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Mote than 97% ofthe products supplied by R&D Systems are developed and manufactured at our own acts This enables us o maintain strc manufacturing and quality control standards, ensuring that our reagents meet the high eves of performance and consistency expected by out customers, OFFERING MORE THAN 15,000 QUALITY PRODUCTS including: Proteins Antibodies ELISAs Multiplex Immunoassays Stem Cell Products & More! - Request a catalog online: - wrw.RnDSystems.com/go/Catalog RACIST Nake ccs Selection expanding weeky—visit www RnDSystems.com/go/request to sign up for week!y new product updates.g \ (> ER {Q) imagination at workEDITORIAL LETTERS oa ing Science and Society 957 Sustainable Foresting Peter ane ond ln tester Easier Said Than Done Ep. sche and Schule NEWS OF THE WEEK Responsible Researchers Requited 928 Race for Cellulosic Fuels Spurs Brarian Research Program 929 Ehles’s Retirerent Called ‘Big Loss for Science 931 Fear of MRI Scans Tips Up Brain Researchers 931 From Science's Ontne Daily News Site 952 Biologists Rush to Protect Great Lakes From Onslaught of Carp 993. Prominent iranian Scientist Blocked From attending Physics Meeting 933 From the Science Policy Blog 34 EMmbatled UK. Scents Defends ‘ack Record of imate Center NE tevnger and 8 ser Ol and Water Do min Jt Koenaa 1989 CORRECTIONS AND CLARIFICATIONS BOOKS ET AL, 960 The Nature of Technology 1,8. uh, vend by MM Alerander 961 Medicine and At Imagining a Future for Life and Love—Leonarde da Vin, Okyo, Damien Hirst Faro and Aral, uanors POLICY FORUM 962 NIM Guidelines for Stem Cell Research ‘and Gamete Donors 935 Behavioral Addictions Debut ional ‘nfeepored AY PERSPECTIVES NEWs FOCUS 964 Rise of the Rival 938 Branding Over Mammography 959 Leprosy’ Last Stand—or Early Days ofa War of Aiton? ‘940 Improbable Partners Am to Bring Biotechnology toa Himalayan Kingdom 942 Jolt Mathematics Meetings ‘A. Norland 8. Mabon >> Reports pp. 1000 ana 1008 985 Cooperativity Tames Reactive Catalysts PR Scteiner >> Report 986 986 When the Beginning Marks the End A. Meg on68.Botow page 936 Politis as Unusual >> Research Article p. 923 page 962 peered 988 On Giant Filter Feeders oH ora SPECIAL FEATURE >> rors pp. 9900 93 945 2009 VISUALIZATION CHALLENGE ike, TS LE Rione bm Hehy Petar Alenor >For rbd one cote tae ‘mune sciencemag.ong/speciatvis2009/, itil See Farce CONTENTS continued >> cover DEPARTMENTS “araching Morphogenesis” an station made rom mre han 917 ThisWeekin Science 75,000 weronece cable i ies iste the pede oes 925. Eats choice eerste by human ing endothe esther neers 926 Science Stat “within an extracellular matrix over time. Winners of the 2009 927 Random Samples 1026. New Products “ScinceiNS International cence & Engineering Visualization 1027. Science Careers Challenge ae featured in a special secon starting on page 945, mage: Jemy Sabin instalation: Petr ly Janes, Jemy Sabin, ‘Andrew Luci, mete FleraSabinsJonesLabstudl, Unies, of Pennsylvania wuwsciencemag.org SCIENCE VOL327 19 FEBRUARY 2010 onChoose QIAGEN for detection Detection platforms, assays, and analysis software a by QIAGEN Use QIAGEN? solutions from sample to result, and benefit from sensitive and reliable detection systems: 1 Quantitative, real-time PCR detection HE Automated analysis of DNA fragments and RNA IE Pyrosequencing® sequence-based DNA detection and quantification i M1 Oplimized, ready-to-use assays and reagents e0cecee lar eaten ae ee QIAGEN® Tel lM mL aM CKauired Ce}www.sciencedigital.org/subscribe For just USS$g9, you can join AAAS TODAY and start receiving Science Digital Edition immediately!www.sciencedigital.org/subscribe For just USS$g9, you can join AAAS TODAY and start receiving Science Digital Edition immediately!CONTENTS i 479 Cane UndesandCusWithout 996 Regulation of Atermatve Sling 972 Rettospective: Marshall Waren Wienberg wi s ie (4927-2010) ing macnely. “es Peder “ “_ RESEARCH ARTICLE byte se citer 7 erin actin ot cares [S20 Greaes Specific Degradation Signa cs Han tefabalc function in buman ther ici 1008] Acetylation of metabolic Enaymes vou ermal scpte ep Coordinates Carbon Source Uitzton cn 3 and Metabolic ux Ong al REPORTS Reversible actation of metabolic enzymes 977 Kepler Planet-Detection Mission: belps bacterla adit to changes nfo Inroduton and Fs Results Wh tate Ntolcnetstuescnfiontheenene 1008 ‘Sonifcant Acetiaion age 960 ot planes wi eis over han thse Jnsor Chinese Coplands Inensiying apt in China nh 980. Turing the Dimensional of the sina China i = +H. Shishido etal a f Aavetumpherewonsion\sshewdby 1010 Peptdomintic Antics Target . \ tying the imensin of hey fermion Outer Membrane Blagenesis he er in Preudomones aeruginosa a i Mines tal 84 The Sicate Mediated Formose Reston: Asytesed baits pee <= Bottom-Up Synthesis of Sugar silicates JA. lombertetal Sete Paitnbcs pthogste, Sagar growth and stbization ata 1014 NMR Structure Determination for Larger — ty slate one te a possible prelate Proteins Using Backbone-Only Data les fide Protein structures can be determined by using gee 969 & 980 986 Asymmetric Cooperative Catalysis the limited nuclear magnetic eon of Strong BranstedAcid-Promoted Seren ish for erp Reactions Using Chiral reas 1018 Limits of Predicabitty in Human Mobility Hduetal, Songetal hiralco-atalyt complements acid ysis of he trajectories of peopl 990 | 100-Miltion-Year Dynasty of Giant Plantivorous Bony Fishes inthe Mesezoe Seas 1M, Finan ta CONTENTS contnuad >> poge 1014 993 | Climate Critters, and Cetaceans: Cenozoic Drivers of the Evolution af Modern Whales FG Morand M.D. hon The erty of whales during the Cenezai wwwsciencemag.org SCIENCE VOL327 19 FEBRUARY 2010 913Call for Papers Chief Scientific Editor Michael B. Yaffe, M.D., Ph.D. Associate Professor, Department of Biology ‘Massachusetts Institute of Technology Editor Nancy R. Gough, Ph.D. ANAS Sifejaeel ae Science Signaling Science Signaling, from the publisher of Science, AAAS, features top-notch, peer- reviewed, original research weekly. Submit your manuscripts in the following areas of cellular regulation: * Biochemistry * Molecular Biology * Bioinformatics * Neuroscience * CellBiology — * Pharmacology * Development * Physiology and * Immunology Medicine Microbiology * Systems Biology Science Signaling is indexed in CrossRef and MEDLINE. Submit your research at: www.sciencesignaling.org/ about/help/research.dtl Subscribing to the weekly Science Signaling ensures that you and your lab have the latest cell signaling resources. For more information visit www.ScienceSignaling.orgSCIENCEONLINE CONTENTS SCIENCEXPRESS ‘wer aclncexpress org Design of Polymethine Dyes with Large Tird-Order Optical Nonlinearies and Loss Figures of Merit LM Halse a Nerinear optical materi ave destned and characterized for potetial applications inaltaptia suteing so 3126loence 1185117 SCIENCENOW vr actonconou.org Highlights From Our Daily News Coverage Is That Elephant Running? Don't Bet oni New study argues that, regardless ofthe speed, elephants don’ actually ron, What Doesn’ Kil cre, Makes Them Stronger Low levee of antibiotic trigger mutations that cause resistance Ancient Human Sequenced for Fist Time strands of hale pla etal pltre of 4000 year-old Greenlander SCIENCESIGNALING ‘aclencengnating.org ‘ha Sign! Transduction Kramledge Envirenment AESEAROH ARTICLE: Microbial Hacking of Complement ike Receptor costal Meng Pathogen nstated canta ten he complet (Saveceper anda ie reepor 2 dele ate AESEARCH ARTICLE: Deciphering Protein Kinase Spicy Through large Scale Analysis of Yeast Phosphorlaton Site Matis tots Atighshoughpu peptide ray approach reves insight ine Kase subatesae speiicy CONNECTIONS WAP: Jasmonate Biochemical Pathay Agel Jnomonate ae potent pylon development Tepes ha aya press CONNECTIONS MAP: Arabidopsis Jasmonate Signaling Pathway ‘A Gir stl Jasmonate replat aloes apc of physllogy tnd development n Aad that, Poocast 6: Hapenengaleond AM anooe The pathogen Porphyramons onsets eves the lnate mene ten by oii galing 8 cross tah a wuwsciencemag.org SCIENCE VOL 327 SCIENCECAREERS won seiencecareets.ora/career magazine Fret Career Rerources for Scents “Tooting Up: The Applications Scents Career Tack DJensen to other industy careers Protecting Poland's Rapeseed Crop Acuy Malgorataerealsyeno dtecting ‘top pathogens has became a at Industry sponsored program Perspective: Audacity Is Overrated Diamonds The audacious approach to scence snot the best spptach,especlly ft sclentts In Wain, can ik the Boneh SCIENCETRANSLATIONAL MEDICINE ‘ve slancetranlationsimedcine.org Integrating Medicine and Science COMMENTARY: Ten Years of the Immune Tolerance Network—An Integrated Clinical Research Organization A Bison eta Nw insights frm old programs yield the equation fora successful esearch program COMMENTARY: Medical Education Research a5 Translational Science WE meGoghie Earl Sncnpoaton of ransatonal sence {in physician education wl improve patent cae RESEARCH ARTICLE: Long-Term ‘Thermostabilzation of Live Powial and ‘Adenoviral Vaccine Vectars at Supraphysological “Temperatures in Carbohydrate Glass Rocka Asicoce-teelase lass fit die ono iter ‘an preserve the acy of saci vital vectors. RESEARCH ARTICLE: S-Nitrosylation from GSNOR Deficiency Impairs DNA Repair and Promotes Hepatocarcinogenesis Wo ssetaL ‘An eye that removes harmful mars fom cluar proteins guards apne Wer cance SCIENCESIGNALING Insight coan oral pathogen SCIENCEPODCAST wen sciencemag,org/multinedia/sodeast Free Weekly Show Download the 19 February Scence Podcast to hear shout predicting human mobil patterns, posable mechanism of prebiotic sugar formation, the winners ‘of the 2009 SclnceM Vsulzaten Challeng, nd more. SCIENCEINSIDER blogs siencemag.org/sciencelnsider Soence aiey News ane Asie AVAAAS 19 FEBRUARY 2010 15“The fastest separations and the gold standard method. That’s electroforward thinking.” Next-generation precast gels. No special buffers required. Introducing lang she life Mini-PROTEAN™ TGX (Tiis-Giycina eXtended) Gals trom Bio-Rad, ‘ha wees TEX iin sree, Gel with us at www.minipratean.com, Research. Together. ‘We at wrmbioc.com BIO-RADema RCM eR ECs Whales are the largest animals today, and many feed on the abur: dant plankton, particularly diatoms, in the oceans. Whales arose and diversified in the Cenezoic, about 30 to 40 million years ago nd Uhen (p. 993) show that their diversity parallels the diversity of diatoms and changes in ‘ocean temperature, Whether there were large predators of plank n before whales has been enigmatic, because the fossil record during the Mesozoic (245 to 65 million years ago) is sparse. Fried ‘man et al. (p. 990) now show that a group of large fish filled this eae CMC es nus ean eed large as whales, these globally distributed fish were still several meters long. Their extinction at the Cretaceous-Paleogene boundary 65.5 million years ago may have cleared the seas for the Gee the Perspective by Cavin). Marx To Degrade or Not to Degrade Regulating the turnover of patens within the cell isof fundamental imaortance to almost every phys iological process. Hwang eta. (. 