic changes. , Evolution of distances between cysteine pairs (the distance between the Cβ atoms of two residues, Cβ–Cβ distances) for specific residues (numbers at right-hand end of traces) observed in molecular dynamics simulations in the absence (dotted lines) and presence (solid lines) of simulated transport. , Superposition of snapshots from the molecular dynamics equilibrations of the crystal structure of LeuT (grey) and the inward-open structure (orange) indicating the conformational rearrangements predicted from the molecular dynamics simulations in the transport mechanism (see Supplementary Movies 1 and 2). In this molecular dynamics frame, the descending leucine substrate is shown near the site of intracellular opening, where the proposed TM1a rearrangement is indicated by arrows. The left and right panels show views parallel to the membrane and from the intracellular side, respectively. The dotted lines comprising the intracellular ends of the same TMs that surround the ex! iting substrate in the open-inward structure obtained through simulation (orange) yield a larger circumference than in the crystal structure (grey). * Figure 4: Effects of mutation and CMI on FRET histograms of LeuT-H7C/R86C and LeuT-239C/480C. FRET histograms from single-molecule traces obtained in the presence of 200 mM KCl for LeuT-H7C/R86C (left) and LeuT-239C/H480C (right) are shown in the context of the mutations R5A, Y268A or R30A, or the presence of 0.5 mM CMI. For clarity, fluorophore dark states have been computationally removed from all histograms (see Methods). Error bars are the standard deviation of 1,000 bootstrap samples of the FRET traces. * Figure 5: Long single-molecule trajectories reveal FRET transitions. Representative single-molecule traces from 160-ms images are shown for LeuT-H7C/R86C (left), LeuT-H7C/R86C/Y268A (centre), and LeuT-H7C/R86C/R5A (right) in 200 mM KCl (, , ), 200 mM NaCl (, , ) or 200 mM NaCl and 20 µM leucine (, , ). Cy3 (donor) and cy5 (acceptor) fluorescence are shown in green and red, respectively. FRET efficiency is shown in blue. One-dimensional histograms (, , ) represent the population data obtained in the presence of 200 mM KCl (grey bars), 200 mM NaCl (blue line), or 200 mM NaCl and 20 µM leucine (red line). Error bars are the standard deviation of 1,000 bootstrap samples of the FRET traces. Author information * Abstract * Author information * Supplementary information * Comments Primary authors * These authors contributed equally to this work. * Yongfang Zhao & * Daniel Terry Affiliations * Center for Molecular Recognition, Columbia University College of Physicians and Surgeons, 630 W. 168th, New York, New York 10032, USA * Yongfang Zhao & * Jonathan A. Javitch * Departments of Psychiatry, Columbia University College of Physicians and Surgeons, 630 W. 168th, New York, New York 10032, USA * Yongfang Zhao & * Jonathan A. Javitch * Division of Molecular Therapeutics, New York State Psychiatric Institute, New York, New York 10032, USA * Yongfang Zhao & * Jonathan A. Javitch * Department of Physiology and Biophysics, Weill Cornell Medical College, 1300 York Avenue, New York, New York 10021, USA * Daniel Terry, * Lei Shi, * Harel Weinstein & * Scott C. Blanchard * HRH Prince Alwaleed Bin Talal Bin Abdulaziz Alsaud Institute for Computational Biomedicine, Weill Cornell Medical College, 1300 York Avenue, New York, New York 10021, USA * Lei Shi & * Harel Weinstein * Department of Pharmacology, Columbia University College of Physicians and Surgeons, 630 W. 168th, New York, New York 10032, USA * Jonathan A. Javitch Contributions Y.Z. expressed, purified, labelled and functionally characterized the LeuT mutants. Y.Z. and D.T. designed, carried out, and analysed the single-molecule experiments. L.S. and H.W. designed and analysed the computational studies, which were carried out by L.S., S.C.B. and J.A.J. helped to design the biochemical and single-molecule experiments and, with L.S. and H.W., helped to interpret the data. All the authors contributed to writing and editing the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Jonathan A. Javitch (jaj2@columbia.edu) or * Scott C. Blanchard (scb2005@med.cornell.edu) Supplementary information * Abstract * Author information * Supplementary information * Comments Flash files * Supplementary Movie 1 (9.1M) This flash movie shows a side view of conformational changes in the simulated transport mechanism from a ~180 ns molecular dynamics simulation that induces and equilibrates the inward-open structure (orange) starting from the LeuT crystal structure (gray). The conformational changes agree with and are reflected in the results from the smFRET measurements. * Supplementary Movie 2 (9.1M) This flash movie shows an intracellular view of conformational changes in the simulated transport mechanism from a ~180 ns molecular dynamics simulation that induces and equilibrates the inward-open structure (orange) starting from the LeuT crystal structure (gray). The conformational changes agree with and are reflected in the results from the smFRET measurements. PDF files * Supplementary Information (1.2M) This file contains a Supplementary Discussion, Supplementary Figures 1-8 with legends, Supplementary Tables 1-3, Notes for Supplementary Movies 1-2 and a Reference. Additional data
• A white dwarf cooling age of 8 Gyr for NGC 6791 from physical separation processes
  - Nature 465(7295):194 (2010)
  Nature | Letter A white dwarf cooling age of 8 Gyr for NGC 6791 from physical separation processes * Enrique García-Berro1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Santiago Torres1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Leandro G. Althaus1, 3, 4 Search for this author in: * NPG journals * PubMed * Google Scholar * Isabel Renedo1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Pablo Lorén-Aguilar1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Alejandro H. Córsico3, 4 Search for this author in: * NPG journals * PubMed * Google Scholar * René D. Rohrmann5 Search for this author in: * NPG journals * PubMed * Google Scholar * Maurizio Salaris6 Search for this author in: * NPG journals * PubMed * Google Scholar * Jordi Isern2, 7 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:NatureVolume:465,Pages:194–196Date published:(13 May 2010)DOI:doi:10.1038/nature09045Received23 November 2009Accepted22 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 NGC 6791 is a well studied open cluster1 that it is so close to us that can be imaged down to very faint luminosities2. The main-sequence turn-off age (~8 Gyr) and the age derived from the termination of the white dwarf cooling sequence (~6 Gyr) are very different. One possible explanation is that as white dwarfs cool, one of the ashes of helium burning, 22Ne, sinks in the deep interior of these stars3, 4, 5. At lower temperatures, white dwarfs are expected to crystallize and phase separation of the main constituents of the core of a typical white dwarf (12C and 16O) is expected to occur6, 7. This sequence of events is expected to introduce long delays in the cooling times8, 9, but has not hitherto been proven. Here we report that, as theoretically anticipated5, 6, physical separation processes occur in the cores of white dwarfs, resolving the age discrepancy for NGC 6791. View full text Subject terms: * Astronomy * Astrophysics Figures at a glance * Figure 1: Colour–magnitude diagrams of the white dwarfs in NGC 6791. , A typical Monte Carlo realization of the colour–magnitude diagram of NGC 6791. Blue dots, synthetic white dwarfs obtained using the procedure outlined in the main text and, thus, incorporating the photometric errors. A total of ~850 white dwarfs with magnitude smaller than mF606W = 28.55 mag have been generated, the same number of white dwarfs observationally found1, 2, 10. Black dots, a theoretical white dwarf isochrone for 8 Gyr. Note the blue hook caused by the most massive white dwarfs of the cluster. Black lines, the observational selection area10; white dwarfs outside this area are not considered. , The observational white dwarf colour–magnitude diagram (ref. 2 and L. Bedin et al., personal communication). * Figure 2: White dwarf luminosity function of NGC 6791. Filled squares, the observational white dwarf luminosity function (error bars, ±1σ; ref. 2). Solid line, the average of 104 Monte Carlo realizations corresponding to the age (8 Gyr), metallicity (0.04) and distance modulus (13.44 mag) of NGC 6791. To illustrate the importance of physical separation processes, we also show the white dwarf luminosity function for the same age and assuming that no 22Ne sedimentation and no phase separation upon crystallization occur (dotted line). The theoretical luminosity function is shifted to lower luminosities (larger magnitudes) to an extent that is incompatible with the observational data. The distance modulus required to fit the observations would be 13.0 mag, a value considerably smaller than those observationally reported1, 2, 22. This distance modulus would imply a main-sequence turn-off age of 12 Gyr, worsening the age discrepancy24. Also shown at the top of the figure are the photometric error bars. Changes in the exponen! t of the initial mass function (of ±0.1) translate into small changes in the positions of the peaks (≤0.02 mag), well below the photometric errors (0.15 mag). As for the relationship between the mass of white dwarfs and the mass of their progenitors, the differences are also small (≤0.04 mag) when other recent relationships are adopted25. The same holds for reasonable choices of main-sequence lifetimes26 (in which case the differences are smaller than 0.02 mag) or the duration of the burst of star formation (≤0.04 mag when the duration of the burst is decreased to 0.1 Gyr). Author information * Author information * Comments Affiliations * Departament de Física Aplicada, Universitat Politècnica de Catalunya, c/Esteve Terrades 5, 08860 Castelldefels, Spain * Enrique García-Berro, * Santiago Torres, * Leandro G. Althaus, * Isabel Renedo & * Pablo Lorén-Aguilar * Institut d'Estudis Espacials de Catalunya, Ed. Nexus-201, c/Gran Capità 2-4, 08034 Barcelona, Spain * Enrique García-Berro, * Santiago Torres, * Isabel Renedo, * Pablo Lorén-Aguilar & * Jordi Isern * Facultad de Ciencias Astronómicas y Geofísicas, Universidad Nacional de La Plata, Paseo del Bosque s/n, 1900 La Plata, Argentina * Leandro G. Althaus & * Alejandro H. Córsico * Instituto de Astrofísica de La Plata (CCT La Plata), CONICET. 1900 La Plata, Argentina * Leandro G. Althaus & * Alejandro H. Córsico * Instituto de Ciencias Astronómicas, de la Tierra y del Espacio, CONICET, Av. de España 1512 (Sur) CC 49, 5400 San Juan, Argentina * René D. Rohrmann * Astrophysics Research Institute, Liverpool John Moores University, 12 Quays House, Birkenhead CH41 1LD, UK * Maurizio Salaris * Institut de Ciències de l'Espai (CSIC), Facultat de Ciències, Campus UAB, Torre C5-parell, 2a planta, 08193 Bellaterra, Spain * Jordi Isern Contributions E.G.-B. and L.G.A. conceived the study. E.G.-B. wrote the paper. J.I., P.L.-A. and E.G.-B. computed the theoretical expressions for the time delays introduced by the different separation processes. L.G.A., I.R., A.H.C. and E.G.-B. computed the cooling sequences. R.D.R. provided the appropriate colours. S.T., E.G.-B. and J.I. did the Monte Carlo simulations. M.S. computed the main-sequence evolutionary ages and errors. All authors discussed the results and made substantial contributions to the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Enrique García-Berro (garcia@fa.upc.edu) Additional data
• Time-resolved observation of coherent multi-body interactions in quantum phase revivals
  - Nature 465(7295):197 (2010)
  Nature | Letter Time-resolved observation of coherent multi-body interactions in quantum phase revivals * Sebastian Will1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Thorsten Best1 Search for this author in: * NPG journals * PubMed * Google Scholar * Ulrich Schneider1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Lucia Hackermüller1 Search for this author in: * NPG journals * PubMed * Google Scholar * Dirk-Sören Lühmann3 Search for this author in: * NPG journals * PubMed * Google Scholar * Immanuel Bloch1, 2, 4 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:NatureVolume:465,Pages:197–201Date published:(13 May 2010)DOI:doi:10.1038/nature09036Received01 February 2010Accepted17 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 Interactions lie at the heart of correlated many-body quantum phases1, 2, 3. Typically, the interactions between microscopic particles are described as two-body interactions. However, it has been shown that higher-order multi-body interactions could give rise to novel quantum phases with intriguing properties. So far, multi-body interactions have been observed as inelastic loss resonances in three- and four-body recombinations of atom–atom and atom–molecule collisions4, 5, 6. Here we demonstrate the presence of effective multi-body interactions7 in a system of ultracold bosonic atoms in a three-dimensional optical lattice, emerging through virtual transitions of particles from the lowest energy band to higher energy bands. We observe such interactions up to the six-body case in time-resolved traces of quantum phase revivals8, 9, 10, 11, using an atom interferometric technique that allows us to precisely measure the absolute energies of atom number states at a lattice sit! e. In addition, we show that the spectral content of these time traces can reveal the atom number statistics at a lattice site, similar to foundational experiments in cavity quantum electrodynamics that yield the statistics of a cavity photon field12. Our precision measurement of multi-body interaction energies provides crucial input for the comparison of optical-lattice quantum simulators with many-body quantum theory. View full text Subject terms: * Applied physics * Engineering Figures at a glance * Figure 1: Signature of multi-body interactions in quantum phase revivals. , A BEC loaded into a weak optical lattice forms a superfluid in which each atom is delocalized over several lattice sites. The quantum state at each site can be expressed as a superposition of Fock states, |n, with amplitudes cn. The number of blue balls in each state indicates the number of atoms, n. , For repulsive interactions, virtual transitions to higher lattice orbitals broaden the ground-state wavefunction at a lattice site depending on the atom number (orange solid lines) relative to the wavefunction in a non-interacting system (grey dashed lines). This gives rise to characteristic Fock state energies, which can be described by effective