973, published inline 28 January, see the Perspective by Mogk and Bukau) now find that acesyated N-terminal methionine Met) isa degradation signal This ddegron is recognized by Saccharomyces cerevisiae 'Daat0, a transmembrane 63 ubiquitin ligase that resides inthe endoplasmic reticulum and inner nuclear membrane, The removal of N-terminal Met bby Met-aminopeptidases generates N-terminal residues that are often N-terminally acetyated, 'Daai0 sletvely binds tothe resulting N-degrons, wich may represent the most prevalent cas of Cellular protein degradation signal 2D Quantum Critical Transitions (Quantum critical transitions occur at near-2er0 temperatures when the properties of quantum matter are tuned by an external parameter such asthe magnetic field or pressure. Heavy fermion ‘materials, which have effective charg carr ‘masses hundreds of times heavier than the bare electron mass, have emerged as a prototypical system for studying these transitions. Now, Shishido etal. (p 980; see the Perspective by Coleman) use 3 heavy fermion compound to experimentally realize a new type of quantum phase transition wher the tuning parameters the dimensionality ofthe system, They engineer @ Family of superlattices made up of fixed number eerie decrease, the system gradually changes chara ter from tree to two-dimensional, with corre sponding changes nits transport properties Detecting Distant Planets |More than 40 planets have ben detected out side the solarsstem, most of which have masses similar to that ofthe gas giant pane, upt Boruck eta. (p. 977, published onlite 7 Janu ay) summarize the planetary findings derived from theft sx wees of observations withthe Kepler mission whose objective isto search for and detecine the frequency of Earth-like planets inthe habitable zones of ther stars. The results ‘ncude the detection of five new exolonets, ic confirm the existence of planets with des ties substantially ler than those predicted for 2s ian plants. Silicate in the Primordial Soup Direct evidence for how prebitic synthesis of complex organic molecules paved the way for the origin af ite is extremely scarce. Thus, stud ies are mainly limited to controled simulations of ikely reactions in early Earth conditions. Similarly, chemical reactions inthe laboratory ‘may generate the products necessary for biosynthesis, but may nevertheless be geo- chemically relevant. Lambert et a. (p. 984) show that silicate ions, present in Earth's sur face waters at relatively high concentrations, catalyze the formation offour- and siecarbon sugars from simple sugars via the formose reac- also circumvents the need forthe formose reac tion to proceed at high temperatures, thus extending the range of possible environments in which fe could have originate. Acid Assistance Protons are quite versatile catalysts of organic reactions, but because they are achiral they can- nat induce streoselectity an their own, One productive way around this problem has been to ‘here the bases | remain tightly hoe substrates. Xu et al (0. 986; see the Perspective by the mechanism of an alternative approach in which an achical acid was used in conjunction with catalysis. High selectivity was attained with this ‘method in the coupling of ary imines wit olefins showed thatthe acd and its chiral partner acted cooperatively in binding the substrates, optimiz- se chiral conjugate bases and perform Paster reactions in med attracted to protonated ~ ~~ Schreiner) thoroughly explored . a second, cial molecule (aurea erative) for Extensive hneti and computational ties ing the tradeoff between speed and select Metabolic Regulation Through Acetylation Covalent modification of tysine residues in var (us proteins inthe nuceusis a recognized mech- anism for contrat of transcription, Now two 8 ctlyesorteconentoratmetltan,andvay | fon The sting comple: sie he | apes sugestht cation may epee 2n FE ingrumberso ae heey ten mate sag moles lng sas ccmulte B ralcan, Athemnbertlyersot cans | iaretr abundance tate sbizton Cina ope 515 vawusciencemag.org SCIENCE VOL327 19 FEBRUARY 2010 97xCELLigence System: CIM-Plate 16 Monitor Cell Migration in Real-Time Exceed the Limits of Cellular Analysis Continuous monitoring of HT1080 cell migration using 2 fibronectin-coated CIM-Plate 16 with 103% serum serving 88 the chemoattractant in the lower chamber. Calis in ibronectin-eoated wells (red trace) show enhanced migration compared to cells grown without fbronectin-coating (green trace) Cells maintained in serum-free media served 88 9 contrat (blue trace). SANACEA ‘Take a step forward in cancer research with the new CIM-Plate 16 for the xCELLigence System, featuring electronic sensing to directly study cell migration and invasion. Replace exogenous labeling with impedance-based technology. |= Monitor cell invasion and migration continuously in real-time ‘over the entire time course of the experiment = Obtain unbiased data using label-free measurements, that do not perturb cel ‘= Experience the full flexibility of the RTCA DP Instrument: Combine E-Plate 16 to measure cell proliferation and CIM-Plate 16 to quantify cell migration and invasion Go beyond techniques requiring cell labeling, Focus on physiologically relevant cell responses using the xCELLigence System ~ now with cell invasion and migration electronic sensing. For more information, visit: www.xeelligence.roche.com. Roche Diegnostics GmbH Roche Applied Science Werk Penzberg 82872 Penzberg, GermanyThis Week in Science Continued fom poge 917 important regulatory mechanism controlling the function of metabolic enzymes (se the Perspective by Norvell and McMahon). Zhao eta. (9.1000) found that a large proportion of enzymes in various ‘metabolic pathways were acetylated in human liver cell, Acetylation regulated various enzymes by dis- tinct meckanisms, directly activating some, inhibiting one, and controling the stability of another. Contra of metabalism by acetylation appears to be evolutionarily conserved: Wang et al. (9. 1004) found thatthe abiity ofthe bacterium Salmonelaentericum to optimize grom on distinct carbon Sources required differential acetylation af key metabolic enzymes, thus controling flux through meta- bolic pathinays. Cropland Acidification in China Chinas experiencing increasing problems with acid ai, groundwater pollution, and nitrous oxide emissions. Rapid development of industry and transportation has accelerate nitrate (W) emisions tothe atmosphere. Consequenty, sol degradation, water shortage, and pollution, in addition to atmospheric quality dete are becoming major public concerns acoss China Since the 1990s, China has become bath the largest consumer of comical Ntertizers andthe highest cereal reducer inthe word, which has consequences for arabe soil acidification. Guo ea (p. 1008, published online 11 February) present a meta-analysis ofa regional acaifcation phenomenon in Chinese arable soils that i largely associated wth higher N fertilization an higher crop production Such large-scale soi acidification istikely to threaten the sustainability of agriculture and afect the bio- geochemical cycles of nutrients and also tonic element in oil Predictable Travel Routines Wile people rarely perceive thei actions tobe random, tartent models of human activity ae fundomentaly sto- hast. Processes that lyon human mobility pattems, tke the prediction of new epidemics, afc engineering, or iy planning, coud benefit ram highly acute predictive models, To avestigate the peediability of human dynam- 4c, Song eta. (p. 1018) used the recorded trajectories of millions of mobilephone ses, oleted by mobile phone ‘companies and anorymized for esearch purposes. They hypotnesized tat give the wide range of travel pater that éitferet users follow, there would be sgrifcanteiterences between their predictability 2: wel: Users who travel less shouldbe easier to pred than those who ae constantly on the road. Suprs- ingly, there was 934% predictability across the whale use base, and individuals predictability did not in genera fl significantly below 80%. Killing Pseudomonas Gram-negative Pseudomonas bacteria ate opportunistic pathogens, and drug-resistant strains present 2 serious health problem. Srinivas etal. (p. 1010) synthesized a family of peptidomimetic antibiotics thats active only against Pseudomonas. These anibatis do nt ise the cell membrane, but instead target an essential outer membrane protein, LptD, which plays a roe inthe assembly of lipopolsac caride inthe outer cell membrane. Activity ina mouse infection model suggest that the antibiotics ‘might nave therapeutic potential In addition, Lot is widely distributed in gram-negative bacteria and ois validation asa target haste potential to drive development of antibiotics witha broader Seat of actity against gram-negative pathogens Examining the Backbone Determination of trtary protein structures by nuclear magnetic resonance (NMR) current relies heavily on side-chain NMI data Te assignment of side-chain atoms is challenging. In adition, proteins larger than 15 kilodaltons (kD) must be deuterated to improve resolution and this elimi- nates the possibility of measuring long-ange intrpraton distance constraints. Now Raman et al (0.1014, published online 4 February) use backbone-only NMR data—chemical shifts, residual dipolar coupling, and backbone amide proton distances—avalabe from highly deuterated pro- tein to guide conformational searching in the Rosetta structure prediction protocol Using this new protocol, they were able to generate accurate structures or proteins of up to 25 KD. wuwesciencemag.org SCIENCE VOL327 19 FEBRUARY 2010 Science Careers in Translation Want to bull eationships with clinical or basic scientists? Get advice on the best way to conduct a clinical and transitional Science career? There's no beter place 9 explore these ideas, and to buld new Scientific relationships, than CTSCNet, the new online community from Science, Science Careers, and AAAS made possible bythe Burroughs Wellcome Fund. ‘There's no charge for joining, and you'l enjoy access t © Practical and specific information on ragatingacaee in clbicalor ‘ransatonl research (© Opportunites connect with ther scientists ining peers, mentors, and (© Access tothe resources ofthe woes leading multisciptinary professional Soxiety and those of our prtaer Connect with CTSciNet now at: ‘Community ScenceCareers.org/CTSciNet CTSciNet sz Clntcal ard Transtlonal lence Network reed MVAAASRare Mutation Matters! Find it by deep sequencing of whole exome or candidate genes BGI'S GENOMICS BAR tech@genomics.cn www.genomics.cn 86-755-25273395 v K —= aN gpa Would 128 Illumina HiSeq 2000 Sequencing Systems, 500 bioinformaticians and the use of cutting-edge software programs developed by BGI satisfy your taste? One of the world's biggest genome centers is looking for collaborators!Bridging Science and Society Poter Aare ithe THE THEME OF THIS YEAR'S ANNUAL MEETING OF THE AMERICAN ASSOCIATION FOR THE ADVANCEMENT President of ARAS and of Science (AAAS) seems especially timely: Bridging Science and Society. Virtually every major dinecor ofthe Malaria issue now confronting society has a science and technology component anc! this means that “the Research nse at need for general scientific understanding by the publi has never been lager, andl the penalty for Johns Hopkins Uni- scientific illiteracy never harsher"™ Today, science and technology are receiving unprecedented versity, financial and policy support worldwide, as more countries invest in science and science education with dhe belie at these investments will enhance economic strength and improve theives oftheir citizens. Inthe United State, the current national leadership frequently focuses on seience, ei y, ence education, and science-based policy-making. As well, the US. National Science Board just reported in Stience Indicators 2070 that the gencral citizenry continues to hold scientists in high J] zit, second only to firefighters in prestige. But this confidence and prestige depend on a belief U] inthe integrity and creeibilty of science, as wel sin the sciemifie commu nity’ ability to help solve global problems. A spate of rocent incidents has threatened the public's trust and argues that greater attention is essential to ‘maintaining a strong bridge betwoen science and the rest of society = ‘The ability of science wo deliver on its promise of practical and timely solutions tothe world'sproblems does not depend solely on esearch accom- plishments but also on the receptivity of society to the implications of sci- entitie discoveries, That receptivity depends on the public auitude about ‘what seienee is Finding and on how it perceives the behavior of scientists themselves. The past decade saw substantial tension inthe science/society Nan teshneris the relationship emerge when scientific advances and theories conflicted with chief executive officer certain cultural values or religious beliefs. Much ofthe turmoil surround OF AAAS and executive ing the teaching of evolution in public ‘or example, derives from publisher of Science, conflict betwoen a modern understanding of eveltion and religious belifs increation. Likewise, objections to embryonic stem cel research arise ftom the belie of some religions that life begins atthe moment of conception, These kinds of tensions tre best addressed by engaging with the public onthe issues and secking common ground when- ver possible. This approach requires seientits to listen and respond to the public's concems and ‘ocducate their fellow citizens about scienlfic advances, Public engagement isincreasingly being facilitated by governmental and nongovernmental institutions, and it has become a high priority for many individual scientists around the word, A focuson creatinga genuine dialogue has cons tently been mere productive than unidirectional attempis at “public education” about scienc Inappropriate behavior by scientists also weakens the bridge between science and society, at times to a degree out of proportion tothe incidents. Widely publiized examples of seien- tific misconduct, or even mere accusations of misconduct, can tarnish the image and diminish the eredibility ofthe entire scientific enterprise. Likewise, undisclosed conflicts of interest, ‘whether real or apparent, can call into question the integrity ofthe whole scientific community Scientists also jeopardize the ereibiliy of science by overinterpreting or misstating scientific Fats, Revent examples inelude misinformation onthe prospests of Himalayan glaciers and the effects of climate change there, and newly discovered problems with a {998 report liking vac- eines to autism, These types of revelations are highly problematic for poliey-makers, the pub- lic, and the scientific community. Every such case should be investigated, with a follow-up public explanation. Scientists should not tolerate threats tothe integrity of science, whether they come from outside the scientific community or from within it “The scientific community can strengthen the bridge between science and society by ensur- ing vigorous enforcement of scientific behavioral norms and standards, augressively focusing problems of global importance, and actively engaging with the public. As scientists and policy-makers convene in San Diego this week at the AAAS meeting, we all should commit ‘o pursuing these goals. = Peter Agre and Alan I, Leshner 8 pe, Ne Bang Speech, 2003, Rep ablpe gabe prasheriny aura 2ODaage eee wwnsciencemagorg SCIENCE VOL327 19 FEBRUARY 2010 921Only days left until bio begins mee are ae© a secondary spectrum mar View from the Ground Ce cnet Pome tee ry be pu ee ee et ee St tere ee eee ene es roe ore eee eee ceed Se er er eer ens er er eee a pene es eee a ee ee ert as ee rn ee ee Reet eee en ee ne ct Pee eer reer Tira nner mere in ene or ee en) eee eer ce eee ers m0 Sh ee een eee er eee eet eee ee Nie ee ete Cee ae eee neta ee, improve secondary markets have had a positive impact. the volume of trading has become com= Increasing Turnover parable tothe volume of intial allcations. Also, Used cr salesmen and yard sale bargai depend on secondary markets, in which the seller ofa good snot the first to have sold the (good. These markets ae cuca for the efficient reallocation of resources to meet changing demands, Yet regulations onthe use and trade of winters | the average time neaded to approve trades has diminished from 150 days fra persona com munications service license in 1998 tole 50 days in 2005. — BW some goods can influence the efficiency with which a secondary market functions. uch isthe | cx v ase with the US. wireless spectrum market (muchas bee munications Commission's (FCC's) se of auc width Make or Break written about the Federal Com In the past several years, increasingly select chemical methods have emerged to append dit ferent groups to specific sites along the Two recent tions to make initial allocati lessisknawn about how licenses are later bought ob periphery of aromatic ring and sold, Mayo and Wall- studies focus instead on manipulating the sten analyzed FCC records ring framewock itself, Donahoe and Bow on icense transfer since show thata widely used olefin metathesis catalyst can direct the formation of furan rings by stitching together an enone and 5 influenced th an allytc alcohol. An advantage ofthis 1994 to assess how requ try contr and approval femergence and function of process isthe ease with which diverse func tional groups canbe introduced throug subsitation ofthe precursors. The immed product of m ‘is pushed along torte final aromatic cycle by ether a sepa ket, Their findings suggest that steps taken by the FCC inthe early 20005 to wwwesciencemag.org SCIENCE VOL 327 19 FEBRUARY 2010 rate acid catalyst ora Heck protocol that appends an additional aryl substituent. Sattler and Parkin work from the ather end of he spectrum, showing that an intact quinoxatine falls rather dramatically apart on contacting a ungsten complex. Aromat among the strongest in organic mole ons, yeti this roduct—formed at 90° haracterized crystallograpicaly atoms previously bounding an edge ofthe ring are separated and linked independently to the angsten center. Their hydrogen substituents are also last inthe process. Though the mechanism onds ae ar skele arben ‘emains uncertain, the authors postulate that insertion ofthe tungsten into the CH bonds racedes CC scission, — SY 0913466107 2010 USA 107, 10.1073 463, 5 Remember, Repeat After Me (Quantum information processors in which information is encodet ‘quire the development of multiple compe- ents, to generat the single pho hem a single photons s, store 1 then eeliaby read them from men 923ESOF20|Osss-ane “@ On July 2-7, aera Torino, Italy, will host the fourth edition of ESOF national meeting held every two years, dedicated to scienti novatior tunity fo present and discuss the frontiers of scientific and technological advancement, Conferences, workshops, and keynote lectures [part o ic Programme! wll be held at the impressive Lingotto venue, where ihe Exhibition will also be set up. There will also be plenty of activities ically dedicated to young res hers (Career Programme} ‘and potential entrepreneurs end inve ly innovative businesees (Science to Business Programme). In addition, Torino will showcase many original events aimed ot the general public (Seienee in the City Programme], held in historical palaces, squares, ofthe city The Scientific Pregranmess main feature of ESOF2010 We have chosen ten main themes as 0 focus f ent and adaptation of organism: 1nd up from Responding to global needs ronliers in energy research and belief ‘ory and learning in organisms, social and artifical systems guages, culures, and variability -¢ with health care integrating si The Scientific Programme at a glance 10! speakers from 39 counities, 7 Nobel laureates among the speakers, 1 Opening Lecture by Julia Fischer, nary lectures by Peter Agre, Kurt Withrich, Ada Yonath and Anton Zeilinger, 18 Keynote Lectures by prominent scientist ESOF2010 is organized by TopESOF - Torino per 2010, an association set up by:Continued from page 923 ‘ry and 9ass them on. Light, however, is always fn the move, usually quickly, and so solid state quantum memories representa crucial component, 3 they would allow the bits of information to be stored while other bits can bbe manipulated, Afeetus ef al. present a solid~ state quantum memory in which an absorbed photon is stored as an electronic excitation within a rare earth ion-doped crystal. The energy levels and optical transitions of the ‘aystal can be contialled and manipulated by a series of laser pulses, so thatthe absorbed pDhoton can be stored for several tens of microseconds. The process can then be reversed to retrieve the photon from the stored electronic tate, Such a robust on- demand quantum memory and repeater should prove invaluable for long-distance quantum communication networks. — 150 Phys, Re. et 104, 40503 (2010). The Fantastic Fourth No Longer The number of exogenously expressed factors that can reprogram somatic cells into what are called induced pluripotent stem celis PSC) has gronn over the past 3 years. The original quartet of transcription fators—Oct4, Sox2, KU, and e-Mye—heads alist that includes familial relatives of the last three and other proteins such asthe transcription factor Nanog and the RNA-binding protein Lin28. Oc4 had appeared tobe indispensable and thus had bbeen considered an essential pat of the reprogramming code. However, Heng eta. report that Oct@ can be replaced. They screened 19 nuclear receptors and found that exogenous expression ofthe nuclear receptor NrSaz in mouse embryonic fibroblasts could enhance reprogram ming efficiency by a factor of 4 as compared to the famous four fac- tors.lone. Moreover, NeSa2 could infact place Oct and act with Sox2, tf, and c-Myc to reprogram somatic cells. The gene expression and chromatin modification profiles ofthese iPSCs (like the iPSCs generated by expression of the orginal four factors) were more similar to those of embryonic stem calls than those of mouse embryonic ibro- blasts. Furthermore, the target genes of NrSa2 overlap with targets of Sox2 and KM that are Jmportant in embryonic ster cell identity, such as Nanog, —LOC Cell Stem Cel 6,367 (2010. wwsciencemagorg SCIENCE VOL 327 EDITORS’CHOICE One-Way Ticket ‘Within the nucleus, chromatin is spatially organ ‘ned, and the intranuclear locations of genes can be correlated with ther transcriptional activity ‘Ahmed eta. have identified two DNA sequences ‘in chromatin that target genomic toc to the nuclear erwelope in yeast. Peripheral targeting ‘of genes, particulary those activated by stresses such as heat shack or nutrient deprivation, such a5 INO, is linked to active transcription. Using lished population. ‘The strategie plan also includes $13 million for the corps to speed completion of a third electrical barrier, now expected by October. Another $13.2 million would accelerate