multi-body interactions. , Quantum phase revivals of a coherent state of interacting atoms in the multi-orbital system of a deep lattice well (blue solid line). The beat signal indicates coherent multi-body interactions. The dynamics are markedly different from the monochromatic evolution expected in a single-orbital model with a s! ingle two-body interaction energy, U (grey solid line). * Figure 2: Multi-orbital quantum phase revivals of atom number superposition states. , Collapse and revival dynamics of number-squeezed superposition states in a deep optical lattice. A BEC of about (1.9 ± 0.3) × 10587Rb atoms was adiabatically loaded into a VL = 8Erec lattice within 100 ms. Quantum phase evolution was induced by a non-adiabatic jump to a deep, VH = (41.0 ± 1.3)Erec, lattice, preserving superposition states with finite number fluctuations, an ensemble-averaged mean atom number of  ≈ 1.0 and a central mean atom number of  ≈ 2.5. Simultaneously with the lattice jump, the underlying harmonic confinement was instantaneously minimized to optimize the coherence time. The quantum phase dynamics show a beat-note signature resulting from coherent multi-body interactions. Each data point corresponds to a single run of the experiment. The solid line interpolates the data and serves as a guide to the eye. , Spectral analysis of the time trace () reveals the contributing frequencies. The solid line shows Gaussian fits to the ! peaks. Grey dashed lines display the frequencies corresponding to the single-orbital interaction energies U and 2U at a lattice depth of 41Erec. a.u., arbitrary units. * Figure 3: Multi-orbital energies and effective multi-body interactions. , Long collapse and revival traces were recorded under identical loading conditions (VL = 8Erec) but variable lattice depths, VH, during phase evolution. Numerical Fourier transforms of the time traces reveal the contributing frequencies of orders U/h (red circles) and 2U/h (blue circles), which have a typical uncertainty of ±50 Hz. The shading of the data points reflects the relative spectral weight (lighter, lower; darker, higher). The solid lines with grey shading indicate the theoretically expected frequencies at an s-wave scattering length of as = (102 ± 2)a0 (a0, Bohr radius), as derived for a basis set with 43 orbitals. Calculation using a smaller basis set, with 33 orbitals, yields slightly higher energies (dashed lines). The black dotted lines show the single-orbital interaction energies U and 2U of the Bose–Hubbard model. At low lattice depths, only the strongest peaks can be resolved, mainly as a result of smaller peak spacings. , Effective two-body (top! ), three-body (middle) and four-body (bottom) interaction strengths as derived from experiment and theory (as = (102 ± 2)a0, 43 orbitals). Error bars, 1 s.d. * Figure 4: Global number statistics on approaching the Mott insulator transition. , Multi-orbital quantum phase revivals in a deep, VH = 40Erec, lattice after adiabatic loading of (3.3 ± 0.3) × 10587Rb atoms into lattices with depths ranging from VL = 3Erec to 13Erec. The mean atom number of the individual traces differed by as little as ±1%. Although the coherence time in shallow lattices is significantly reduced, the visibility reliably shows dynamics down to the per cent level (inset). , The corresponding Fourier spectra reveal frequency contributions from Fock states containing up to six atoms. The peak positions agree with the theoretical predictions (dashed vertical lines) and are independent of VL. Number squeezing manifests itself both in reduced peak amplitudes and in a narrowing of the spectra for increasing VL. The solid lines show Gaussian fits to the peaks. Author information * Author information * Supplementary information * Comments Affiliations * Institut für Physik, Johannes Gutenberg-Universität, 55099 Mainz, Germany * Sebastian Will, * Thorsten Best, * Ulrich Schneider, * Lucia Hackermüller & * Immanuel Bloch * Fakultät für Physik, Ludwig-Maximilians-Universität, 80799 München, Germany * Sebastian Will, * Ulrich Schneider & * Immanuel Bloch * Institut für Theoretische Physik, Universität Hamburg, 20355 Hamburg, Germany * Dirk-Sören Lühmann * Max-Planck-Institut für Quantenoptik, 85748 Garching, Germany * Immanuel Bloch Contributions S.W. and T.B. carried out the measurements, S.W. performed the data analysis and D.-S.L. performed the numerical calculations. S.W. and I.B. wrote the manuscript with substantial contributions by all authors. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Sebastian Will (sebastian.will@lmu.de) Supplementary information * Author information * Supplementary information * Comments PDF files * Supplementary Information (275K) This file contains Supplementary Information and Data 1-4, Supplementary Figures 1-4 with legends and References. Additional data
• A proximity-based programmable DNA nanoscale assembly line
  - Nature 465(7295):202 (2010)
  Nature | Letter A proximity-based programmable DNA nanoscale assembly line * Hongzhou Gu1 Search for this author in: * NPG journals * PubMed * Google Scholar * Jie Chao2 Search for this author in: * NPG journals * PubMed * Google Scholar * Shou-Jun Xiao2 Search for this author in: * NPG journals * PubMed * Google Scholar * Nadrian C. Seeman1 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:NatureVolume:465,Pages:202–205Date published:(13 May 2010)DOI:doi:10.1038/nature09026Received30 October 2009Accepted12 February 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 Our ability to synthesize nanometre-scale chemical species, such as nanoparticles with desired shapes and compositions, offers the exciting prospect of generating new functional materials and devices by combining them in a controlled fashion into larger structures. Self-assembly can achieve this task efficiently, but may be subject to thermodynamic and kinetic limitations: reactants, intermediates and products may collide with each other throughout the assembly time course to produce non-target species instead of target species. An alternative approach to nanoscale assembly uses information-containing molecules such as DNA1 to control interactions and thereby minimize unwanted cross-talk between different components. In principle, this method should allow the stepwise and programmed construction of target products by linking individually selected nanoscale components—much as an automobile is built on an assembly line. Here we demonstrate that a nanoscale assembly line can ! be realized by the judicious combination of three known DNA-based modules: a DNA origami2 tile that provides a framework and track for the assembly process, cassettes containing three independently controlled two-state DNA machines that serve as programmable cargo-donating devices3, 4 and are attached4, 5 in series to the tile, and a DNA walker that can move on the track from device to device and collect cargo. As the walker traverses the pathway prescribed by the origami tile track, it sequentially encounters the three DNA devices, each of which can be independently switched between an 'ON' state, allowing its cargo to be transferred to the walker, and an 'OFF' state, in which no transfer occurs. We use three different types of gold nanoparticle species as cargo and show that the experimental system does indeed allow the controlled fabrication of the eight different products that can be obtained with three two-state devices. View full text Subject terms: * Materials science * Biochemistry * Biophysics * Methods * Materials Figures at a glance * Figure 1: The molecular assembly line and its operation. , The basic components of the system are the origami tile (shown as a tan outline), programmable two-state DNA machines inserted in series into the file (shown in blue, purple and green) and the walker (shown as a trigonal arrangement of DNA double helices in red). The machines have cargoes consisting respectively of a 5-nm gold particle (C1), a coupled pair of 5-nm particles (C2) and a 10-nm particle (C3) (indicated by green–brown dots), and their state can be PX (meaning ON or 'donate' cargo) or JX2 (meaning OFF or 'do not donate' cargo). In the example shown, the walker collects cargo from each machine. , Atomic force micrographs of the system corresponding to the process steps sketched as states (i)–(vi) in . Atomic force microscopy (AFM) was performed by tapping in air; this mode of AFM results in only the nanoparticles and the origami being visible, and the individual nanoparticle components are not individually resolved. Owing to the washing procedures bet! ween steps, the AFM images are not of the same individual assembly line. Scale bars, 50 nm. * Figure 2: Details of the walker, movement and cargo transfer. , Walker structure. The drawing on the left is a stick figure indicating the three hands (H1–H3) and four feet (F1–F4). The image on the right shows the strand structure. , Movement. Walker reactions are shown in the upper two images, and movement on the origami is shown in the lower two images. A-k binds Fk to the origami and FA-k is a fuel strand that removes A-k, undoing the corresponding binding. Foot-binding sites on the origami are labelled such that in its nth binding to the origami, Fk binds to site kn. Supplementary Fig. 5 shows the complete walker transit. , Cargo transfer. The PX state brings the arm of cassette one close to H1 (left), the brown toehold binds its complement (red; centre) and branch migration transfers the cargo strand to H1 (right). * Figure 3: The eight products of the assembly line. Roman numerals indicate the different pathways illustrated. , The eight possible products that can be generated through appropriate programming of the states of the three DNA machines. The walker is shown on the left, without cargo. Each DNA machine is shown twice: in the upper row in the OFF state, in which no cargo transfer takes place, and in the lower row in the ON state, in which cargo can be transferred to the walker. The different assembly trajectories are colour-coded as black (i), dark blue (ii), rose (iii), brown (iv), yellow/green (v), light blue (vi), green (vii) and purple (viii), giving the respective products shown on the right. , Schematics of the final state the system reaches for each of the eight assembly pathways. The states of the cassettes and the dispositions of the cargo species (attached to the robot arms or attached to the walker) are shown. , Transmission electron microscope images of the products generated in each of the assembly pathways. (Note t! hat transmission electron microscopy resolves the individual gold nanoparticles.) In each image, several products generated by the given pathway are visible. Scale bars, 50 nm. Author information * Author information * Supplementary information * Comments Affiliations * Department of Chemistry, New York University, New York, New York 10003, USA * Hongzhou Gu & * Nadrian C. Seeman * State Key Laboratory of Coordination Chemistry, School of Chemistry and Chemical Engineering, Nanjing National Laboratory of Microstructures, Nanjing University, Nanjing 210093, China * Jie Chao & * Shou-Jun Xiao Contributions H.G. and J.C. did the research, analysed data and wrote the paper. S.-J.X. analysed data and wrote the paper. N.C.S. designed the project, analysed data and wrote the paper. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Nadrian C. Seeman (ned.seeman@nyu.edu) Supplementary information * Author information * Supplementary information * Comments PDF files * Supplementary Information (4.1M) This file comprises Supplementary Figures S1- S12 with legends and Supplementary Table S1 as follows: origami design (figure S1) and sequence; cassette design and sequence (figures S2-S4); walker locomotion and sequence design (figure S5); nondenaturing gels of the cassettes and walker (S6); agarose gels of the DNA-nanoparticle conjugates (figure S7), AFM images of the movement of the walker on the origami (figure S8); AFM images of the dynamic switching of each cassette on the origami (figure S9); zoomed TEM images for statistical analysis (figures S10 and S11); the distance distribution between particles on the walker (figure S12), and the statistical analysis for the different additions (table S1). Additional data
• Molecular robots guided by prescriptive landscapes
  - Nature 465(7295):206 (2010)
  Nature | Letter Molecular robots guided by prescriptive landscapes * Kyle Lund1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Anthony J. Manzo3 Search for this author in: * NPG journals * PubMed * Google Scholar * Nadine Dabby4 Search for this author in: * NPG journals * PubMed * Google Scholar * Nicole Michelotti3, 5 Search for this author in: * NPG journals * PubMed * Google Scholar * Alexander Johnson-Buck3 Search for this author in: * NPG journals * PubMed * Google Scholar * Jeanette Nangreave1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Steven Taylor6 Search for this author in: * NPG journals * PubMed * Google Scholar * Renjun Pei6 Search for this author in: * NPG journals * PubMed * Google Scholar * Milan N. Stojanovic6, 7 Search for this author in: * NPG journals * PubMed * Google Scholar * Nils G. Walter3 Search for this author in: * NPG journals * PubMed * Google Scholar * Erik Winfree4, 8, 9 Search for this author in: * NPG journals * PubMed * Google Scholar * Hao Yan1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorsJournal name:NatureVolume:465,Pages:206–210Date published:(13 May 2010)DOI:doi:10.1038/nature09012Received31 March 2009Accepted11 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 Traditional robots1 rely for their function on computing, to store internal representations of their goals and environment and to coordinate sensing and any actuation of components required in response. Moving robotics to the single-molecule level is possible in principle, but requires facing the limited ability of individual molecules to store complex information and programs. One strategy to overcome this problem is to use systems that can obtain complex behaviour from the interaction of simple robots with their environment2, 3, 4. A first step in this direction was the development of DNA walkers5, which have developed from being non-autonomous6, 7 to being capable of directed but brief motion on one-dimensional tracks8, 9, 10, 11. Here we demonstrate that previously developed random walkers12—so-called molecular spiders that comprise a streptavidin molecule as an inert 'body' and three deoxyribozymes as catalytic 'legs'—show elementary robotic behaviour when i! nteracting with a precisely defined environment. Single-molecule microscopy observations confirm that such walkers achieve