con- struetion of physical barriersom the Des Plaines River and a canal to prevent fish from moving through with floodwaters. Additional funds ‘would go toward developing selective “bub barriers to keep fish from spawning ares inthe (Chicago-area waterways or, ifecessary inthe Great Lakes. There's also $1.5 million for USGS to work om formulating fish poison that ‘targets only Asian carp and $1 milion o study pheromones that might help tap or deter carp. ‘The surest way to prevent carp from getting established, sientsts isto achieve“ecolou- ‘cal separation” by permanently closing the Tocks in Chicago and creating physical barriers {0 water flow in the other entry points to Lake Michigan. That might pose problems for the 0,000 ors recreational and commercial boats ‘hat passthrough the Chicago River Lock cach year. One option, say advocates, isto lift the ‘boats over, but the American Waterways Oper- ators opposes any substantial changes. In December, Michigan’s attorney general sued the state of linois, demanding thatthe canal Jocks be closed, but the US. Supreme Court seclined to hear the case Lodge and others says that an investment in separating the waterways would pay off by also preventing other invasive species, such a the northern snakehead fish, from reaching the Great Lakes and reducing the odd that any of the more than 180 invaders in the lakes will ‘cave inland via rivers. “Its far more expensive ‘always be reacting” to invasions, he says, Apart of the new plan, the Army Compshas| ‘moved up the deadline for its comprehensive study of how to prevent the movement of inva- sive species between the Great Lakes and the “Mississippi River Basin to 2012, That's not fast enough for Gaden and others. "We don’t have the time," he says. There will be quicker action, according the strategie plan: By 30 April, the corps could begin modifying Chicago River Lock operations—opening it for only a few days a week, for example—to reduce the chance of carp geting through, ERIK STOXSTAD 19 FEBRUARY 2010 VOL327 SCIENCE snw.sciencemag.orgU.S. IMMIGRATION POLICY Prominent Iranian Scientist Blocked From Attending Physics Meeting ian physicist Farhad Ardalan has spent nouih time inthe United States to regard tas his second home, But a case of apparent mis- taken identity may prevent him from ever vise iting his adopted country again. Last fal, Ardalan was named a fellow o the American Physical Society, in part bbecause of his efforts to connect Iran to the global scientific community and strengthen bonds hetween tran and the United States, The new class of fellows will be honored at the society’ meeting next month in Portland, Oregon. But U.S, consular officials have derailed Ardalan’s application fora visa after telling him that U.S. government records show he was arested in the United States in 1983 for an unspecified offense. They also say that he may have been involved in depor tation proceedings 20 years earlier AArdalan denies both charges, and his US. colleagues say that the State Department is ‘making big mistake, “He isprecisely the kind of person who should be welcomed to the Visa problems. Farhad Avdalan suspects a “doppe anger” has tainted his application, US.," says Stanford University physicist Herman Winick about Ardalan, a string theo- Fist atthe Institute for the Study of Fundamnen- tal Sciences in Tehran, Ardalan fist came to the United States in 1958 and attended Columbia University, where he received his bachelor's and masters degrees. Aer earning his Ph.D. in 1970, he returned to Ian to teach at Sharif University, ‘where he helped create the first Iranian doe: ‘oral program in physics. Ardalan spent sabbatical years at Yale and Stony Brook universities in 1974 and 1977 and, ‘male short visit to the United Statesin 1986, He claims that, like most Iranians, he wasn't alloweel to Ieave the country for several years after the 1979 Iranian Revolution. “I simply www.sciencemag.org SCIENCE VOL327 could not have been in the US. in 1983," he says about the government's charge, which he first learned about when he appeared for his visa interview on 29 January at the U.S. Embassy in Bern, Switzerland, (Iranians must goabroad fora US. vis.) When Ardalan disputed the charge, he says, the official told him that his name and Fingerprints matched the record of the arrest and that he would have to come back for ‘another appointment to give officials time 10 Took into the ease. “I sai, ‘Forget it? And I sys Ardalan, (On 2 February, an e-mail from the U, Embassy leveled a new charge, “A chock of US. records appears to suggest that you were involved in deportation proceedings before the New York oifice of the Immigration and Naturalization Service on July §, 1962. Please confirm or dispute this information,” the e-mail said, “I don't know what they are talking about” says Ardalan, He says neither of these two charges were raised in 1993 when he and his wife received permanent residency in the United States. For the next decade, Ardalan spent several months every year asa visiting sciemtist at the University of Cincinnati in Ohio, working with his friend Freydoon “Mansouri, Ardalan moved back to Iran after “Mansouti died in 2003, Ardalan guesses that the problem isa dop~ pelginger. “There was a person with the same ‘name who was. leader ofthe Kurdish guerrilla ‘movement as result, for yeas I was routinely stopped and interrogated at the Tehran airport,” Ardalan says. I took a meeting with the head of aisport security to elear his name. Ardalan says he deserves an apology for how he has been treated, and he refuses to go back to the embassy or reapply for a vist. State Department officials did not return calls for comment, The department has been criticized for botching visa applications of prominent scientists, including Goverdhan Mehta, an Indian chemist whose visa applica tion was denied in 2006 (Science, 17 February 2006, p. 933) “We hope the issue ean be resolved and Ardalan can come to the March meeting," says John Clark, a physicist at Washington Univer sily in St Louis, Missouri and former chair of APS's Forum on International Physies. The ‘meeting runs from 15 to 19 March ~YUDHIT BHATTACHARIEE NEWS OF THE WEEK i Sciencelnsider | From the Science Policy Blog The United Nations hs formed joint task force of experts trom Hait and around the world to provide advice on recovering ‘rom last month's disaster. The team hopes to generate a preiminary forecast of seismic activity and provide day-to-day advice toast eit and recovery efor. psy 9USsek The new ci of Europe's tp science pol- Icy office says she's “passionate” about using research and innovation to spur eco nomic development and push governments to invest 3% of ther gross domestic prod ucton science. Keland’s Mite Geoghegan ‘Quinn thnks that nt being a scientist may actualy help her to “beak dow the barr 5" to gaining greater public support. psi yah First Lady Michelle Obama has launched 8 major initiative on childhood obesity that features a now nonprofit foundation. Let's Move embraces beter school lunches, ‘more exercise, and more accessible healthy food, including financial incentives to bing farmers’ markets and full-service groceries to urban areas. htps/bit.yaguuy’ Alang-tunning battle among farmer conservation groups, and UK. scene advisers took anew tum when a team fom the UK. Medial Research Council reported that systematic culling of badgers does redluce outbreaks of bovine tuberculosis in Brits cattle but not enough to justify the cost of the cull. The finding likely wont lend the controversy http: yH7NS Foreigners ving in one ofthe word's mast polluted cities nom have more information about the quality ofthe air in Beijing The US. Embassy, which hasbeen using Tater to publish average hourly readings of fine particulate mater, has also begun tweetng hourly data on ground-level ozone, the prime component of smog. But local residents wil have to use a proxy server to access the data because Titer is blacked in China, hpit.ycQX0Pn Forthe ful postings and mare, goto blogs sciencemag.orgcienceinsider 19 FEBRUARY 2010 933a NEWS OF THE WEEK 934 NEWSMAKER INTERVIEW. Embattled U.K. Scientist Defends Track Record of Climate Center Phil Jones is at the center ofa swirling con- troversy over e-mails stolen ot leaked from the Climatic Research Unit (CRU) at the University of East Anglia in the United Kingdom, which he has direeted since 2004 In the 3 months since those messages came to light, Jones has been battered by erticism thatthe e-mails reveal a failure to share cli- ‘mate data publicly and an effort to prevent certain papers from being cited in interna tional climate change reports. He's stepped down temporarily from his job to allow for an independent inguiry, and he’s been treated for depression, But Jones hasn't walked away from the batileield. “I've got no agenda here, I'm not ‘politician, I'm just a scientist” he told Science during an interview last week from the University of East Anglia Jones declined to talk about allegations concerning the hacked e-mails, citing the ongoing investiga tion, But he forcefully defended the quality of the center’ efforts to create a global tem- perature database, which points to an average 0.1°C warming of Earth’s land areas per decade since 1900, That work earned him a 2002 medal from the European Geosciences Union, among other acclaim. Here are some highlights froma the inter view,a longer version of which appears online. ELL KINTISCH Q: Ifthe CRU data set were set aside, are there ther data that corroborate your find- ings about rising temperatures? PJ There’ the two othr data sts produced in the U.S. [at NASA and the National Oceanic and Atmospheric Administration). But there's alo a lot of othe ev dence showing thatthe world’s swarming, by just ooking ouside and seeing glaciers retreating the reduction of sea ice. vere all heredicton of snow areas in the Northern Hemisphere, the earlier [annual] breakup of sea ice and some fand ice, and rive ice around the world, and the fact that spring seems to be coming ear lierin many par of the word 0: Critics say that your producing the same trends as the NASA and NOAA data sets is insignificant given that you start with the same raw data. Online sciencemag.org ‘Ao extended vce sion of this inter ew canbe found hee PJ There are differences, The two American suse a larger number of flemperature] sta- tions than we do. They both use about 7200 stations, and we use about 5000 stations, But we look at that data in different ways and have different techniques for deciding \hether the stations are used oF not Q: One of the real challenges is going from the available raw data to the final tempera- ture sets that you retease. Do you feel that you have released enough information that someone could repeat that exercise? js Yes, I feel they have. [Our papers] have bbeen peer-reviewed; we've been doing this \work for almost 30 years now. [NOAA] has something called the Global Historical Cli- matology Network, and people cean download the station data it's essentially the same data, it may not be exactly the same— they could go and take that data, make their own choices about what stations to use, ... they could reproduce their own gridded tempera- ture data. A lot ofthe people at the moment criticize what we do but fare not doing) any thing constructive and new Q:A sticking point with some of your crit- ics has been how much of the data isn’t available. Pl}: We've been putting up more of the data online on the U.K, Met Office site [eover- ing] 80% of the stations we use. You can download the data and you can download the program we use to produce the data sets. Q: One concern of your critics is whether there are adequate procedures in place to assure the quality of this data Pj: That’s the sort of work we've dane inthe past and published in the papers. You've emphasized that you have a small staff. Would more people checking these data be a useful thing? Pi: could be useful, but then we've got to bring them up to speed in terms of what we're looking fo... The national meteoro- logical services [which provide the raw numbers] are doing quality control on this data before it even reaches us, : When duting your career has pressure from outsiders to criticize or, as you would put it, “distort” your work become significant? J: In 2007, [as] the blog sites started then, {had responded to some ofthese people in years earlier but had given up... [just didn't have the time 1 respond. They didn’t soem to want to understand, Q: One ofthe skeptics who wanted station data was Warwick Hughes. What did you mean when you wrote in an e-mail to him that “even if WMO [World Meteorological Organization} agrees | wil stil not pass on the data, We have 25 or so years invested in the work.” PJ. Til rather not go there. It was an ‘witen in haste. : When did the pressure become severe? Piz In July 2009, we received 60 Freedom ‘of Information requests ina few days request was for Five counties" worth of data, We probably should've responded to these requests ina different way. We stand by the science that we were doing. Maybe we need {o be more progetive and open about releas- ing data, But the 60 requests were just too snuch to deal with at that one time, Q: Do you have any regrets about how you hhandled the chapter you've co-authored in the 2007 IPCC report? PJ.: No regrets, but I don't really want (0 20 talking about IPCI stand by that chapter. Q: Why are you speaking out now? Pj. ltjust soems like the righ time, Itwas too difficult back in November and December. 19 FEBRUARY 2010 VOL327 SCIENCE snw.sciencemag.orgPSYCHIATRY NEWS OF THE WEEK i Behavioral Addictions Debut in Proposed DSM-V Inpsychiaty, the only disorders that have been considered addictions are those involving alco- hol or other drugs. Now, proposed revisions for the American Psychiatric Association (APA) Diagnostic and Statistical Manual of Mental Disorders (DSM) include for the First time ‘pchavioral addictions" —a change some say is long overdue and others say is still premature (Science, 12 February, p. 770), So fa, only one behavior has made the cut gambling, which under the new proposal ‘Would join substance-use disorders as ful fledged addiction, DSM has recognized pathological gambling for decades, but it has been consigned to a grab bag of “impulse con: trol disorders not otherwise specified” along ‘with klepromania, hair-pulling, and fire-seting, “Many scientists have long believed that com- pulsive gamblers closely resemble alcohol rot only from the outside destroying jobs, fi hips in pursuit their jobsession—but, increasingly, on the inside as ‘well. Brain imaging and neurochemical tests have made a “pretty strong ease that [gam- bling] activates the reward systera in much the same way that a drug does,” says psychiatrist Charles O'Brien ofthe University of Penney vania and chair ofthe addictions work group for DSM-V. Gamblers report craving and highs in response to ther stimulus of choice: gambling also runs along with other addie tons in the same families. Psychologist Gerhard Meyer ofthe Univer- sity of Bremen in Germany says he's been argu- ing since 1982 that pathological gambling should be classified asa behavioral addiction, Research by his group has shown that problem, casino gamblers show inereases in heart rate and salivary stress hormones as well a blood. levels of norepinephrine compared with non. ices, and ratio (eV AE ke Boe aA Co Serene Se eet at www.sciencemag.org SCIENCE VOL327 problem gamblers. The former also show increases in dopamine, the key player in the brain's “reward circuit” Mare Potenza, a psychiatrist and gambling researcher at Yale University, says his group's brain-imaging studies show that when exposed to gambling videos, problem gamblers” brains show “important similaritios” to changes in the brains of cocaine addicts when viewing a video about cocaine Other behaviors may eventus ally follow gambling into DSM as addictions, as more is revealed about their neurobiology and ‘genetics. The top contender at present is“Inter- net addiction,” which now has its foot in the DSM door i ill be listed in the appendix, a atch-all category for disorders that don’t meet criteria for full-fledged diagnosis. Some researchers have argued that people whose ernet use seems to be out of control show ‘many hallmarks of addiction such as toleranee and withdrawal. But theres still no consensus ‘on what constitutes so-called Internet addic~ tion, Some argue that it is not the computer thas the issue but the content mainly sex, gambling, and games-and that people hhooked on the Internet suffer primarily from afflictions such as depression, personality dis- orders, and substance addictions, Some researchers saya case could be made for classifying some eating disorders, bulimia. in particular, as addictions, Family studies show that bulimia clusters with alcoholism. and drug abuse. “Binge eating disorder,” pulled out of the DS4TV’ appendix and now proposed as.a diagnosis in the eating disorders category, similarly has much in common DSM-v ten nN a eer) Se ea ese eee eee with binge drinking. Columbia University physician Timothy Walsh, char ofthe eating disorders work group, says he suspects ‘we'll discover underlying abnormalities in ‘brain pathways" shared by addic- tions and some eating disorders. But as yet, diagnoses in his field are “still really descriptive” as ‘opposed to biology-based “Sex addiction” has received a Jot of press lately, but O"Brien says his work group found “no scientific evidence” that sex qual- ifies. APA psychiatrist Darrel Regier, co-chair ofthe DSM task force, says “it's not clear that reward circuitry is operative in the same way as in addictive areas.” Nonetheless, a near equivalent may make it into the sexual dis ‘orders section of DSM: That work group is proposing a controversial new diagnosis of “hypersexual disorder: ‘The DSM teams have also tussled with the often-blurry line between addictions and com pulsions. “I used to think [addictions] over lapped with OCD [obsessive-compulsive di order)” says O'Brien, But new data fom both brain-imaging and treatment studies suggest ‘moce dissimilarities than similaiies”” In another major change, O’Brien’ group ing at and labeling all problems ‘majorand minor as substance “use disorde (or “disordered gambling”). Since the late 1980s, says "Brien, “numerous large poptt k 2” have shown there's no “break point” where “abuse” becomes something ‘more serious. He also says the term “depend- only implies physiological depend sme as the psycho- logical obsession of addiction, Some longtime addiction researchers, such as psychiatrist Vietor Hesselbrock ofthe University of Connecticut, Farmington, have qualms about the direction DSM is moving, Hesselbrack believes behavioral addictions are dicey territory and prefers to limit the term “addiction” to substances, which are ‘pathogens we can identify.” He also objects to fusing all drinking problems into "alcohol use disordet” Hesselbrock says he and others think there are proven subcategories oF aleo holism that would aid both in treatment and dis- ccvering causes, “When you doa one-size-fits- alliype of classification system," he says, “that ‘ill ita lot oFpeaple but not so well CONSTANCE HOLDEN 19 FEBRUARY 2010 935936 Brawling Over Mammography Ascientific study of the benefits and harms of screening women in their 40s got buried by politics The Obama Administration is a self described champion of science. But it was put on the spot last fall when it received a scientific report that questioned the value of sereening women routinely for breast cancer before age just as Democrats wore trying to push 0. The timing was bad, coming health reform through Congress. To oppo- nents ofthe legislation, the report smacked of “medical ration by bureaucrats, the kind they elaimed to see in the bills. It became a huge distraction, and in the end the Administration quie! from the findin The Map continued, loud and angry, for days. Exports and nonexperts lobbed cri tiques and rebuttals, at times questioning ‘one another's motives. Authors ofthe report distanced its rejeoted the “rationing” charge, s never took cost into account. The nonprofit American Cancer Society, which had endorsed early breast cancer screening (at age 40), announced it wasn't going to change its recommendation. But a leader of 9 BRUARY 2010 VOL327 SCIENCE the American College of Radiology, Daniel Kopans of Massachusetts General Hospital in Boston, vas less restrained, He circulated ‘critique saying thatthe sponsor of the new report—the U.S. Preventive Services Task Foree (USPSTF)—“does not think it is, worth saving women in their 4055... it thinks that women should be allowed to die from their breast cancers. Secretary of Health and Hi vices (HHS) Kathleen Sebelius weighed in ‘on 18 November, Het ageney is the official hhome of USPSTE, b report. Instead, she ran for cover. She noted that USPSTF members are independent not HHS staff-and were appointed in this by George W. Bush. On CBS News, Sebelius advised women to “do what you've Asked Sebelius an Ser- she didn’t defend its always done; .. talk to your doctor.” ifshe was “refuting” the task for said mo, but added tists and health officials who have waded into an area where the recommendations have gone back and forth for years itis one panel of scien- Ri USPSTF guidelines wer ‘earchers who had worked on the disappointed that their analysis was being dismissed out of hhand, “Politis got in the way ofthe science and the best public health practice,” says Jeanne Mandelblatt, an M.D.-epideriolo: tat Georgetown University in Washing ton, D.C., and first author of an analysis USPSTF by si cls to find the best sereet oups that compared mod 1 adds Fleidi Nelson, trate an M.D.-epidemiologist at the Oregon Health & Science University in Portland, ‘whe led a separate team that gather donee for USPSTF Karla Kerlikowske, a breast cancer researcher at the University of California, San Francisco, was “amazed” that “people were reacting in I day” to studies that took ‘Tam a big believer thought peop ‘would really embrace the new science and say, “Wow, his is really good.’ ” One lesson she learned is that it's hard (0 take away her a Tong time to read in science and evidence, something that has been promoted in the past—and mammography at 40 has been widely promoted. Second, Kerlikowske says, “people just didn’t like the answer hat’ what it came down to, unfortunately.” Fury over the 40s The argument over mammography was in many ways an echo of elashes over the same 96 and 20402, when USPSTF, an ‘group whose adviee is intended independ vnwwsciencemag.org‘The closer you look. Experts are debating the false postive in mammograms of younger wore, for primary care doctors and patients, issued its previous statements (Science, 21 February 1997, p. 1056; 1 March 2002, p. 1624), Indeed, many ofthe same protag ponists came back to champion the same arguments. There was a difference, though: The 2002 USPSTF panel said that sereen- ing should begin at age 40; 7 years later, USPSTE said age 50 is when women with rno known tisk factors for breast cancer should begin screening. ‘Asked how the data changed in 2009, USPSTF task force co-chair Diana Petit, an M.D.-biostatistician at Arizona State University, Tempe, said, “No change.” She added: “The evidence has pretty much been the same all along; i’ the packaging and the policy that goes with that packaging, and people's perceptions, ... which seem to con- tinually cause the controversy” ‘The task force did alter the packaging: It took a eloser look than did the earlier report atthe potential “harms” of mammography. ‘These include downsides such as false posi tive x-ray results, exira doctor visits, biop- sies, surgery, and the anxiety that zoes with result that indicates a possibility of cancer, Many clinical trials have indicated that sereening with mammography benefits ‘women over 50, mainly because they have a fast-rising risk of developing breast cancer, Abnormalities picked up by x-rays are more likely to be dangerous; treating them as ean cer is more likely to save lives, and the “price” in false alarms seems worth paying, The panel concluded, however, that for women in their 40s, the benefits oF routine screening by mammography don't outweigh the harms, When to begin screening is complex. No one proposes that teenagers be sereened, for exam= ple. Young women aren't good. candidates because their tissue may be more opaque (o x-rays and because abnormalities, if found, are not likely to be life- 300 Parallels Kala Kerlkowske is develop- ‘ing markers to distinguish risky trom benign OCIS tumors; since the 1980s, detection of DCIS exploded with the rise ‘of mammography. featuring young women who said that a mammogram had sad thei if. Such tes timony is powerful but not relevant, Berry says, “We are all duped by observations,” Berry said in an e-mail, “and much of the medical community buys into sereening. Some (exemplified by Dan Kopans) have their professional reputations onthe line.” ‘The paradox, explains epidemiologist Steven Goodman of Johns Hopkins Univer- yin Baltimore, Maryland, is that “every ‘woman who has been diagnosed through ‘mammography Fels that she benefited, but, wwe know that even without the sercening her cancer would have had a very high chance of successful treatment.” That is particulary true ofa subcategory of small breast tumors known as ductal eareinoma in situ (DCIS), says Kedlikowske, Detection of DCIS has skyrocketed with mammoaraphy: incidence has increased 270% since 1987 Although nearly 100% of women survive DCIS, it’s treated as cancer, often with sur- gery and chemotherapy, because it's not well understood, and—who MAMMOGRAPHY AND TUMOR DETECTION _ threatening (Science, 9 Septem- ber 2005, p. 1664), ‘Common sense and individ- g § t t t : : : a a i § 3 i i § 5 z g i 3 5 3 ual experience —even a doctor's experience of treating cancer isn’t a guide, either. So says bio- statistician Donald Berry of the M.D. Anderson Cancer Center s ‘Ye Change from 1987-2008 in Houston, Texas. His warning applies especially to media reports like those aired last fall ww sciencemag.org 18 195 1987 99 199 206 22 zt SCIENCE VOL327 NEWSFOCUS t face anes Maen arena NG U.S. PREVENTIVE SERVICES TASK FORCE Screen ages 50-75 every 1-2 years Screen ages 50-69 every 1-2 years Screen ages 40-69 every 1-2 years Against routine screening ‘ages 40-49 years, but screen ages 50-74 every 2 years knows?—it nosis of eancers that won't progress, known as “overdiagnosis,” is a “diabolical phe- nomenon,” according to Goodman, because it ean be seen only “through the prism ofa population view.” ‘The best guide to the benefits of sereen- ing, statisticians say, is to look at deaths among women who sign up for sereening in trials and compare this with deaths among those who don’ sign up. Nelson, who pulled together the data for the 2009 USPSTF review, conducted a meta-analysis combin- ‘ng the eight best randomized clinical trials since 1986, This review includes information not in the 2002 USPSTF study from older ‘Swedish trials and from a new trial designed to look at women in their 40s, a U.K. study ‘completed in 2006 known asthe “Age” tra (The Age study found a small benefit breast cancer deaths were down 17% in the sereening group-—but it wasn't significant.) ‘Women in the sereening group had about 15% less likelihood of dying from breast cancer. Nelson's group calculated that this corresponds 10 prevent one death from breast cancer for every 1904 women screened in their 40s, By the same index, ser ing 30-year-olds prevents one death per 1339 women sercened: the panel accepted. ‘That small benefit is no surprise to Peter Gatzsche, an epidemiologist who heads the Nordic Cochrane Center in Copenhagen, Denmark. Gotzsche and Ole Olsen co-authored analyses a decade ago that dismissed mammography-screening pfograms at any age as having no benefit for healthy women, Organized mammogra- phy is hyped, Gatesche has said—a“med- ‘cal seandal” that has “seduced” thousands, Even Gotzsche, however, recently adjusted his reading of mortality data. A 2009 review he co-authored acknowledges that trials have detected a slight decline—about 15% in deaths among women ofall ages ratio 19 FEBRUARY 20107 NEWSFOcUS 938 screened by mammography. But the review finds this is negated by false alarms and unneeded medical procedures At the opposite pole is Kopans. He sees a scandal, 00, but for him it a conspiracy to obseure the good news that mammogra- phy works, Kopans has basic objections to the way that USPSTF and others slice the data, He claims that the 2009 USPSTF analysis used “the lowest possible” number There is “no scientific ridiculed.” She added later that, “People are saying it’s something we should ignore; 1 guess they've never been through a false- positive mammogram.” Goodman, who followed the debate closely, was also troubled by the gibes atthe “harms” tally. He says he keeps a file of “ear- toons that parody the reasoning of the task “much of the medical com- Mangled message ‘The 2009 review aimed for a “nuanced” view of the risks and benefits of screening, Petit says, that would predict what might hhappen under different screening strategies For this, USPSTF turned to “the best mod elers from the top research institutions, says Mandelblatt. A group led by Berry, called the Cancer Intervention and Surveillance Modeling Network, drew on experience from trial data to represent Support” for limiting munity buys into screen- io estimate how different the average benefit of mam- mammography screening | ing. ... We are all duped by screening frequencies and ‘ography for women in their a on Starting points would affect 40s. He insists that experts €0 those over 50. [individual] observations.” je number of x-ray exams, should have cited a range of DANIEL KOPANS, DONALD BERRY, deaths avoided, and false po results from the best trials — MASSACHUSETTS M. D.ANDERSON _jtives, The models plugged from 15% to 44%. Or, he sug- gests, why not use the 44% benefit from the “Gothenburg” trial or the 35% benefit from the “Malm&" trial, both in Sweden, That's what he would do, and he credits mammography with reducing breast cancer deaths in the United States “by at least 30%" over 2 decades. And he insists that there is “no scientific support” for drawing a line at age 50. “The body doesn’t know it's 50," he says. Nelson has a terse response: “Fishing around to find an answer you like is inappropriate.” The dark side Few dispute the USPSTF panel's (GENERAL HOSPITAL force, .. really terrible stuff.” He thinks USPSTF was right; indeed, he urged the panel tobe specific in a critique of its 2002 report. USPSTF authors, he wrote back then, wore too “oblique” inadding up costs, and he called for hard data on the number of Iumpectomies and mastectomies that follow false alarms, Goodman thinks the adverse effects are sill sof-pedaled in the 2009 report, He says that he’s “totally shocked” that “the word ‘mastectomy’ doesn’t appear” in the list of PEACE! CEA tetimate of he benefits of eguniemnd At i, ae ie screening, bu sending ot EAs mse So thers mein 2003, USPSTE eee produced «deta i of we Te Tre ovloones The new Goa (a co a men from he US. Brest Caner Sue vellane Contam, calibe-_ mie ose eae tation of Us. community groups: For every 1000 women Sissy nau! a8 a oo & fered nice dy, he review (ar as found 978 fase-pstve mame ast ‘mograms per round of screening, (up to 10 times a decade), 84.3 call-back doctor visits, and 9.3 biopsies. (Exposure to radiation {isn’t flagged as a major concern.) Expressed another way, for every case of invasive ean: cer detected, $56 women have mammo. grams and five have biopsies. ‘The false positives, the panel said, pro- dduce untold anxiety in addition to needless follow-up procedures, The report's attention to anxiety prompted snickers. In testimony for a House of Representatives panel in December, Petitti said, “The mention of anxiety and psychological distress as a hharm of a false-positive test has ... been ‘Churning For evry 1000 women in ther 403 screened with mam- ography, abovt 100 initial get 2 false positive result, potential harms. The USPSTF evidence team considered getting into unnecessary surgery, according to Nelson, but she says .eo-author at the Oregon Health & Science University, oncologist Arpana Naik, recommended ‘against it. The reason, Nelson says: Naik ‘argued that women opt for a mastectomy out of fear even when test results don't indicate 's needed, and Naik “talks people out of Imastectomies every day.” The team felt that the high surgery rate would reflect US, cul- ‘ural norms, not medical norms. ‘CANCER CENTER gaps in the (rials, Among other things, Berry says, they confirmed the sketchy data on women in their Os and “gave confidence” that screen ing recommendations would be solid. particularly that having a mammogram {every second year was fine for women over 550, They also confirmed that new treatments have not made older trial data obsolete ‘The models led USPSTF to endorse the ‘most efficient plan: Begin sereeningat age 50, soteen every other year, and continue at least toage 74, Petit later acknowledged thatthe ‘echnical language used in the message made it sound more nogative than intended. When the furor erupted, USPSTF added a red-ink cortection on the HHS Web site, making it Clear tha experts did not want wonten to avoid ‘mammography and that women should "con sult your doctor” She concedes, “We commu nicated what we really meant poorly’ Goodman agrees. USPSTE's presenta tion of findings was stiff and failed to con- sider how to communicate risks toa lay pub- lic. The task force, Goodman says, “was not prepared for what ensued” when it fell into the “tindorbox” of the congressional debate ‘on health care, Despite the intensity of the public di agreement that followed the release of the USPSTF recommendations, it’s clear that people in all camps agree on