directional movement by sensing and modifying tracks of substrate molecules laid out on a two-dimensional DNA origami landscape13. When using appropriately designed DNA origami, the molecular spiders autonomously carry out sequences of actions such as 'start', 'follow', 'turn' and 'stop'. We anticipate that this strategy will result in more complex robotic behaviour at the molecular level if additional control mechanisms are incorporated. One example might be interactions between multiple molecular robots leading to collective behaviour14, 15; another might be the ability to read and transform secondary cues on the DNA origami landscape as a means of implementing Turing-universal algorithmic behaviour2, 16, 17. View full text Subject terms: * Biochemistry Figures at a glance * Figure 1: Deoxyribozyme-based molecular walker and origami prescriptive landscape. , The NICK3.4A3+1 spider consists of a streptavidin core, with a 20-base single-stranded DNA (green) that positions the spider at the start, and three deoxyribozyme legs. , The 8-17 deoxyribozyme cleaves its substrate at an RNA base, creating two shorter products (respectively 7 and 11 bases in length). Dissociation from these products allows legs to associate with the next substrate. , Spider actions: after release by a 27-base single-stranded DNA trigger, the spider follows the substrate track, turns and continues to a STOP site (red). , Schematic of the DNA origami landscape with positions A–E labelled; track EABD is shown with I indicating a topographical imaging marker. , A representative origami landscape showing the START position (green), the substrate track (brown), STOP and CONTROL sites (red), and a topographical imaging marker (blue). rA, ribonucleotide position at which cleavage occurs; dA, deoxyribonucleotide within non-chimeric and non-cleavable analogue of ! substrate at a STOP position. * Figure 2: Spider movement along three tracks and AFM images of the spider at the START, on the TRACK and at the STOP site. , ABD track. , EABC track. , ABD and EABC spider statistics before and 30 min after release. , EABD track. , EABD track with spider on control. , EABD product-only track. , EABD spider statistics before and 15, 30 and 60 min after release, and 60 min after release on the product-only track. All AFM images are 144 nm × 99.7 nm and the scale bars are 20 nm. N, number of origami with a single spider that were counted for the given sample. * Figure 3: AFM movie of spider movement. –, Schematics and AFM images of the spider moving along the EABD track 5 min (), 16 min (), 26 min () and 31 min () after TRIGGER was added. AFM images are 300 nm × 300 nm and the scale bars are 100 nm. * Figure 4: Spiders imaged on origami tracks in real time using super-resolution total-internal-reflection fluorescence microscopy. , Position–time trajectory of a selected spider (EAC 2, Cy3-labelled) on the EAC substrate track. The position as a function of time is represented by colour-coded dots (see Supplementary Information for details). A small green dot (upper right) represents the START site and a large red oval (lower left) represents the Cy5-labelled STOP site. ZnSO4 was added at time zero. , Displacement of the spider trajectory in panel from its initial position as a function of time. The green line represents displacement calculated from raw position measurements, and the black line represents the displacement calculated from a rolling 4-min average (Supplementary Information). σ, standard error of the mean of the raw displacement with respect to 4-min time bins. , Ensemble r.m.s. displacement of exemplary spiders on the EAC substrate track in the presence (red, corresponding to the 15 Tier 1 Spiders in Supplementary Fig. 29) and absence (black, 7 spiders) of Zn2+, with the corresponding! displacements used to calculate each ensemble r.m.s. displacement for each buffer condition (similarly coloured line graphs). , Ensemble r.m.s. displacement of spiders on EAC tracks satisfying simple filtering criteria. Curves are shown for spiders on the EAC substrate track (red, 85 spiders), the EAC product track with TRIGGER introduced to the sample 10–15 min before imaging (blue, 18 spiders) and the EAC product track with TRIGGER introduced 30–60 min before imaging (black, 29 spiders). The first two plots are fitted with a power-law function and the third is fitted with a straight line. Individual displacements are shown with colours corresponding to the respective ensemble r.m.s. displacement plots. All data were obtained in SSC buffer. Author information * Author information * Supplementary information * Comments Affiliations * Department of Chemistry and Biochemistry, Arizona State University, Tempe, Arizona 85287, USA * Kyle Lund, * Jeanette Nangreave & * Hao Yan * The Biodesign Institute, Arizona State University, Tempe, Arizona 85287, USA * Kyle Lund, * Jeanette Nangreave & * Hao Yan * Department of Chemistry, Single Molecule Analysis Group, University of Michigan, Ann Arbor, Michigan 48109, USA * Anthony J. Manzo, * Nicole Michelotti, * Alexander Johnson-Buck & * Nils G. Walter * Computation & Neural Systems, California Institute of Technology, Pasadena, California 91125, USA * Nadine Dabby & * Erik Winfree * Department of Physics, University of Michigan, Ann Arbor, Michigan 48109, USA * Nicole Michelotti * Division of Experimental Therapeutics, Department of Medicine, Columbia University, New York, New York 10032, USA * Steven Taylor, * Renjun Pei & * Milan N. Stojanovic * Department of Biomedical Engineering, Columbia University, New York, New York 10032, USA * Milan N. Stojanovic * Computer Science, California Institute of Technology, Pasadena, California 91125, USA * Erik Winfree * Bioengineering, California Institute of Technology, Pasadena, California 91125, USA * Erik Winfree Contributions AFM experiments were performed by K.L. (majority), J.N. and N.D.; analysis was performed by N.D., K.L., J.N. and S.T. and supervised by E.W. and H.Y. Fluorescence microscopy and particle tracking analysis were performed by A.J.M., N.M. and A.J.-B, supervised by N.G.W. Spiders were synthesized and purified, and their integrity was confirmed and monitored, by S.T. Surface plasmon resonance experiments were performed by R.P. Research coordination was by M.N.S. and materials transfer coordination was by S.T., J.N. and K.L. Experimental design and manuscript preparation received input from all authors. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Milan N. Stojanovic (mns18@columbia.edu) or * Nils G. Walter (nwalter@umich.edu) or * Erik Winfree (winfree@caltech.edu) or * Hao Yan (hao.yan@asu.edu) Supplementary information * Author information * Supplementary information * Comments PDF files * Supplementary Information (8.7M) This file contains a Supplementary Discussion, Supplementary Materials and Methods, Supplementary Figures 1-32 with legends, Supplementary Tables 1-4 and References. Additional data
• Long-term stability of global erosion rates and weathering during late-Cenozoic cooling
  - Nature 465(7295):211 (2010)
  Nature | Letter Long-term stability of global erosion rates and weathering during late-Cenozoic cooling * Jane K. Willenbring1 Search for this author in: * NPG journals * PubMed * Google Scholar * Friedhelm von Blanckenburg1 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorsJournal name:NatureVolume:465,Pages:211–214Date published:(13 May 2010)DOI:doi:10.1038/nature09044Received07 August 2009Accepted22 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 Over geologic timescales, CO2 is emitted from the Earth's interior and is removed from the atmosphere by silicate rock weathering and organic carbon burial. This balance is thought to have stabilized greenhouse conditions within a range that ensured habitable conditions1. Changes in this balance have been attributed to changes in topographic relief, where varying rates of continental rock weathering and erosion1, 2 are superimposed on fluctuations in organic carbon burial3. Geological strata provide an indirect yet imperfectly preserved record of this change through changing rates of sedimentation1, 2, 4. Widespread observations of a recent (0–5-Myr) fourfold increase in global sedimentation rates require a global mechanism to explain them4, 5, 6. Accelerated uplift and global cooling have been given as possible causes2, 4, 6, 7, but because of the links between rates of erosion and the correlated rate of weathering8, 9, an increase in the drawdown of CO2 that is predict! ed to follow may be the cause of global climate change instead2. However, globally, rates of uplift cannot increase everywhere in the way that apparent sedimentation rates do4, 10. Moreover, proxy records of past atmospheric CO2 provide no evidence for this large reduction in recent CO2 concentrations11, 12. Here we question whether this increase in global weathering and erosion actually occurred and whether the apparent increase in the sedimentation rate is due to observational biases in the sedimentary record13. As evidence, we recast the ocean dissolved 10Be/9Be isotope system as a weathering proxy spanning the past ~12 Myr (ref. 14). This proxy indicates stable weathering fluxes during the late-Cenozoic era. The sum of these observations shows neither clear evidence for increased erosion nor clear evidence for a pulse in weathered material to the ocean. We conclude that processes different from an increase in denudation caused Cenozoic global cooling, and that global ! cooling had no profound effect on spatially and temporally ave! raged weathering rates. View full text Subject terms: * Earth sciences * Geology * Geophysics * Climate science * Evolution Figures at a glance * Figure 1: Terrigenous sediment input into the oceans through the late-Cenozoic era and atmospheric CO2. Yellow bars show global terrigenous sediment accumulation in oceans5. Values are separated into bins with an interval of 5 Myr and appear to increase abruptly during the past 5 Myr. The CO2 data compilation is derived from a number of independent proxies and shows steady atmospheric concentrations from the mid-Miocene epoch to today (pre-industrial values) despite the observed increase in terrigenous sediment accumulation. Records include the stable boron isotopes in planktonic foraminifera (purple band), the stomatal distribution in the leaves of C3 plants (green band), the stable carbon isotopes in alkenones (blue band) and air trapped in ice cores from Antarctica (thin black line). Each colour band spans the associated data points and their uncertainties. See Supplementary Information for additional details, uncertainties and references. * Figure 2: Sediment accumulation rates and erosion rates as functions of geological time. Four representative environments in which erosion and sedimentation rates change with time. Values are plotted at age midpoints. Insets, same data plotted on a log–log scale. , Global values for ocean basin sediment accumulation5. The ellipse in the inset shows the modern riverine flux range22, which matches that expected for the flux plotted on a ~105–106-yr timescale. , Volumetric erosion rates for the past 10 Myr from the European (Eastern and Western) Alps19. Rates were estimated from measurements of sediment accumulation in basins around the Alps and were corrected for compaction. In the inset, average cosmogenic isotope measurements23 provide an estimate for recent millennial-scale denudation rates. , Mass accumulation rates from 18 mostly offshore sedimentary basins in Asia after the initial India–Asia collision20. , Global Pliocene-to-Cambrian land and ocean accumulations (by volume)21 and Quaternary ocean accumulation5. All rock volumes were translated to de! nudation rate by normalizing for the area of continents that were exposed above sea level at that time22. All four environments share a common exponent of -0.5 (equation (1)), which is consistent with processes of stochastic deposition and erosion within the sedimentary deposits10, 13. * Figure 3: Palaeo-ocean dissolved 10Be/9Be ratios as weathering proxies. Data from Pacific Ocean marine cores (RC12-65, violet circles) and hydrogenous ferromanganese crusts (F7-86-HW/CD29-2, blue diamonds; F10-89-CP/D11-1, white squares; VA13-2/KD237, green triangles; Nova IX/D137-01, grey squares; F10-89-CP/D27-2-1, brown/green diamonds) () and from the Atlantic Ocean hydrogenous ferromanganese crusts (ALV-539, yellow triangles; BM-1969.05, orange squares) and the Arctic Ocean marine core (ACEX, pink circles) (). See Supplementary Information for data and references. Most individual measurements have a maximum 2-σ analytical error of ~10%, with lower uncertainty for recent measurements. Several outlier measurements within the data set have greater uncertainties. Because 10Be decays with a half-life of 1.39 Myr, the original 10Be/9Be ratio at the time of deposition was calculated by assuming constant crust growth rates or sediment accumulation rates and correcting for decayed 10Be for each sample interval14. An apparent circularity in this ap! proach can be discounted because 10Be-derived Pacific Fe–Mn crust growth rates agree with crust growth rates from Os isotope stratigraphy; decay-corrected Pacific Fe–Mn crust 10Be/9Be ratios agree with the corrected deep-sea core RC12-65 10Be/9Be ratios, where magnetostratigraphy yields an independent age estimate; and ratios agree with each other within an ocean basin and also with young Fe–Mn surfaces within these basins28. These high-fidelity records of dissolved 9Be and meteoric cosmogenic 10Be in the open oceans imply ratios that fluctuate about a mean of 1 × 10-7 for Pacific sites and 0.5 × 10-7 for Atlantic and Arctic sites28 (shown as horizontal bands shaded blue in and pink in ). A fourfold increase in the recent flux of 9Be-bearing terrigenous material (Fig. 1) would cause a decreasing trend in the ratio towards recent time, which is not observed in the three ocean basins sampled through time here. See Supplementary Information for additional detai! ls. Author information * Author information * Supplementary information * Comments Affiliations * Deutsches GeoForschungsZentrum GFZ, Section 3.4: Earth Surface Geochemistry, Telegrafenberg, D-14473 Potsdam, Germany * Jane K. Willenbring & * Friedhelm von Blanckenburg Contributions J.K.W. and F.v.B. contributed equally to every aspect of the study. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Friedhelm von Blanckenburg (fvb@gfz-potsdam.de) or * Jane K. Willenbring (jane.willenbring@sas.upenn.edu) J.K.W. is at the Department of Earth & Environmental Sciences, University of Pennsylvania, from July 2010. Supplementary information * Author information * Supplementary information * Comments PDF files * Supplementary Information (1.3M) This file contains Supplementary Data, which includes a schematic diagram, References, Supplementary Figures A1-A5 with legends and Supplementary Tables A1-A2. Additional data