at least one point: Research is nceded to understand the biology of early-stage breast cancer better. Kerlikowske and others conclude that even the best analysis may aot help the public understand risks ifthe disease is scary and the underlying biology is confused A better understanding of DCIS and other early changes in breast tissue might make it possible to differentiate risk better and earlier It might even help reduce the ‘number of explosive policy brawls on mam- ography. ELIOT MARSHALL 19 FEBRUARY 2010 VOL327 SCIENCE yw sciencemag.or iCHINA Leprosy’s Last Stand—or Early Days of a War of Attrition? China thought it had licked leprosy 10 years ago, but the disease is stubbornly hanging on JANGYAN, CHINA—Shunned by his home village, You Ya-Gan has lived in a moat- inged compound here in eastern China's Jiangsu Province for 40 years. In 1970, the farmer was diagnosed with leprosy and bundled off to Jiangyan Leprosarium, Treatment with the antibiotic dapsone tamed his infection, but his hands and feet ‘were irrevocably disfigured and You, like ‘many leprosy vietims, languished in quar- antine until a drug cocktail in the 1980s brought a long-elusive cure, Free to leave the leprosarium, You opted to stay. The other 118 residents are “like family.” explains You, 65, who earns a small income by helping in the clinic, Besides, he con- fesses, “I have nowhere else to £0.” Some 20,000 disabled people still reside in 617 leprosaria or leprosy villages across China. After decades of neglect, the central government has launched a campaign to renovate decaying leprosy facilities. The overhaul is one prong of a reinvigorated strategy against an age-old malady that China nearly vanquished a decade ago but which persists mainly in remote parts ofthe southwest. To researchers’ consternation, the number of new leprosy cases reported teach year in China has held steady since the Tate 1990s, A fresh worry here is a possible uptick in resistance to Fifampicin, the big gun in multidrug therapy. Four years ago, conferees at a World Health Organization (WHO) forum con- cluded that leprosy was “on the verge of defeat” But the recalcitrant foe has not sur- rendered. “The transmission of leprosy is not totally interrupted yet,” says Denis ‘Daumerie, WHO'S project manager for neg lected tropical diseases. “Based on our c\ rent tools and knowledge, itis impossible to eradicate the disease,” adds Wang Baoxi director of the Chinese Center for Disease Control and Prevention’s National Center for Leprosy Control (NCLC) in Nanjing. [Nations have made great strides in bring: ing the disease to heel, Since a cocktail of rifampicin, dapsone, and clofazimine became standard treatment 30 years ago, the slobal disease burden has declined from 5.2 million cases in 1985 to just over 213,000 wwo.sciencemag.org SCIENCE VOL327 last year. Leprosy continues to bedevil afew ccountries—for example, more than half of ‘new eases in 20019 accurted in India, But by WHO'S definition, the disease was climi- nated as a public health threat a decade ago when prevalence fell under one ease per 10,000 people worldwide, ‘Chinese officials have set a mor. tious target: They define elimination as a prevalence of less than one case per 100,000 people atthe county level. nationwide epi- demiological survey in 1957 netted 300,000 ubie Nolding on. NCIC'sYan Liangbin and a anayan patent he recenly operated on, Leprosy is still a ‘scourge in suthnest China, PEs 5S ew ie cases, and by the early °70s, an army of vil- NEWSFOCUS I But the endgame is shaping up as a long march, Early infections of Mycobacterium leprae easily evade detection; it can take upto 20 years before symptoms from the slowly ‘multiplying pathogen show. New eases in China have hovered at about 1600 a year for the past decade, with most occurring in poor, rural areas of three southwestern provinces, Guizhou, Sichuan, and Yunnan (see map), Elsewhere in China, the majority of eases last ‘year was among the [80 million workers who ‘migrated tothe industrialized east and south in search of jobs. “Leprosy detection in this floating population is a severe challenge, Zhang says. An added complication is that in rural China, leprosy remains a social stigma, For that reason, Wang says, “many poor peo- ple refuse to be diagnosed Rifampicin resistance may pose a new threat. Whereas the other drugs in the cock tail hobble I, leprae, rifampicin slays it Rifampicin-resistant strains emerged in the 1980s but may now be gaining traction in China. “We're seeing increasing numbers of cases with rifampicin resistance,” says Chen Xiangsheng, a senior epi demiologist at NCLC, The cer {er is now setting up a national surveillance system for monitor ing resistant strains. in the meantime, China is, striving to make amends to the paariahs who have spent most of their adult lives in ramshackle wards. In December, Jiangyan Leprosarium completed a reno- vation ofits 40-year-old faciltics that included installing indoor plumbing, solar water heaters, and cable TV. The government is spending $32 million to refurbish leprosaria; after the leprosy victims di officials say, the facilities will be used as hospices for AIDS patients of retreats for treating drug addicts. Leprosy vietims are relishing the ereature comforts, “It's much better here now,” says Gu Jin Fa, who at 54 years old isthe youngest resident of Jiangyan Leprosarium, He smiles at NCLC’s Yan Liangbin, who performed surgery on his feet a couple of years ago, ‘enabling him to walk again after years in a ‘wheelchair. Gu’s affection seems genuine, ‘but because the bacterium ravaged his facial nerves his expression is strained and his eft ‘yeis tearing, Like many with severe disabi ities or who would be shunned if they retumed to thei villages, Gu has no interest in reintegrating into society, “This is my home," he says. RICHARD STONE 19 FEBRUARY 2010 939BHUTAN Improbable Partners Aim to Bring Biotechnology to a Himalayan Kingdom A British mycologist and a local businessperson have launched a pharmaceutical venture grounded THIMPU—In a quaint wooden house on o ill overlooking Bhutan’ rustic eapital, dozens of test tubes lie on a table bathe! inthe morning sun, “This is the worst way to store fungi, growls Nigel Hywel-Jones, The mycologist anda pair of young apprentices cooked up the growth medium themselves potato dextrose agar by chopping up and boiling spuels and adding dextrose, antibiotics, and agar powder. ‘They filled test tubes with the homebrew and set them ina slope to maximize the surface area for culturing fungi, some possibly new to science, that they collected in Bhutan’s high ‘country It would have been better to keep the fungi ftozen in suspended animation, but for now that’s not an option, says Hywel-Jones, who is waiting fora 80°C freezer to ative. The challenges of carrying out high-caliber science are daunting in an isolated country known for tacking its standard of living with Gross National Happiness index, Hywel- Jones, a lab chief at the National Center for Genetic Engineering and Biotechnology (BIOTEC) in Bangkok, arrived here last Sep tember to help a biotech company take root in a land with a mere handful of Ph.D. scientists. “Lovas ready fora new challenge," he says. HywelJones, ona temporary assignment from BIOTEC, is serving as an adviser to Bhutan Pharmaccutials Private Ltd, (BPPL), company founded by local entrepreneur Wangdi Jamyang, Besides prospecting for fungi that attack inscots and culturing isolates 19 FEBRUARY 2010, ‘a Buddhist reverence for the environment that may have medicinal value, Hywel Jonesis ing a business model for biotech in the kingdom, There is no dearth of rw materials. Bhutan’ highlands area prime habitat for a least one prized commodity: an insoet fungus, Condyceps sinensis, that fetches outrageous prices as a Chinese medicine. Hywel-Jones believes that Bhutan, a biodiversity hot spot, ‘may harbor‘ wealth of novel compounds for Western medicine, too, “There's fantastic potential," he says. Wang puts it this wa a) oer i ba ee ar Pees Bhutan biopharma brain trust. Entrepreneur Wangai Jamyang (left) and mycologist Nigel Hywel-ones, Joone knows much about Bhutan, and that’s selling point Brand Bhutan: “The duo insists that they are not embarking ‘on abiological Gold Rush. For starters, BPPL is collaborating with the government's National Biodiversity Centre and has approval for now to collet only Cordceps and related species. (The company hopes to expand to ‘other fungi and plants after Bhutan develops ‘expertise in these areas.) Wangdi emphasizes that thei activities will be guided by a Bud > JOINT MATHEMATICS MEETINGS | Politics as (Un)usual Pits, ssid the art of the possible. And for decades, mathematical voting theorists have pointed to the possibility that different voting systems—from simple plurality to instant run-off to the rank-ordered Borda ‘count could produce vastly different results Donald Saar, director ofthe Institute for Math- ‘ematical Behavioral Sciences at the University of California, Irvine, and a leading voting ‘theorist, ikes to boast that if you tell him the esl you want (say, fora teubook election oF anew hie for your department) and your vot- crs’ actual preferences, he'll find a perfectly fairway of guaranteeing the desired result, ‘But how common are such paradoxes in practice? Not very, if recent case study is typical, Ina session on voting theory at the joint meeting of the American Mathematical ‘Society and the Mathematical Association of America, Anna Pope, a graduate stent in psychology at the University of Ilinois, Urbana-Champaign, described an analysis of ranked ballots from 8 years of voting forthe presidency of the American Psychological Association (APA). She and her adviser, Michel Regenwetter, found that different Perfection in a Box Here's a good way to keep mathematicians busy for eenturies: Make up an object, call it “perfect.” and then try to find out if it exists. Take so-ealled perfect numbers: ‘whole numbers such as 6 or 28, which equal the sum of their divisors (6= 1'+2+3;28= 1 +2474 14), l’shard enough to find jones that are even, but no one has ever spot- ted a single one that’s odd. Things are even worse with “perfect ceuboids”: rectangular brieks whose three different edges and four different diagonals (three on faces of the brick and one angling through its “body”) all have lengths that are exact integers, Nobody knows whether they exist at all. But something similar has just turned up: At the meeting, researchers reported the first sightings of “perfect parallelepipeds.” A parallelepiped is basically a brick whose sides are allowed to be parallelograms rather than strict 90° rectangles. Like a cuboid, a parallelepiped has three different edges, but it has 10 different diagonals: two on voting methods gave the same result much more often, than not. ‘One classic voting para- dx is known as a Condorcet cycle. An extreme example ‘occurs wen a third ofthe vot- ers prefer Alice to Bob to Cathy, another third prefer Bob to Cathy to Alice, ‘nd a final third preter Cathy to Alice w Bob, In such a ease, an outright majority prefers Alice to Bob, a different outright majority prefers Bob to Cathy, and yet another outright majority prefers Cathy to Alice. Subtler examples, with the numbers not exactly ‘equal, produce cases in which a reasonable