• Ordovician faunas of Burgess Shale type
  - Nature 465(7295):215 (2010)
  Nature | Letter Ordovician faunas of Burgess Shale type * Peter Van Roy1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Patrick J. Orr2 Search for this author in: * NPG journals * PubMed * Google Scholar * Joseph P. Botting3 Search for this author in: * NPG journals * PubMed * Google Scholar * Lucy A. Muir4 Search for this author in: * NPG journals * PubMed * Google Scholar * Jakob Vinther1 Search for this author in: * NPG journals * PubMed * Google Scholar * Bertrand Lefebvre5 Search for this author in: * NPG journals * PubMed * Google Scholar * Khadija el Hariri6 Search for this author in: * NPG journals * PubMed * Google Scholar * Derek E. G. Briggs1, 7 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorsJournal name:NatureVolume:465,Pages:215–218Date published:(13 May 2010)DOI:doi:10.1038/nature09038Received22 January 2010Accepted22 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 renowned soft-bodied faunas of the Cambrian period, which include the Burgess Shale, disappear from the fossil record in the late Middle Cambrian, after which the Palaeozoic fauna1 dominates. The disappearance of faunas of Burgess Shale type curtails the stratigraphic record of a number of iconic Cambrian taxa. One possible explanation for this loss is a major extinction2, 3, but more probably it reflects the absence of preservation of similar soft-bodied faunas in later periods4. Here we report the discovery of numerous diverse soft-bodied assemblages in the Lower and Upper Fezouata Formations (Lower Ordovician) of Morocco, which include a range of remarkable stem-group morphologies normally considered characteristic of the Cambrian. It is clear that biotas of Burgess Shale type persisted after the Cambrian and are preserved where suitable facies occur. The Fezouata biota provides a link between the Burgess Shale communities and the early stages of the Great Ordovician ! Biodiversification Event. View full text Subject terms: * Earth sciences * Evolution * Palaeontology * Zoology Figures at a glance * Figure 1: Exceptionally preserved Burgess Shale-type organisms from the Early Ordovician Fezouata biota. , Demosponge Pirania auraeum19, top of Lower Fezouata Formation (CAMSM X 50156.1a). , Choiid demosponge, top of Lower Fezouata Formation (YPM 226567). , Annelid worm, top of Lower Fezouata Formation (YPM 226538). , Organism showing possible similarities to halkieriids, Upper Fezouata Formation (YPM 227515). , Possible armoured lobopod, Upper Fezouata Formation (YPM 227516). , Thelxiope-like arthropod, Upper Fezouata Formation (YPM 226544). , Marrellomorph arthropod, probably belonging to the genus Furca, Upper Fezouata Formation (MHNT.PAL.2007.39.80.1). , Skaniid arthropod, Upper Fezouata Formation (YPM 226539). , Spinose arthropod appendage apparatus consisting of six overlapping elements, top of Lower Fezouata Formation (YPM 226559). * Figure 2: Exceptionally preserved post-Cambrian elements of the Fezouata biota. , Aglaspidid arthropod Tremaglaspis, Upper Fezouata Formation (MHNT.PAL.2007.39.92.1). , Cheloniellid arthropod, Upper Fezouata Formation (NMS G 2004.2.1). , Possible stalked barnacle, Upper Fezouata Formation (YPM 227519). , Xiphosuran with fully segmented opisthosoma, top of Lower Fezouata Formation (MHNT.PAL.2007.39.43.2). , Xiphosurid with fused preabdomen, Upper Fezouata Formation (YPM 227586). Author information * Author information * Supplementary information * Comments Affiliations * Department of Geology and Geophysics, Yale University, PO Box 208109, New Haven, Connecticut 06520, USA * Peter Van Roy, * Jakob Vinther & * Derek E. G. Briggs * UCD School of Geological Sciences, University College Dublin, Belfield, Dublin 4, Ireland * Peter Van Roy & * Patrick J. Orr * Leeds Museum Discovery Centre, Carlisle Road, Leeds LS10 1LB, UK * Joseph P. Botting * 42 Birkhouse Lane, Moldgreen, Huddersfield HD5 8BE, UK * Lucy A. Muir * UMR CNRS 5125 PEPS, bât. Géode, Université Lyon 1, Campus de la Doua, 2 Rue Dubois, F-69622 Villeurbanne cedex, France * Bertrand Lefebvre * Département Sciences de la Terre, Faculté des Sciences et Techniques-Guéliz, Université Cadi Ayyad, Avenue Abdelkrim el Khattabi BP 549, 40000 Marrakech, Morocco * Khadija el Hariri * Yale Peabody Museum of Natural History, Yale University, New Haven, Connecticut 06520, USA * Derek E. G. Briggs Contributions All authors carried out field work and contributed to the interpretation of the fossils. P.V.R., P.J.O., J.P.B. and D.E.G.B. wrote the paper with input from the other authors. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Peter Van Roy (peter.vanroy@yale.edu) or * Derek E. G. Briggs (derek.briggs@yale.edu) Supplementary information * Author information * Supplementary information * Comments PDF files * Supplementary Figures (9.3M) This file contains Supplementary Figures S1-S3 with legends. Additional data
• A formal test of the theory of universal common ancestry
  - Nature 465(7295):219 (2010)
  Nature | Letter A formal test of the theory of universal common ancestry * Douglas L. Theobald1 Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:NatureVolume:465,Pages:219–222Date published:(13 May 2010)DOI:doi:10.1038/nature09014Received28 August 2009Accepted17 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 Universal common ancestry (UCA) is a central pillar of modern evolutionary theory1. As first suggested by Darwin2, the theory of UCA posits that all extant terrestrial organisms share a common genetic heritage, each being the genealogical descendant of a single species from the distant past3, 4, 5, 6. The classic evidence for UCA, although massive, is largely restricted to 'local' common ancestry—for example, of specific phyla rather than the entirety of life—and has yet to fully integrate the recent advances from modern phylogenetics and probability theory. Although UCA is widely assumed, it has rarely been subjected to formal quantitative testing7, 8, 9, 10, and this has led to critical commentary emphasizing the intrinsic technical difficulties in empirically evaluating a theory of such broad scope1, 5, 8, 9, 11, 12, 13, 14, 15. Furthermore, several researchers have proposed that early life was characterized by rampant horizontal gene transfer, leading some to que! stion the monophyly of life11, 14, 15. Here I provide the first, to my knowledge, formal, fundamental test of UCA, without assuming that sequence similarity implies genetic kinship. I test UCA by applying model selection theory5, 16, 17 to molecular phylogenies, focusing on a set of ubiquitously conserved proteins that are proposed to be orthologous. Among a wide range of biological models involving the independent ancestry of major taxonomic groups, the model selection tests are found to overwhelmingly support UCA irrespective of the presence of horizontal gene transfer and symbiotic fusion events. These results provide powerful statistical evidence corroborating the monophyly of all known life. View full text Subject terms: * Evolution * Molecular biology * Organismal biology * Mathematics * Statistics Figures at a glance * Figure 1: Selected class I evolutionary hypotheses, excluding HGT. , The model ABE, representing UCA of all taxa in the three domains of life. , A competing multiple-ancestry model, AE+B, representing common ancestry of Archaea and Eukarya, but an independent ancestry for Bacteria. Trees shown are actual maximum likelihood estimates, with branch lengths proportional to the number of sequence substitutions. * Figure 2: Selected class II evolutionary hypotheses, including HGT. , The reticulated model [ABE]II, representing UCA. , A competing network model of multiple ancestry, [AE+B]II, representing common ancestry of Archaea and Eukarya, but a separate ancestry for Bacteria. Models are shown as phylogenetic networks (reticulate trees). The phylogenetic networks are derived from the maximum likelihood estimates of the 23 individual protein phylogenies using the evolutionary model parameters shown for ABE and AE+B in Table 1. Author information * Author information * Supplementary information * Comments Affiliations * Department of Biochemistry, Brandeis University, Waltham, Massachusetts 01778, USA * Douglas L. Theobald Competing financial interests The author declares no competing financial interests. Corresponding author Correspondence to: * Douglas L. Theobald (dtheobald@brandeis.edu) Supplementary information * Author information * Supplementary information * Comments PDF files * Supplementary Information (352K) This file contains Supplementary Tables S1-S12, Supplementary Equations and Discussion 2.1-2.3, Supplementary Figures S1-S2 with legends, Supplementary Methods and Results 3.1-3.4, Supplementary Notes 4.1-4.3 and References. Additional data
• Mutations of optineurin in amyotrophic lateral sclerosis
  Maruyama H Morino H Ito H Izumi Y Kato H Watanabe Y Kinoshita Y Kamada M Nodera H Suzuki H Komure O Matsuura S Kobatake K Morimoto N Abe K Suzuki N Aoki M Kawata A Hirai T Kato T Ogasawara K Hirano A Takumi T Kusaka H Hagiwara K Kaji R Kawakami H - Nature 465(7295):223 (2010)
  Nature | Letter Mutations of optineurin in amyotrophic lateral sclerosis * Hirofumi Maruyama1 Search for this author in: * NPG journals * PubMed * Google Scholar * Hiroyuki Morino1 Search for this author in: * NPG journals * PubMed * Google Scholar * Hidefumi Ito2, 17 Search for this author in: * NPG journals * PubMed * Google Scholar * Yuishin Izumi3 Search for this author in: * NPG journals * PubMed * Google Scholar * Hidemasa Kato4 Search for this author in: * NPG journals * PubMed * Google Scholar * Yasuhito Watanabe5 Search for this author in: * NPG journals * PubMed * Google Scholar * Yoshimi Kinoshita2 Search for this author in: * NPG journals * PubMed * Google Scholar * Masaki Kamada1, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Hiroyuki Nodera3 Search for this author in: * NPG journals * PubMed * Google Scholar * Hidenori Suzuki6 Search for this author in: * NPG journals * PubMed * Google Scholar * Osamu Komure7 Search for this author in: * NPG journals * PubMed * Google Scholar * Shinya Matsuura8 Search for this author in: * NPG journals * PubMed * Google Scholar * Keitaro Kobatake9 Search for this author in: * NPG journals * PubMed * Google Scholar * Nobutoshi Morimoto10 Search for this author in: * NPG journals * PubMed * Google Scholar * Koji Abe10 Search for this author in: * NPG journals * PubMed * Google Scholar * Naoki Suzuki11 Search for this author in: * NPG journals * PubMed * Google Scholar * Masashi Aoki11 Search for this author in: * NPG journals * PubMed * Google Scholar * Akihiro Kawata12 Search for this author in: * NPG journals * PubMed * Google Scholar * Takeshi Hirai12 Search for this author in: * NPG journals * PubMed * Google Scholar * Takeo Kato13 Search for this author in: * NPG journals * PubMed * Google Scholar * Kazumasa Ogasawara14 Search for this author in: * NPG journals * PubMed * Google Scholar * Asao Hirano15 Search for this author in: * NPG journals * PubMed * Google Scholar * Toru Takumi5 Search for this author in: * NPG journals * PubMed * Google Scholar * Hirofumi Kusaka2 Search for this author in: * NPG journals * PubMed * Google Scholar * Koichi Hagiwara16 Search for this author in: * NPG journals * PubMed * Google Scholar * Ryuji Kaji3 Search for this author in: * NPG journals * PubMed * Google Scholar * Hideshi Kawakami1 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:NatureVolume:465,Pages:223–226Date published:(13 May 2010)DOI:doi:10.1038/nature08971Received17 August 2009Accepted02 March 2010Published online28 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 Amyotrophic lateral sclerosis (ALS) has its onset in middle age and is a progressive disorder characterized by degeneration of motor neurons of the primary motor cortex, brainstem and spinal cord1. Most cases of ALS are sporadic, but about 10% are familial. Genes known to cause classic familial ALS (FALS) are superoxide dismutase 1 (SOD1)2, ANG encoding angiogenin3, TARDP encoding transactive response (TAR) DNA-binding protein TDP-43 (ref. 4) and fused in sarcoma/translated in liposarcoma (FUS, also known as TLS)5, 6. However, these genetic defects occur in only about 20–30% of cases of FALS, and most genes causing FALS are unknown. Here we show that there are mutations in the gene encoding optineurin (OPTN), earlier reported to be a causative gene of primary open-angle glaucoma (POAG)7, in patients with ALS. We found three types of mutation of OPTN: a homozygous deletion of exon 5, a homozygous Q398X nonsense mutation and a heterozygous E478G missense mutation within its ! ubiquitin-binding domain. Analysis of cell transfection showed that the nonsense and missense mutations of OPTN abolished the inhibition of activation of nuclear factor kappa B (NF-κB), and the E478G mutation revealed a cytoplasmic distribution different from that of the wild type or a POAG mutation. A case with the E478G mutation showed OPTN-immunoreactive cytoplasmic inclusions. Furthermore, TDP-43- or SOD1-positive inclusions of sporadic and SOD1 cases of ALS were also noticeably immunolabelled by anti-OPTN antibodies. Our findings strongly suggest that OPTN is involved in the pathogenesis of ALS. They also indicate that NF-κB inhibitors could be used to treat ALS and that transgenic mice bearing various mutations of OPTN will be relevant in developing new drugs for this disorder. View full text Subject terms: * Neuroscience * Genetics * Genomics Figures at a glance * Figure 1: Exon 5 deletion, nonsense and missense mutations of the OPTN gene. , Family 1. The filled circle or square indicate the affected individuals; the arrows indicate the probands. , Agarose gel electrophoretogram. Subject 1 (V-1) and subject 2 (V-2) showed lack of exon 5 PCR product and shortened product of exon 4 to intron 5. , Chromatogram with OPTN deletion of exon 5 and schematic structure of deleted gene. , Families 2 and 3. Dots indicate heterozygous carriers. , Chromatograms from index subjects with OPTN mutation of c.1502 C>T. Homozygous mutation is in red, and the mutation is indicated by using the single-letter amino-acid code. , Family 4. *DNA sample could not be obtained. Numerals show the age at death. , Chromatograms from index subjects with the OPTN mutation of c.1743A>G. The heterozygous mutation is marked by the square. * Figure 2: Influence of OPTN mutations. , Luciferase assay to assess the ability of various OPTNs to inhibit activation of NF-κB. The wild type and E50K have a similar NF-κB activation-inhibiting effect, whereas mock, Q398X and E478G types lack this effect. Error bars, standard deviations of triplicate assays. , Localization of OPTN. Flag is the white signals in a–c and red signals in g–i. GM130 is the white signals in d–f and green signals in g–i. The wild type shows many fluorescent granules closely localized with the Golgi apparatus. E478G OPTN shows a reduced number of granules, and rarely co-localized with the Golgi apparatus. E50K OPTN granules have become large and closely localized with the Golgi apparatus. Scale bar, 10 μm. * Figure 3: Identification of OPTN in distinctive intracytoplasmic inclusions of subjects with ALS. –, Neuropathology of the lumbar spinal cord from subject 5. Klüver-Barrera () show loss of myelin from the corticospinal tract (arrow) and loss of motor neurons from the anterior horn (arrowhead). The cytoplasm of the remaining motor neurons contains an amorphous eosinophilic region (, arrow). H&E, haematoxylin and eosin. The same neuron was re-stained with the anti-OPTN antibody (, arrow). The eosinophilic retention occasionally appears to form a hyaline inclusion (, arrow), which is intensely immunolabelled with the anti-OPTN antibody (, arrow). –, Round hyaline inclusions of subjects with SALS (, ) are immunolabelled with anti-OPTN-C and anti-OPTN-I antibodies ( and , respectively). The sections were re-stained with anti-ubiquitin (Ub) antibodies (, ). –, Skein-like inclusions of patients with SALS are reactive with the anti-OPTN-I and anti-OPTN-C antibodies (, ). Re-staining of with the anti-ubiquitin antibody () and with anti-TDP-43 antibody (). –, Lewy-body-li! ke hyaline inclusion of a patient with FALS, stained with haematoxylin and eosin (), anti-OPTN-C antibody () and SOD1 antibody (). Scale bars, 200 µm (), 20 µm (–). Author information * Author information * Supplementary information * Comments Affiliations * Department of Epidemiology, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima 734-8553, Japan * Hirofumi Maruyama, * Hiroyuki Morino, * Masaki Kamada & * Hideshi Kawakami * Department of Neurology, Kansai Medical University, Moriguchi 570-8506, Japan * Hidefumi Ito, * Yoshimi Kinoshita & * Hirofumi Kusaka * Department of Clinical Neuroscience, University of Tokushima Graduate School, Tokushima 770-8503, Japan * Yuishin Izumi, * Masaki Kamada, * Hiroyuki Nodera & * Ryuji Kaji * Division of Developmental Biology, Research Center for Genomic Medicine, Saitama Medical University, Saitama 350-1241, Japan * Hidemasa Kato * Laboratory of Integrative Bioscience, Hiroshima University Graduate School of Biomedical Sciences, Hiroshima 734-8553, Japan * Yasuhito Watanabe & * Toru Takumi * Faculty of Human Science, Hiroshima Bunkyo Women's University, Hiroshima 731-0295, Japan * Hidenori Suzuki * South Osaka Neurosurgical Hospital, Osakasayama 589-0011, Japan * Osamu Komure * Department of Genetics and Cell Biology, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima 734-8553, Japan * Shinya Matsuura * Department of Neurology, Kobatake Hospital, Fukuyama 720-1142, Japan * Keitaro Kobatake * Department of Neurology, Okayama University, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama 700-8558, Japan * Nobutoshi Morimoto & * Koji Abe * Department of Neurology, Tohoku University School of Medicine, Sendai 980-8574, Japan * Naoki Suzuki & * Masashi Aoki * Department of Neurology, Tokyo Metropolitan Neurological Hospital, Fuchu, Tokyo 183-0042, Japan * Akihiro Kawata & * Takeshi Hirai * Department of Neurology, Haematology, Metabolism, Endocrinology and Diabetology, Yamagata University Faculty of Medicine, Yamagata 990-9585, Japan * Takeo Kato * Department of Pathology, School of Medicine, Shiga University of Medical Science, Ohtsu 520-2192, Japan * Kazumasa Ogasawara * Division of Neuropathology, Department of Pathology, Montefiore Medical Center, New York, New York 10467-2490, USA * Asao Hirano * Department of Respiratory Medicine, Saitama Medical University, Saitama 350-0495, Japan * Koichi Hagiwara * Present address: Department of Neurology, Kyoto University Graduate School of Medicine, Kyoto 606-8507, Japan. * Hidefumi Ito Contributions H. Kawakami designed and supervised the study. H.Mo. and K.H. extracted candidate genes. H.Ma. and M.K. performed sequencing analysis. H.Ma., H.Mo., Y.W., T.T., S.M., H. Kawakami and H.S. conducted molecular biological analysis. H.I., Y.K., H. Ku., H. Kato, K.O. and A.H. performed pathological analysis and provided pathological samples. Y.I., H.N., R.K., O.K., N.M., K.A., A.K., T.H, T.K., M.A., N.S. and K.K. collected clinical information and samples. H. Kawakami, H.Ma., H.I. and K.H. wrote the paper. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Hideshi Kawakami (hkawakam@hiroshima-u.ac.jp) Supplementary information * Author information * Supplementary information * Comments PDF files * Supplementary Information (7.1M) This file contains Supplementary Tables 2 and 4, Supplementary Figures 1-9 with legends, Supplementary Patient Information and Supplementary Notes and References. * Supplementary Table 1 (253K) This file contains the SNP typing of subjects 1, 2, 3 and 4. * Supplementary Table 3 (641K) This file contains the SNP typing of subjects 5, 6, 7 and 8. Additional data
• Induction of tumour immunity by targeted inhibition of nonsense-mediated mRNA decay
  - Nature 465(7295):227 (2010)
  Nature | Letter Induction of tumour immunity by targeted inhibition of nonsense-mediated mRNA decay * Fernando Pastor1 Search for this author in: * NPG journals * PubMed * Google Scholar * Despina Kolonias1 Search for this author in: * NPG journals * PubMed * Google Scholar * Paloma H. Giangrande2 Search for this author in: * NPG journals * PubMed * Google Scholar * Eli Gilboa1 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:NatureVolume:465,Pages:227–230Date published:(13 May 2010)DOI:doi:10.1038/nature08999Received21 October 2009Accepted02 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 main reason why tumours are not controlled by the immune system is that, unlike pathogens, they do not express potent tumour rejection antigens (TRAs). Tumour vaccination aims at stimulating a systemic immune response targeted to, mostly weak, antigens expressed in the disseminated tumour lesions. Main challenges in developing effective vaccination protocols are the identification of potent and broadly expressed TRAs1, 2, 3 and effective adjuvants to stimulate a robust and durable immune response4, 5, 6. Here we describe an alternative approach in which the expression of new, and thereby potent, antigens are induced in tumour cells by inhibiting nonsense-mediated messenger RNA decay (NMD)7, 8, 9, 10. Small interfering RNA (siRNA)-mediated inhibition of NMD in tumour cells led to the expression of new antigenic determinants and their immune-mediated rejection. In subcutaneous and metastatic tumour models, tumour-targeted delivery of NMD factor-specific siRNAs conjugated t! o oligonucleotide aptamer ligands led to significant inhibition of tumour growth that was superior to that of vaccination with granulocyte–macrophage colony-stimulating factor (GM-CSF)-expressing irradiated tumour cells11, and could be further enhanced by co-stimulation. Tumour-targeted NMD inhibition forms the basis of a simple, broadly useful, and clinically feasible approach to enhance the antigenicity of disseminated tumours leading to their immune recognition and rejection. The cell-free chemically synthesized oligonucleotide backbone of aptamer–siRNAs reduces the risk of immunogenicity and enhances the feasibility of generating reagents suitable for clinical use. View full text Subject terms: * Cancer * Immunology * Molecular biology * Therapeutics Figures at a glance * Figure 1: Expression of Upf2 or Smg1 shRNA in CT26 tumour cells leads to immune-mediated inhibition of tumour growth. , Intratumoral accumulation of OVA-specific OT-I T cells in response to NMD inhibition. B16/F10 tumour cells transduced with shRNA-encoding lentiviral vectors (described in Supplementary Fig. 1a) were stably transfected with an NMD reporter plasmid (described in Supplementary Fig. 1b) containing the class I-restricted epitope of chicken ovalbumin (OVA). Mice were implanted subcutaneously with parental tumour cells (wild-type (WT) B16) or with the lentivirus-transduced tumour cells, and either received or did not receive doxycycline in their drinking water. When tumours became palpable, mice were injected with either OT-I or Pmel-1 transgenic CD8+ T cells (three mice per group). Six days later, tumours were excised and analysed for OT-I and Pmel-1 T-cell content by flow cytometry. Ctrl, control. n = 2 , Balb/c mice were implanted subcutaneously with CT26 tumour cells stably transduced with the shRNA inducible lentiviral vector encoding Smg1, Upf2 and control shRNA (ten mice p! er group). Each group was divided into two subgroups receiving (filled circles) or not receiving (open circles) doxycycline in the drinking water. n = 2. , Same as except that tumour cells were injected into immune-deficient nude mice. n = 1. * Figure 2: Inhibition of tumour growth in mice treated with PSMA aptamer targeted Upf2 and Smg1 siRNAs. , Balb/c mice were implanted subcutaneously with PSMA-CT26 tumour cells and 3 days later injected via the tail vein with PBS (filled circles) or with PSMA aptamer–siRNA conjugates (open circles, control siRNA; open squares, Upf2 siRNA; filled squares, Smg1 siRNA) (5 mice per group). n = 2. , C57BL/6 mice were implanted with PSMA-B16/F10 tumour cells by tail vein injection, and 5 days later were injected with PSMA aptamer–siRNA conjugates (ten mice per group). Metastatic load was determined by measuring lung weight at the time of euthanization. n = 2. , Combination immunotherapy using NMD inhibition and 4-1BB co-stimulation. PSMA-CT26 tumour-bearing mice (five mice per group) were treated with various combinations of PSMA aptamer conjugated to Smg1 or control siRNA and an agonistic or co-stimulation-deficient 4-1BB aptamer dimer26 (mut4-1BB) and monitored for tumour growth. n = 1. * Figure 3: PSMA aptamer–Smg1 siRNA rejection of PSMA-expressing, but not parental, CT26 tumour cells. , Mice were co-implanted subcutaneously with PSMA-expressing (left flank) and parental (right flank) CT26 tumour cells and injected with PSMA aptamer–Smg1 siRNA via the tail vein. , Fifteen days after tumour inoculation, 32P-labelled aptamer–siRNA was injected, and 3 or 24 h later tumours were excised and the 32P content determined. n = 3. , Three days after tumour inoculation, mice were injected with aptamer–siRNA conjugate (eight mice per group) as described in Fig. 2a and tumour growth was monitored. Open circles, parental CT26; filled circles, PSMA-CT26. n = 2. * Figure 4: Comparison of PSMA aptamer–Smg1 siRNA treatment to vaccination with GM-CSF expressing irradiated tumour cells. C57BL/6 mice were injected intravenously with B16/F10 tumour cells and treated with PSMA aptamer–siRNA conjugates starting at day 5 as described in Fig. 2b, or vaccinated with GM-CSF-expressing irradiated B16/F10 tumour cells (GVAX) starting at days (D) 1 or 5 using the protocol described previously29. n = 1. Author information * Author information * Supplementary information * Comments Affiliations * Department of Microbiology & Immunology, Dodson Interdisciplinary Immunotherapy Institute, University of Miami Miller School of Medicine Miami, Florida 33134, USA * Fernando Pastor, * Despina Kolonias & * Eli Gilboa * Department of Internal Medicine and Department of Radiation Oncology, Molecular and Cellular Biology Program, University of Iowa, Iowa City, Iowa 52242, USA * Paloma H. Giangrande Contributions F.P. suggested the approach and was responsible for designing the aptamer–siRNA conjugates and interpreting the results, D.K. was responsible for the mouse studies, P.H.G. helped design the aptamer–siRNA conjugates, and E.G. oversaw experimental design, data analysis, and wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Eli Gilboa (egilboa@med.miami.edu) Supplementary information * Author information * Supplementary information * Comments PDF files * Supplementary information (1.4M) This file contains Supplementary Figures 1-8 with legends, a Supplementary Discussion and References. Additional data
• Native GABAB receptors are heteromultimers with a family of auxiliary subunits
  - Nature 465(7295):231 (2010)
  Nature | Letter Native GABAB receptors are heteromultimers with a family of auxiliary subunits * Jochen Schwenk1, 7 Search for this author in: * NPG journals * PubMed * Google Scholar * Michaela Metz2, 7 Search for this author in: * NPG journals * PubMed * Google Scholar * Gerd Zolles1, 7 Search for this author in: * NPG journals * PubMed * Google Scholar * Rostislav Turecek2, 6, 7 Search for this author in: * NPG journals * PubMed * Google Scholar * Thorsten Fritzius2 Search for this author in: * NPG journals * PubMed * Google Scholar * Wolfgang Bildl1 Search for this author in: * NPG journals * PubMed * Google Scholar * Etsuko Tarusawa4 Search for this author in: * NPG journals * PubMed * Google Scholar * Akos Kulik4 Search for this author in: * NPG journals * PubMed * Google Scholar * Andreas Unger4 Search for this author in: * NPG journals * PubMed * Google Scholar * Klara Ivankova2 Search for this author in: * NPG journals * PubMed * Google Scholar * Riad Seddik2 Search for this author in: * NPG journals * PubMed * Google Scholar * Jim Y. Tiao2 Search for this author in: * NPG journals * PubMed * Google Scholar * Mathieu Rajalu2 Search for this author in: * NPG journals * PubMed * Google Scholar * Johana Trojanova6 Search for this author in: * NPG journals * PubMed * Google Scholar * Volker Rohde3 Search for this author in: * NPG journals * PubMed * Google Scholar * Martin Gassmann2 Search for this author in: * NPG journals * PubMed * Google Scholar * Uwe Schulte1, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Bernd Fakler1, 5 Search for this author in: * NPG journals * PubMed * Google Scholar * Bernhard Bettler2 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorsJournal name:NatureVolume:465,Pages:231–235Date published:(13 May 2010)DOI:doi:10.1038/nature08964Received10 December 2009Accepted24 February 2010Published online18 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 GABAB receptors are the G-protein-coupled receptors for γ-aminobutyric acid (GABA), the main inhibitory neurotransmitter in the brain. They are expressed in almost all neurons of the brain, where they regulate synaptic transmission and signal propagation by controlling the activity of voltage-gated calcium (Cav) and inward-rectifier potassium (Kir) channels1. Molecular cloning revealed that functional GABAB receptors are formed by the heteromeric assembly of GABAB1 with GABAB2 subunits2, 3, 4, 5. However, cloned GABAB(1,2) receptors failed to reproduce the functional diversity observed with native GABAB receptors6, 7, 8. Here we show by functional proteomics that GABAB receptors in the brain are high-molecular-mass complexes of GABAB1, GABAB2 and members of a subfamily of the KCTD (potassium channel tetramerization domain-containing) proteins. KCTD proteins 8, 12, 12b and 16 show distinct expression profiles in the brain and associate tightly with the carboxy terminus of GA! BAB2 as tetramers. This co-assembly changes the properties of the GABAB(1,2) core receptor: the KCTD proteins increase agonist potency and markedly alter the G-protein signalling of the receptors by accelerating onset and promoting desensitization in a KCTD-subtype-specific manner. Taken together, our results establish the KCTD proteins as auxiliary subunits of GABAB receptors that determine the pharmacology and kinetics of the receptor response. View full text Subject terms: * Neuroscience * Cell biology * Biochemistry Figures at a glance * Figure 1: Identification of four KCTD proteins as subunits of native GABAB receptors. , Input and scheme of the proteomic approach used. SDS–PAGE separation of solubilized (S) and insoluble (P) protein fractions obtained with the CL-91 buffer from brain membranes of wild-type (WT) and the indicated knockout mice western blotted with anti-GABAB1. , Normalized abundance values of proteins in affinity purifications (AP) with anti-GABAB1 and anti-GABAB2. Grey circles denote background proteins, filled circles indicate proteins specifically co-purified with both antibodies, half-filled symbols represent proteins specifically co-purified with one antibody (left side filled with anti-GABAB2, lower half filled with anti-GABAB1). Inset: conserved domains of KCTD proteins. , Two-dimensional gel separation of solubilized GABAB receptors from rat brain western blotted with the indicated antibodies. Size (blue native (BN)–PAGE) and molecular mass (SDS–PAGE) as indicated. , SEC of the GB1-tagged KCTD12-T1; BSA added for reference. Inset: sedimentation equilibrium ana! lysis of the monodisperse SEC fraction of GB1-tagged KCTD12-T1 (molecular mass 20.4 kDa); data were obtained with a protein concentration of 0.5 mg ml-1 at 12,000 r.p.m. The continuous line is a fit of the data yielding a molecular mass of 81.2 kDa. , Left: SEC analysis of the KCTD12-T1 + GABAB2-C745–940 complex; purified KCTD12-T1 added for comparison. Inset: SDS–PAGE of the indicated SEC fraction (yellow bar) stained with Sypro Orange. Right: stoichiometry of the complex constituents obtained from fluorescence intensity measurements (Sypro analysis) or from quantitative mass spectrometry analyses of gel separations as in the inset; data are means and s.d. for three experiments. * Figure 2: Expression profile and subcellular localization of KCTD proteins in the brain. , In situ hybridizations with digoxigenin-labelled antisense probes on horizontal sections of the medial tier (overview (left), hippocampal formation (middle) and cerebellum (right)) of adult mouse brains. DG, dentate gyrus; gcl, granule cell layer; ml, molecular cell layer; arrows and arrowheads denote cerebellar Golgi/Purkinje cells. KCTD12b transcripts are restricted to the medial habenula (rectangular frame, enlarged in inset). Scale bars, 200 μm (overviews) and 40 μm (hippocampus and cerebellum). , Immunoreactivities for the indicated KCTDs and GABAB1 in the stratum radiatum of the hippocampal CA1 region detected by pre-embedding immunogold electron microscopy. Immunogold particles were most abundant at the extrasynaptic plasma membrane (filled arrowheads) of dendritic spines (s, s1, s2) and dendritic shafts (Den) of pyramidal cells, but also found at the presynaptic membranes (open arrowheads) of boutons (b, b1, b2) of putative excitatory cells. , Clusters of GAB! AB1 and KCTD proteins (arrowheads) over the surface of dendrites (Den) of CA1 pyramidal cells assessed by SDS-digested freeze–fracture replica immunolabelling. Insets are enlargements of the indicated frames. Scale bars, 0.2 μm (, ). * Figure 3: G-protein signalling of GABAB receptors is determined by KCTD proteins. , K+ currents recorded at -50 mV in response to GABA applications (1 mM) from whole Xenopus oocytes co-expressing GABAB, Kir3.1/3.2 channels and the indicated KCTD proteins. The extracellular K+ concentration was 100 mM. , Experiments as in but with oocytes co-expressing GABAB, Cav2.2 channels and the indicated KCTD proteins; the bath solution contained 10 mM Ba2+. Data points are maxima of currents elicited every 3 s by voltage steps from -100 mV to 0 mV (shown on the right for the indicated time points), successive GABA applications are denoted by the horizontal bars. The current scale is 0.1 μA. , Bar graphs summarizing the relative desensitization determined for the indicated subunit combinations; wo KCTD, without KCTD. Data points are means and s.d. for 6–12 (GABAB + Kir3.1/3.2), 8–13 (GABAB + Cav2.2) and 3–7 (GABAB + Cav2.1) experiments. Top right: time constants (τdesens) and their relative contributions obtained from double-exponen! tial fits to the Kir3.1/3.2 current decay recorded in experiments as in with KCTD12; data points are means and s.d. for 8 measurements. * Figure 4: KCTDs alter kinetics and agonist potency of the GABAB response in CHO cells and neurons. , Left: K+ currents through Kir3.1/3.2 channels recorded at -50 mV in response to baclofen applications (0.1 mM) from whole CHO cells co-expressing GABAB, Kir3.1/3.2 channels and the indicated KCTD proteins; wo KCTD, without KCTD. The extracellular K+ concentration was 2.5 mM. Right: summary plot as in Fig. 3c; data are means and s.d. for 6–31 experiments. , The 20–80% rise time of Kir3.1/3.2 currents activated by GABAB without or together with KCTD proteins. Data points are means and s.d. for 6–17 experiments. Inset: representative current onsets. , Apparent dose–response relations obtained with GABAB (open circles) or with GABAB and KCTD proteins 12 (filled red circles) and 16 (filled black circles). Data points are means and s.d. for 7–13 experiments; lines are fits of a logistic function with values for EC50 values and Hill coefficients of 68.7 μM and 1.02 (GABAB), 21.0 μM and 0.75 (GABAB + KCTD16), 9.9 μM and 0.63 (GABAB + KCTD12), respe! ctively. , Top: currents through Kir3 channels recorded in experiments as in from cultured hippocampal neurons without (native) or after transfection with KCTD12 (red trace) or KCTD16. Bottom: bar graphs summarizing relative desensitization (left) and onset (right) of the Kir3 currents as in and ; data points are means and s.d. for 7–38 experiments. Author information * Author information * Supplementary information * Comments Primary authors * These authors contributed equally to this work. * Jochen Schwenk, * Michaela Metz, * Gerd Zolles & * Rostislav Turecek Affiliations * Institute of Physiology II, University of Freiburg, Engesserstrasse 4, 79108 Freiburg, Germany * Jochen Schwenk, * Gerd Zolles, * Wolfgang Bildl, * Uwe Schulte & * Bernd Fakler * Department of Biomedicine, Institute of Physiology, Pharmazentrum, University of Basel, Klingelbergstrasse 50/70, CH-4056 Basel, Switzerland * Michaela Metz, * Rostislav Turecek, * Thorsten Fritzius, * Klara Ivankova, * Riad Seddik, * Jim Y. Tiao, * Mathieu Rajalu, * Martin Gassmann & * Bernhard Bettler * Logopharm GmbH, Engesserstrasse 4, 79108 Freiburg, Germany * Volker Rohde & * Uwe Schulte * Institute of Anatomy and Cell Biology, University of Freiburg, Albertstrasse 23, 79104 Freiburg, Germany * Etsuko Tarusawa, * Akos Kulik & * Andreas Unger * Center for Biological Signaling Studies (bioss), Albertstrasse 10, 79104 Freiburg, Germany * Bernd Fakler * Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, Videnska 1083, 14220-Prague 4, Czech Republic * Rostislav Turecek & * Johana Trojanova Contributions J.S., M.M., G.Z. and R.T. are equally contributing first authors. J.S., W.B., V.R., U.S. and B.F. performed proteomic analysis, biochemistry and evaluation of mass spectrometry. M.M., J.Y.T., M.G., J.T., T.F., M.R. and K.I. performed in situ hybridization, cellular biology, mouse work and KCTD antibody generation. G.Z., R.T., R.S. and M.R. conducted the electrophysiological recordings on oocytes, cultured cells and neurons. A.U., E.T. and A.K. performed electron microscopy. B.F., B.B. and U.S. initiated, designed and supervised the study. B.F. and B.B. wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Bernd Fakler (bernd.fakler@physiologie.uni-freiburg.de) or * Bernhard Bettler (bernhard.bettler@unibas.ch) Supplementary information * Author information * Supplementary information * Comments PDF files * Supplementary Information (5.5M) This file contains Supplementary Figures 1-13 with legends and Supplementary Tables 1-2. Additional data
• Self-assembly of spider silk proteins is controlled by a pH-sensitive relay
  - Nature 465(7295):236 (2010)
  Nature | Letter Self-assembly of spider silk proteins is controlled by a pH-sensitive relay * Glareh Askarieh1, 2, 5 Search for this author in: * NPG journals * PubMed * Google Scholar * My Hedhammar3, 5 Search for this author in: * NPG journals * PubMed * Google Scholar * Kerstin Nordling3 Search for this author in: * NPG journals * PubMed * Google Scholar * Alejandra Saenz4 Search for this author in: * NPG journals * PubMed * Google Scholar * Cristina Casals4 Search for this author in: * NPG journals * PubMed * Google Scholar * Anna Rising3 Search for this author in: * NPG journals * PubMed * Google Scholar * Jan Johansson3 Search for this author in: * NPG journals * PubMed * Google Scholar * Stefan D. Knight2 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorsJournal name:NatureVolume:465,Pages:236–238Date published:(13 May 2010)DOI:doi:10.1038/nature08962Received05 June 2009Accepted24 February 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 Nature's high-performance polymer, spider silk, consists of specific proteins, spidroins, with repetitive segments flanked by conserved non-repetitive domains1, 2. Spidroins are stored as a highly concentrated fluid dope. On silk formation, intermolecular interactions between repeat regions are established that provide strength and elasticity3, 4. How spiders manage to avoid premature spidroin aggregation before self-assembly is not yet established. A pH drop to 6.3 along the spider's spinning apparatus, altered salt composition and shear forces are believed to trigger the conversion to solid silk, but no molecular details are known. Miniature spidroins consisting of a few repetitive spidroin segments capped by the carboxy-terminal domain form metre-long silk-like fibres irrespective of pH5. We discovered that incorporation of the amino-terminal domain of major ampullate spidroin 1 from the dragline of the nursery web spider Euprosthenops australis (NT) into mini-spidroi! ns enables immediate, charge-dependent self-assembly at pH values around 6.3, but delays aggregation above pH 7. The X-ray structure of NT, determined to 1.7 Å resolution, shows a homodimer of dipolar, antiparallel five-helix bundle subunits that lack homologues. The overall dimeric structure and observed charge distribution of NT is expected to be conserved through spider evolution and in all types of spidroins. Our results indicate a relay-like mechanism through which the N-terminal domain regulates spidroin assembly by inhibiting precocious aggregation during storage, and accelerating and directing self-assembly as the pH is lowered along the spider's silk extrusion duct. View full text Subject terms: * Structural biology * Materials science * Molecular biology * Biochemistry Figures at a glance * Figure 1: pH-dependent assembly of NT and mini-spidroins. , Self-assembly of mini-spidroins with NT (NT4RepCT or NT4Rep) or without NT (4RepCT or 4Rep) at pH 7 (above time scale) or at pH 6 (below time scale). The arrows indicate when macroscopic structures were first detected. The scanning electron micrographs are representative for early structures of 4RepCT at both pH 6 and 7 (figure above time axis) and of NT4RepCT at pH 6 (figure below time axis), respectively. The self-assembly process results in continuous fibres only for mini-spidroins including the C-terminal domain (NT4RepCT and 4RepCT). , Turbidimetry of NT and NT4Rep at different pH values measured immediately after sample preparation. Mean values (± s.d., n = 3) of NT4Rep (green) and NT (red). , Hydrodynamic radii determined by dynamic light scattering and zeta-potentials determined by electrophoretic mobility of NT at different pH values (± s.d., n = 3). , Tryptophan fluorescence of NT between pH 7.0 and 6.0. The changes are reversible, and in the pres! ence of 0.3 M NaCl no change in fluorescence is observed. a.u., arbitrary units. * Figure 2: Structural and conserved features of spidroin N-terminal domain. , Amino acid sequence of NT. Numbering as in the deposited structure. Secondary structure elements are labelled H1–H5. Strictly conserved residues and residues appearing in >60% of sequences (Supplementary Fig. 4) are shown with green and brown background, respectively. , Overall structure of NT homodimer. Subunits A (grey) and B (blue) are shown in surface and ribbon representations, respectively. Strictly conserved residues are highlighted in green. , Stereo ribbon diagram of the NT dimer showing the distribution of conserved residues (sticks colour coded as in ). , Surface representation of the exposed charged residues on NT dimers, with acidic (red) and basic (blue) residues shown as sticks. (Viewed at 90° rotation compared to ). , Superposition13, 14 of the subunits showing conformational differences in the N- and C-terminal regions (chain A in grey, chain B in blue). The hash-sign indicates structural elements of the neighbouring subunit. Accession codes * Accession codes * Author information * Supplementary information * Comments Primary accessions Protein Data Bank * 3LR2 * 3LR6 * 3LR8 * 3LRD * 3LR2 * 3LR6 * 3LR8 * 3LRD Author information * Accession codes * Author information * Supplementary information * Comments Primary authors * These authors contributed equally to this work * Glareh Askarieh & * My Hedhammar Affiliations * Department of Chemistry, Oslo University, 1033 Blindern, 0315 Oslo, Norway * Glareh Askarieh * Department of Molecular Biology, Uppsala BioCenter, SLU, Biomedical Centre, P.O. Box 590, SE-751 24 Uppsala, Sweden * Glareh Askarieh & * Stefan D. Knight * Department of Anatomy, Physiology and Biochemistry, SLU, Biomedical Centre, P.O. Box 575, SE-751 23 Uppsala, Sweden * My Hedhammar, * Kerstin Nordling, * Anna Rising & * Jan Johansson * Department of Biochemistry and Molecular Biology I & CIBER Enfermedades Respiratorias, Complutense University of Madrid, 28040-Madrid, Spain * Alejandra Saenz & * Cristina Casals Contributions G.A. and M.H. contributed equally to this work. G.A., M.H. performed experiments and wrote the paper, K.N., A.S. performed experiments, C.C., A.R., J.J., S.D.K. discussed experiments and wrote the paper. Competing financial interests M.H., K.N., A.R. and J.J. own stocks and are funded by Spiber Technologies AB, a company that aims to commercialize recombinant spider silk for biomedical applications. Corresponding authors Correspondence to: * Stefan D. Knight (stefan.knight@molbio.slu.se) or * Jan Johansson (jan.johansson@afb.slu.se) X-ray crystallographic coordinates and structure factors have been deposited in the RCSB Protein Data Bank (PDB) with PDB ID codes 3LR2, 3LR6, 3LR8, 3LRD. Supplementary information * Accession codes * Author information * Supplementary information * Comments PDF files * Supplementary Information (607K) This file contains Supplementary Figures 1-8 with legends and Supplementary Tables 1-2. Additional data
• A conserved spider silk domain acts as a molecular switch that controls fibre assembly
  - Nature 465(7295):239 (2010)
  Nature | Letter A conserved spider silk domain acts as a molecular switch that controls fibre assembly * Franz Hagn1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Lukas Eisoldt3 Search for this author in: * NPG journals * PubMed * Google Scholar * John G. Hardy3 Search for this author in: * NPG journals * PubMed * Google Scholar * Charlotte Vendrely3, 5 Search for this author in: * NPG journals * PubMed * Google Scholar * Murray Coles4 Search for this author in: * NPG journals * PubMed * Google Scholar * Thomas Scheibel3 Search for this author in: * NPG journals * PubMed * Google Scholar * Horst Kessler1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorsJournal name:NatureVolume:465,Pages:239–242Date published:(13 May 2010)DOI:doi:10.1038/nature08936Received20 May 2009Accepted11 February 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 A huge variety of proteins are able to form fibrillar structures1, especially at high protein concentrations. Hence, it is surprising that spider silk proteins can be stored in a soluble form at high concentrations and transformed into extremely stable fibres on demand2, 3. Silk proteins are reminiscent of amphiphilic block copolymers containing stretches of polyalanine and glycine-rich polar elements forming a repetitive core flanked by highly conserved non-repetitive amino-terminal4, 5 and carboxy-terminal6 domains. The N-terminal domain comprises a secretion signal, but further functions remain unassigned. The C-terminal domain was implicated in the control of solubility and fibre formation7 initiated by changes in ionic composition8, 9 and mechanical stimuli known to align the repetitive sequence elements and promote β-sheet formation10, 11, 12, 13, 14. However, despite recent structural data15, little is known about this remarkable behaviour in molecular detail. Here w! e present the solution structure of the C-terminal domain of a spider dragline silk protein and provide evidence that the structural state of this domain is essential for controlled switching between the storage and assembly forms of silk proteins. In addition, the C-terminal domain also has a role in the alignment of secondary structural features formed by the repetitive elements in the backbone of spider silk proteins, which is known to be important for the mechanical properties of the fibre. View full text Subject terms: * Biophysics * Structural biology * Molecular biology * Materials science Figures at a glance * Figure 1: Sequence analysis and structure of the non-repetitive (NR) domain of ADF-3. , Hydrophobicity index of various silk proteins. , Sequence alignment of the C-terminal NR domains of the major ampullate silk proteins (MaSp) of Araneus diadematus, Nephila clavipes and Euprosthenops australis, and minor ampullate silk fibroin 1 (ADF-1) of Araneus diadematus (top to bottom). Grey bars indicate the degree of conservation between these sequences. The conserved charged residues are coloured. , Overlay of the 20 best-energy structures of the C-terminal NR domain of ADF-3 having a root mean square deviation (r.m.s.d.) of 0.18 Å. * Figure 2: Assembly and aggregation properties of our spider silk-like proteins. , Salt-induced protein aggregation. Error bars indicate standard deviation. , Binding of hydrophobic ANS by proteins. a.u., arbitrary units; WT, wild type. , Thermal transition experiments indicate molten globule-like behaviour of the D93A variant (red symbols), whereas the wild-type protein binds to the dye only during unfolding (black symbols). A structural transition of the D93A variant can be induced by sodium chloride (open symbols: in presence of 300 mM sodium chloride). Without the NR domain, no transition can be observed (green symbols). , The concentration dependence of the LCST of (AQ)24NR3. , The sodium chloride dependence of the LCST of (AQ)24NR3. * Figure 3: Stability and folding of spider dragline silk constructs. , NMR chemical shift perturbation experiments with urea, sodium chloride and ANS indicate that the structural elements around the salt bridges represent the most labile and hydrophobic region within the protein. , Urea-induced unfolding of NR3 as determined by CD spectroscopy. , Binding of hydrophobic SYPRO orange to NR3 during urea-induced unfolding. , Shear-induced ANS binding of NR3. , Aggregation of our spider silk-like proteins with and without shear stress. Shear stress leads to increased aggregation and is the most likely trigger for the assembly process in nature. Error bars represent standard deviation. * Figure 4: Fibre assembly mechanism of dragline silk proteins. , A well-defined fibre (dry state) of (AQ)12NR3 and ill-defined fibrous aggregate of (AQ)24 formed under shear. , Infrared absorption spectra of (AQ)12NR3 and (AQ)24 fibres recorded at 0° and 90° relative to the long axis of the fibrous aggregates (black vs red line). , Estimated β-sheet content of the aggregates. The values of β-sheet content for the aggregates formed under shear were recorded at 0°, 45° and 90° relative to the long axis of the fibrous aggregates. , The silk proteins are stored as higher oligomeric assemblies. Exposure of these assemblies to shear and salting out leads to partial unfolding of NR3 and controlled fibre assembly. Accession codes * Accession codes * Author information * Supplementary information * Comments Primary accessions Protein Data Bank * 2khm * 2khm Author information * Accession codes * Author information * Supplementary information * Comments Affiliations * Center for Integrated Protein Science (CIPSM) and, * Franz Hagn & * Horst Kessler * Institute for Advanced Study, Department Chemie at the Technische Universität München, 85747 Garching, Germany * Franz Hagn & * Horst Kessler * Lehrstuhl für Biomaterialien, Fakultät für Angewandte Naturwissenschaften, Universität Bayreuth, 95440 Bayreuth, Germany * Lukas Eisoldt, * John G. Hardy, * Charlotte Vendrely & * Thomas Scheibel * Department of Protein Evolution, Max-Planck-Institute for Developmental Biology, 72076 Tübingen, Germany * Murray Coles * Present address: Université de Cergy-Pontoise, 95302 Cergy-Pontoise cedex, France. * Charlotte Vendrely Contributions F.H. designed research, performed cloning work, purified proteins, performed protein folding studies, recorded NMR experiments, performed structure calculations and wrote the manuscript; L.E. performed cloning work, purified proteins and performed aggregation assays. J.G.H. performed aggregation assays, characterized the fibres and wrote the manuscript; C.V. performed cloning work and purified proteins; M.C. was involved in structure calculation and provided software tools for structure calculation and analysis; T.S. designed research and wrote the manuscript; H.K. designed research and wrote the manuscript. All authors discussed the results and commented on the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Horst Kessler (horst.kessler@ch.tum.de) or * Thomas Scheibel (thomas.scheibel@uni-bayreuth.de) The resonance assignment obtained was deposited at the BMRB data bank under accession code 16249 and the atomic coordinates of the best 20 structures plus a regularized average structure have been deposited at the Protein Data Bank under accession code 2khm. Supplementary information * Accession codes * Author information * Supplementary information * Comments PDF files * Supplementary Information (1.5M) This file contains Figures 1-6 with Legends, Supplementary Tables 1-2 and a Reference. Additional data
• Histone H2A deubiquitinase activity of the Polycomb repressive complex PR-DUB
  Scheuermann JC de Ayala Alonso AG Oktaba K Ly-Hartig N McGinty RK Fraterman S Wilm M Muir TW Müller J - Nature 465(7295):243 (2010)
  Nature | Letter Histone H2A deubiquitinase activity of the Polycomb repressive complex PR-DUB * Johanna C. Scheuermann1, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Andrés Gaytán de Ayala Alonso1, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Katarzyna Oktaba1 Search for this author in: * NPG journals * PubMed * Google Scholar * Nga Ly-Hartig1 Search for this author in: * NPG journals * PubMed * Google Scholar * Robert K. McGinty2 Search for this author in: * NPG journals * PubMed * Google Scholar * Sven Fraterman1 Search for this author in: * NPG journals * PubMed * Google Scholar * Matthias Wilm1 Search for this author in: * NPG journals * PubMed * Google Scholar * Tom W. Muir2 Search for this author in: * NPG journals * PubMed * Google Scholar * Jürg Müller1 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:NatureVolume:465,Pages:243–247Date published:(13 May 2010)DOI:doi:10.1038/nature08966Received07 July 2009Accepted25 February 2010Published online02 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 Polycomb group (PcG) proteins are transcriptional repressors that control processes ranging from the maintenance of cell fate decisions and stem cell pluripotency in animals to the control of flowering time in plants1, 2, 3, 4, 5, 6. In Drosophila, genetic studies identified more than 15 different PcG proteins that are required to repress homeotic (HOX) and other developmental regulator genes in cells where they must stay inactive1, 7, 8. Biochemical analyses established that these PcG proteins exist in distinct multiprotein complexes that bind to and modify chromatin of target genes1, 2, 3, 4. Among those, Polycomb repressive complex 1 (PRC1) and the related dRing-associated factors (dRAF) complex contain an E3 ligase activity for monoubiquitination of histone H2A (refs 1–4). Here we show that the uncharacterized Drosophila PcG gene calypso encodes the ubiquitin carboxy-terminal hydrolase BAP1. Biochemically purified Calypso exists in a complex with the PcG protein ASX, a! nd this complex, named Polycomb repressive deubiquitinase (PR-DUB), is bound at PcG target genes in Drosophila. Reconstituted recombinant Drosophila and human PR-DUB complexes remove monoubiquitin from H2A but not from H2B in nucleosomes. Drosophila mutants lacking PR-DUB show a strong increase in the levels of monoubiquitinated H2A. A mutation that disrupts the catalytic activity of Calypso, or absence of the ASX subunit abolishes H2A deubiquitination in vitro and HOX gene repression in vivo. Polycomb gene silencing may thus entail a dynamic balance between H2A ubiquitination by PRC1 and dRAF, and H2A deubiquitination by PR-DUB. View full text Subject terms: * Molecular biology * Biochemistry * Cancer * Genetics * Genomics Figures at a glance * Figure 1: The Polycomb group proteins BAP1 and ASX form a conserved complex in vivo and in vitro. , Domain architecture of the Drosophila Calypso protein and molecular lesions in calypso mutant alleles. UCH, ubiquitin C-terminal hydrolase domain. , Calypso complexes isolated by TAP26 from wild-type (WT) or TAP-calypso transgenic embryos. Input material for purification was normalized by protein concentration, and equivalent amounts of eluate from calmodulin-affinity resin were separated on a 4–12% polyacrylamide gel and visualized by silver staining together with a molecular mass marker (M). Calypso bait protein containing the calmodulin-binding tag (CBP–Calypso), and bands representing ASX fragments were identified by mass spectrometry (Supplementary Table 1 and Supplementary Fig. 2). No band corresponding to full-length ASX (180 kDa) was detected in several independent purifications, even though ASX is present as a single polypeptide of 180 kDa in total embryo extracts (Supplementary Fig. 5). This suggests that ASX is degraded during nuclear extract preparation! or TAP purification. , Reconstitution of recombinant Calypso–ASX and BAP1–ASXL1 complexes. Proteins were extracted by Flag-affinity purification from cell lysates containing the indicated Flag-tagged proteins and HA–ASX(2–337) (left) or HA–ASXL1(2–365) (right). Experiments with full-length ASX are shown in Supplementary Fig. 3. Proteins were visualized by Coomassie staining or western blotting analysis, as indicated. Input material for experiments in lanes 3–6 (left) and 7–10 (right) were probed by western blotting to ensure that comparable amounts of proteins were present in cell lysates. On the Coomassie-stained gel, Flag-tagged proteins are marked with an asterisk, HA–ASX(2–337) and HA–ASXL1(2–365) are marked with a hash symbol. * Figure 2: PR-DUB is bound at Polycomb target genes in Drosophila. , PR-DUB is bound at PREs of PcG target genes in Drosophila. ChIP profiles of PR-DUB subunits ASX (dark blue) and Calypso (light blue), and of Ph18 (grey) and Pho17 (grey) at the Antennapedia HOX gene cluster in imaginal disc and CNS tissues from third instar Drosophila larvae. Hybridization intensities for oligonucleotide probes are plotted as coloured bars above the genomic map (release 5, kilobase coordinates) of Drosophila melanogaster; significantly enriched regions are marked below plots. HOX genes labial (lab), proboscipedia (pb), Deformed (Dfd), Sex combs reduced (Scr), Antennapedia (Antp) and other genes on the plus (above) or minus (below) strand are represented with exons (black boxes) and introns (thin black lines). , Venn diagrams showing the overlap of 879 PR-DUB-bound regions with 1,681 Ph-bound and 670 Pho-bound regions in larval cells. , PR-DUB is bound at the inactive and at the active Ubx gene. ChIP analyses monitoring ASX and Calypso binding in wing and h! altere/third leg imaginal discs from wild-type third instar Drosophila larvae. Graphs show results from independent ChIP reactions (n = 3 ChIP reactions) with ASX or Calypso antibodies. ChIP signals, measured by qPCR, are presented as the mean percentage of input chromatin precipitated at each region; error bars indicate ±s.d. (see Methods). Locations of Ubx PREs (boxes) and other regions relative to the Ubx transcription start site are indicated in kilobases. As control, binding was monitored at two euchromatic (eu) and one heterochromatic (het) region elsewhere in the genome, and at the PREs of the HOX genes Abd-B and Scr that are both inactive in wing and haltere/third leg imaginal discs. Calypso and ASX are bound at Ubx PREs both in wing and in haltere/third leg disc cells; at the -30-kb PRE, Calypso and ASX ChIP signals were comparable in wing and haltere/third leg chromatin, at the +32-kb PRE, the signal in haltere/third leg chromatin is about 2–4-fold lower than i! n wing chromatin, paralleling PRC1 and PRC2 binding at both th! ese PREs19. * Figure 3: Recombinant Drosophila and human PR-DUB deubiquitinate H2A in nucleosomes in vitro. , Cleavage of Ub-AMC by Calypso and Calypso–ASX(2–337) complexes. Reactions (n = 4) contained 25 pmol Ub-AMC and 10 pmol of the indicated protein (complex); release of AMC was monitored by fluorescence spectroscopy at 436 nm; error bars indicate ±s.d. a.u., arbitrary units. , Mononucleosomes were reconstituted with recombinant Xenopus histone octamers and were unmodified (lane 1), monoubiquitinated at H2AK119 (lane 2) (see Methods) or monoubiquitinated at H2BK120 (lane 3) (see Methods). The material was analysed on a 4–12% polyacrylamide gradient gel and histones were visualized by Coomassie staining. , Drosophila and human PR-DUB deubiquitinate H2Aub1 in nucleosomes. Xenopus mononucleosomes (15 pmol) containing 30 pmol of either H2Aub1 (top gels left and right) or H2Bub1 (bottom gels left and right) were incubated without (lanes 2 and 10) or with 30 pmol of the indicated Drosophila PR-DUB complexes (lanes 3–8) or human BAP1 or PR-DUB complex (lanes 11–1! 6), respectively, and deubiquitination was monitored at indicated time points by western blot analysis with anti-H2A (top gels left and right) or anti-H2B (bottom gels left and right) antibody (5 pmol nucleosome per lane). Unmodified mononucleosomes (lanes 1 and 9) served as a control. Comparable results were obtained with Drosophila mononucleosomes containing H2Aub1 (Supplementary Fig. 4). PR-DUB containing full-length ASX(1–1668) also specifically deubiquitinated H2Aub1 but not H2Bub1 in nucleosomes (Supplementary Fig. 4). , K48- or K63-linked hexameric polyubiquitin chains (160 ng; corresponding to maximally 17.5 pmol ubiquitin linkage bonds) were incubated for 40 min with 10 pmol of the indicated protein or protein complex under the same assay conditions as in , followed by western blot analysis with an anti-ubiquitin antibody. * Figure 4: PR-DUB is required for H2A deubiquitination in Drosophila and its catalytic activity is essential for HOX gene repression. , PR-DUB is required for H2A deubiquitination in Drosophila embryos. Serial dilutions (1:3:9) of histone extracts from 16–18-h-old wild-type or Asx22P4 homozygous embryos were separated on 4–12% polyacrylamide gels and analysed by western blotting with the indicated antibodies. H2Aub1 levels in lanes 3 and 4 are comparable, suggesting that H2Aub1 levels are almost tenfold higher in Asx22P4 mutants than in wild type. H2Bub1 levels in Asx22P4 mutants are less than threefold increased compared to wild type (compare lane 3 with lanes 5 and 6). The band detected by an anti-ubiquitin antibody represents the combined signal of H2Aub1 and H2Bub1. H3K4me3 levels appear very slightly increased in Asx22P4 mutants. , Calypso deubiquitinase activity is required for HOX gene repression. Wing imaginal discs with clones of calypso2 homozygous mutant cells from animals that carried no transgene or the indicated hsp70-calypso transgenes. calypso2 mutant cells are marked by the absence of ! GFP and discs were stained with antibodies against UBX or Calypso protein, as indicated. In all cases, clones were induced 96 h before analysis and larvae were repeatedly heat-shocked for 1 h every 12 h over a 96-h period to provide a continuous supply of Calypso protein from the transgene. In the absence of an hsp70-calypso transgene, Ubx is misexpressed in most calypso2 mutant clones in the pouch of the disc but remains repressed in the notum and hinge (left). Wild-type Calypso protein rescues repression of Ubx in mutant clones (middle), whereas the Calypso(C131S) protein fails to rescue (right), even though both transgene-encoded proteins are expressed at comparable levels and show nuclear localization like endogenous Calypso protein (bottom row). Accession codes * Accession codes * Author information * Supplementary information * Comments Primary accessions ArrayExpress * E-TABM-908 Author information * Accession codes * Author information * Supplementary information * Comments Primary authors * These authors contributed equally to this work. * Johanna C. Scheuermann & * Andrés Gaytán de Ayala Alonso Affiliations * European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany * Johanna C. Scheuermann, * Andrés Gaytán de Ayala Alonso, * Katarzyna Oktaba, * Nga Ly-Hartig, * Sven Fraterman, * Matthias Wilm & * Jürg Müller * The Rockefeller University, 1230 York Avenue, New York, New York 10065, USA * Robert K. McGinty & * Tom W. Muir Contributions J.C.S., A.G.A.A., K.O., N.L.-H. and J.M. conceived the project, designed and carried out the experiments, discussed and interpreted the data and prepared the manuscript. R.K.M. synthesized H2Bub1 in the laboratory of T.W.M., S.F. performed the mass spectrometry analysis in the laboratory of M.W. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Jürg Müller (juerg.mueller@embl.de) The microarray data have been deposited in the ArrayExpress database (http://www.ebi.ac.uk/arrayexpress) under the accession number E-TABM-908. Supplementary information * Accession codes * Author information * Supplementary information * Comments Excel files * Supplementary Table 2 (667K) This table shows the chromosomal coordinates of PR-DUB-bound regions and assigned target genes with gene ontology classifications. PDF files * Supplementary Information (2.9M) This file contains Supplementary Figures 1-8 with legends and Supplementary Table 1. Additional data
• Stepwise [FeFe]-hydrogenase H-cluster assembly revealed in the structure of HydAΔEFG
  Mulder DW Boyd ES Sarma R Lange RK Endrizzi JA Broderick JB Peters JW - Nature 465(7295):248 (2010)
  Nature | Letter Stepwise [FeFe]-hydrogenase H-cluster assembly revealed in the structure of HydAΔEFG * David W. Mulder1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Eric S. Boyd1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Ranjana Sarma1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Rachel K. Lange1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * James A. Endrizzi1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Joan B. Broderick1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * John W. Peters1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:NatureVolume:465,Pages:248–251Date published:(13 May 2010)DOI:doi:10.1038/nature08993Received21 October 2009Accepted05 March 2010Published online25 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 Complex enzymes containing Fe–S clusters are ubiquitous in nature, where they are involved in a number of fundamental processes including carbon dioxide fixation, nitrogen fixation and hydrogen metabolism1, 2. Hydrogen metabolism is facilitated by the activity of three evolutionarily and structurally unrelated enzymes: the [NiFe]-hydrogenases, [FeFe]-hydrogenases and [Fe]-hydrogenases3, 4 (Hmd). The catalytic core of the [FeFe]-hydrogenase (HydA), termed the H-cluster, exists as a [4Fe–4S] subcluster linked by a cysteine thiolate to a modified 2Fe subcluster with unique non-protein ligands5, 6. The 2Fe subcluster and non-protein ligands are synthesized by the hydrogenase maturation enzymes HydE, HydF and HydG; however, the mechanism, synthesis and means of insertion of H-cluster components remain unclear7, 8, 9, 10. Here we show the structure of HydAΔEFG (HydA expressed in a genetic background devoid of the active site H-cluster biosynthetic genes hydE, hydF and hydG) r! evealing the presence of a [4Fe–4S] cluster and an open pocket for the 2Fe subcluster. The structure indicates that H-cluster synthesis occurs in a stepwise manner, first with synthesis and insertion of the [4Fe–4S] subcluster by generalized host-cell machinery11, 12 and then with synthesis and insertion of the 2Fe subcluster by specialized hydE-, hydF- and hydG-encoded maturation machinery7, 8, 9, 10. Insertion of the 2Fe subcluster presumably occurs through a cationically charged channel that collapses following incorporation, as a result of conformational changes in two conserved loop regions. The structure, together with phylogenetic analysis, indicates that HydA emerged within bacteria most likely from a Nar1-like ancestor lacking the 2Fe subcluster, and that this was followed by acquisition in several unicellular eukaryotes. View full text Subject terms: * Structural biology * Biochemistry * Genetics * Genomics * Evolution Figures at a glance * Figure 1: Ball-and-stick representation of the H-cluster in [FeFe]-hydrogenase from Clostridium pasteurianum29. The H-cluster is bound to the protein (tube representation) by four cysteine ligands of the [4Fe–4S] subcluster, which is further linked by a cysteine thiolate ligand to a 2Fe subcluster with unique non-protein ligands including CO, CN– and a dithiolate ligand30. A water molecule is coordinated to the distal Fe of the 2Fe subcluster in the presumed-active, oxidized state. Dark red, Fe; orange, S; grey, C; blue, N; light red, oxygen; magenta, central atom of the dithiolate ligand. Protein Data Bank ID, 3C8Y. * Figure 2: X-ray crystal structure of C. reinhardtii HydAΔEFG determined to a resolution of 1.97 Å, compared with HydA from CpI. , Ribbon diagram of the overall HydAΔEFG structure, with a space-filling representation of the associated [4Fe–4S] cluster. The two conserved loop regions thought to undergo major conformational rearrangement are coloured green. , HydAΔEFG active-site area, where a 2Fe subcluster recipient cavity is adjacent to the [4Fe–4S] cluster. , Ribbon diagram of the overall CpI structure in the same orientation as HydAΔEFG, with a space-filling representation of the intact H-cluster. The regions of CpI corresponding to the loop regions of HydAΔEFG shown in are coloured green. , CpI active-site region, with ball-and-stick representation of the H-cluster. Protein representations are coloured according to secondary structure (light blue, α-helices and loops; violet, β-sheets). The atomic colouring scheme is the same as in Fig. 1. * Figure 3: Active-site comparison between C. reinhardtii HydAΔEFG and HydA from CpI. , The [4Fe–4S]-cluster active-site environment in HydAΔEFG. The anomalous-difference Fourier map (blue) is shown contoured at 4.5σ, indicating the positions of the Fe atoms localized to the [4Fe–4S] cluster. An acetate molecule and a chloride ion are modelled into the Fo–Fc map (magenta, contoured at 3.5σ) of the HydAΔEFG cavity. Residues are labelled according to single-letter amino-acid abbreviations and sequence number. The side chain of Cys 129 has increased conformational freedom and can be refined in three conformations (one shown). , H-cluster active-site environment in CpI, shown in the same orientation as HydAΔEFG in . The atomic colouring scheme is the same as in Fig. 1. * Figure 4: Channels for insertion into hydrogenase and nitrogenase during complex Fe–S-cluster assembly. , Surface representation of HydAΔEFG (grey) superimposed on native CpI (green ribbon and ball and stick representations). , HydAΔEFG channel leading to the active-site 2Fe-subcluster recipient cavity. , , Electrostatic surface representations of HydAΔEFG () and the catalytic domain of CpI (), calculated using the PYMOL plug-in APBS. Red, negative (-10kBT/e); blue, (10kBT/e); kB, Boltzmann's constant; e, elementary charge. , Surface representation of the FeMo-cofactor-deficient form of NifDK (Av1ΔnifB) (grey; PDB ID, 1L5H; ref. 22) superimposed on NifDK from native nitrogenase MoFe-protein (Av1) (PDB ID, 3MIN; ref. 20) (green ribbon and ball and stick representations). , Av1ΔnifB NifDK channel leading to the active-site FeMo-cofactor recipient cavity. , , Electrostatic surface representations of Av1ΔnifB NifDK () and Av1 NifDK () calculated with identical APBS parameters as in and and represented using the same colouring scheme. The atomic colouring scheme is the same! as in Fig. 1 and the unknown atom (X) of the FeMo-cofactor in is blue. Accession codes * Accession codes * Author information * Supplementary information * Comments Primary accessions Protein Data Bank * 3C8Y * 3LX4 * 3C8Y * 3LX4 Author information * Accession codes * Author information * Supplementary information * Comments Affiliations * Astrobiology Biogeocatalysis Research Center, * David W. Mulder, * Eric S. Boyd, * Ranjana Sarma, * Rachel K. Lange, * James A. Endrizzi, * Joan B. Broderick & * John W. Peters * Department of Chemistry & Biochemistry, Montana State University, Bozeman, Montana 59717, USA * David W. Mulder, * Eric S. Boyd, * Ranjana Sarma, * Rachel K. Lange, * James A. Endrizzi, * Joan B. Broderick & * John W. Peters Contributions The structural work was conducted by D.W.M. with contributions from R.S. and J.A.E. E.S.B. led the phylogenetic work with contributions from R.K.L. J.W.P. supervised the work with assistance from J.B.B. D.W.M., E.S.B. and J.W.P. led the manuscript preparation with contributions from J.B.B., R.S., R.K.L. and J.A.E. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * John W. Peters (john.peters@chemistry.montana.edu) Coordinates and structure factors of C. reinhardtii HydAΔEFG have been deposited in the Protein Data Bank under the accession code 3LX4. Supplementary information * Accession codes * Author information * Supplementary information * Comments PDF files * Supplementary information (6.9M) This file contains Supplementary Figures 1-5 with legends, Supplementary Tables 1-2, a Supplementary Discussion and References. Additional data
• Fetalogue
  - Nature 465(7295):260 (2010)
  Early learning.