argument can be made and a voting system adopted for electing any ofthe candidates. Popova and Regenwetter obtained datasets fiom APA elections from 1998 to 2005, each .with upward of 20,000 voters giving full oF partial rankings o five candidates for presi- ent. (APA uses form of instant-runofT yot- ing, in which candidates with the fewest first- place votes are successively eliminated and ‘heir supporters’ ballotsare edi é four crisscrossing the body. Perfection is achieved iffall 13 ofthese numbers are exact integers. Now Clifford Reiter of Lafayette College in Faston, Pennsylvania, and Jorge Sawyer, an undergraduate at Lafayette, have found the first examples of perfect parallelepipeds, ‘Thoir search depended on a simple property of all parallclograms, established by simp! A perfect 10. Con you find the _ 10 diagonals that mate the parallelepiped at ight as perfect asthe labeled one at tt? 13-16 JANUARY | SAN FRANCISCO, CALIFORNIA tributed to those voters’ second-place selections.) The research, which compared seven different voting methods, turned up no examples of Condoroet cycles and found only ‘one casein which one method (plurality vote) produced a different winner from the others. Steven Brams, a voting theory expert at ‘New York University in New York City, ques- tions the generality of the psychologists’ findings, noting that balloting in professional societies is often heavily influenced by the process of selecting nominees, “I don'tallege that its nominating committees pick some preferred candidate and then fill inthe rest of the ballot with weak opponents.” he says. algebra: The sum of the squares ofa parallel- ‘ogram’s two diagonals is twice the sum of the squares of its 1wo edges. With that for- ‘mula and a bit of number theory to guide them, Reiter and Sawyer could easily run a systematic scarch for all parallclograms with edges of “short” integer length, say, into the thousands, whose diagonals are also integers. They then looked for combinations é 3 3 5 19 FEBRUARY 2010 VOL327 SCIENCE ww sciencemag.orgFor PRESIDENT oé iS @® ® (ms candice A Y a Condicate 8 ID cnaitee (@ io andicate 0 OOO COCO e ., 900100, @ @ Condit De Oo fh oe ora pret es onde “Nome Who's on First? Optical illusion ese) mimics 2 par aedoxica “everybody wins” outcome that can actually ecu in ranked-hoice voting, ‘But it may be more than coincidence that cone candidate almost always handily beats four opponents.” Saar is similarly unsure. “The only way such an empirical result can hhappen is if people have remarkably similar preferences, much more so than we would expect in general society” he says. Regenwetter agrees there's & lot left to be explained. But it's becoming clear, he says, that “the empirical world is highly different from the picture that has generally emerged out of the mathematical theory of social choice.” erfect paallelograms”to serve as sides of candidate parallelepipeds and devised a computer algorithm to pick out the perfect ones. “The biggest surprise for me was how uickly we got results” Sawyer says. Their first example has edges of length 271, 106, and 103 (sce igure, lef) Inall, the computer search found 30 perfect parallelepipeds, with, edge lengths up to 3920. Some are tantaliz- ingly close to being cuboids, with one or two rectangular sides. Such findings “may spark even more interest in the perfect cuboid problem,” Sawyer says Ezra Brown, a number theorist at Vir- gina Polytechnic Institute and State Univer sity in Blacksburg, agrees. “The size of Reiter and Sawyer’s smallest solution is very surprising,” he says. “Apparently, easing the restrictions that the Faces be rectangles is more crucial than anyone thought.” Nonetheless, he notes, perfect cuboids, if they existat all, area long way off: Comput= ers have looked at all possible bricks with cedge lengths upto 10 illion without finding 1a single one that’s perfect. wwo.sciencemag.org SCIENCE VOL327 What Comes Next? (One of mathematicians” most beloved Web sites is getting ready for a makeover The Online Encyclopedia of Integer Sequences, established by Neil Sloane at AT&T Labs Research in 1996 and run largely as a one-man shop, is poised to 20 50 associate editors taking over ‘much of the workload. The OEIS, or simply “Sloane” as it’s known to sequence fanatics, isa database of nearly 200,000 lists of numbers—a mathe ‘matical equivalent to the FBI's voluminous fingerprint files. Much as fingerprints give police a quick way to link a new erime (0 earlier ones, sequences enable researchers to make connections between mathematical problems that might otherwise go unno- ticed, The innocuous-seeming sequence 1 2,5, 14,42, 132, ..,forexample, arises ina hhuge number of different contexts, from ‘counting the arrangements of nonintersect ing chords inside a cirele to enumerating ssecondaty structure possibilities of RNA. To sequence fanatics, such “Catalan numbers,” they're known, are even more famous than the ubiquitous Fibonacei sequence 1,1 2,3,5,8,13, Sloane began compiling sequences in 1965 as a graduate student at Comell Uni sity, By 1973 he had 2372 of them, which he published as 4 Handbook of Inte ger Sequences. An updated edition, with 5487 sequences, appeated in 1995, with the help of Simon Plouife of the University of ‘Quebee, Montreal, Butby then, Sloane was already moving online. The OEIS made its debut a year later, with a database of 10,000 sequences, The OEIS “is one of the most useful tools available online for the working mathemati- ian,” says Doron Zeilberger, a combinatorial- ist at Rutgers University, New Brunswick. "It also isa great tool for deterrining the novelty ‘of a new sequence. If it is notin Sloane, itis ‘most likely to be new! “The Web site invites users to submit n ‘sequences or comment on existing ones ‘Such contributions have fueled the data- base’s steady growth. In 2009 alone, the total increased by 18,709 sequences, or ‘more than $0 a day. “Sequences are still ppouring in,” Sloane says. Even as he edits the constant stream of new sequences, Sloane takes time to admine some of the contributions, His latest favorite is the “toothpick sequence,” added in 2008 by Omar Pol, an OEIS contributor from Buenos Aires. The toothpick sequence reg- isters the increasing size of a geometric NEWSFOCUS t arrangement of toothpicks, in which @ now batch is added at each stage, centered on and. at right angles (othe exposed tips of the pre atch (see figure). The picture that and his AT&T colleague David Applegate have written paper on the sequences math- matical properties, and Applegate has con- tributed a movie of its geometric growth, linked t its entry in the OFIS. Sloane set up the OEIS Foundation last year and transferred intellectual-property rights to the nonprofit organization. With Applegate’ help, he plans to move the data- Chewy. Database managed by Neil Sloan (top) indludes the “toothpick” sequence (2, 3, 7,14, 15, 23, 35,.) shown herein color-coded steps base toa wiki format, giving each sequence its own Web page, with new submissions moderated by a board of editors. The transi tion has hita snag, however: Search-engine soflware in the “wikiverso" can’t yet handle ‘Once that technicality is overcome. Sloane expects the wiki format to be an improvement. “I's the correet mechanisen for handling the database,” he says. As for his anticipated reduced workload, “it I mostly be a relief. On the other hand, Vl miss all the e-mails.” BARRY CIPRA 19 FEBRUARY 2010 943MLW Naan ite of Systems Biology" free and open eae een er using immer: ere Tor Seco onan Dee eta esVisualization Challenge SOMETIMES SCIENTIFIC INFORMATION CAN BE VIVIDLY REPRESENTED WITH commonplace materials, Take the illusiation on the cover of this issue of Science. tis a toree-dimcasional at installation made from 75,000 cable zip ties depicting the dynamic relationships between endothi Onli lial cals and thor surrounding microenvironment, Or turn to ININE page 950 fora description of how cotton batting. a clothes: sciencemag.org linc, and sewing pins are used in a video to illustrate the Sideshow nd impact ofthe environment on gene expression, The seulpture jd evew and video are among the awardees inthis year's Intemational Science & Engineering Visualization Challenge. Clearly, the convergence of art and science can successfully convey complex phenomena in spectacular ways. For the past 7 years, Science and the U'S. National Seience Foundation (NSF) have co-sponsored annual challenges to promote cuting-edge efforts vis- alize scientific data, principles, and findings. The visualization challenge is led to showcase and encourage ths kind of work year's entries present a bold and unigue look at life, from the broad view of planet Earth, toa more intimate picture of aetivity inside the human brain, We received 130 entries from 14 countries, 23 U.S. states, and the Disteict of Columbia, A committee of stall members from Science and NSF serecned the entries, and an outside panel of experts in scientific visualization reviewed the finalists and selected the winners. The winning entries appear on the following pages We encourage you to submit applications for next years challenge, dota of ‘which will be available at wowwinsf.govinews'scivis, and to join us in colebrat ing this year's winners Tari Joyner of NSF onsanized this year's challenge. Michael Torrice of Science's news staf wrote the text that accompanies the images in this special section, and Martyn Green, Tara Marathe, and Andrew Whitesell put together a special Web presentation at wonsciencemag ory spacial vis20091 JEFF NESBIT, DIRECTOR, OFFICE OF LEGISLATIVE AND PUBLIC AFFAIRS, NSF MONICA BRADFORD, EXECUTIVE EDITOR, SCIENCE Science 4 Judges Donna Cox University of tlinais Urbana IL Thomas Lucas Thomas Lucas Productions ine. Ossining, NY Alisa Zapp Machalek National Intute of General Medical Sciences Bethosds, MD Corinne Sandrone Johns Hopkins Schoo! of Medicine Baltimore, MO Tierney Thys National Gecgyaphic Emerging Explorer Carmel, CA Thomas Wagner Cryosphere Science Program, NASA Washington, DC wawsclencemagors SCIENCE VOL327 19 FEBRUARY 2010 945946 19 FEBRUARY 2010 VOL327 SCIENCE www-sciencemag.orgp you draw through that point? The Greek mathematician Buclid said just one, but for more than 2000 years after his death, mathenia odo prove that he was right based on imagine," Palais says, Other mathematicians beyan build- wiwusciencemagorg SCIENCE VOL 32i Illustration First-Place Winners ing on the discavery and created new kinds of geometry (Albert Einstin sed these geometries when he developed his general theory of they made was th ty.) One important discovery urfaces called pseudospheres, which marallel postulate, could be describ sim. an oguation called sine-Gordon, eet of paper shows sketches surfaces, called Kuen’s surface, and the expression, calleda soliton, that deseribesit,“We wanted to talk about ations in a way that nonmathe- "Palais says. “So surface itself stands as mard rendered Kuen's ng light bounci y folds off the paper and into th that required mote th says panel of judges member € inne Sandone. 19 FEBRUARY 2010Illustration Honorable Mentions Jellyfish Burger Dave Bock, Clarkson University, and Jennifer Jacquet, University of British Columbia Fast-food diners don't expect stringy tentacles ora gelatinous blob ‘when they bite into their burger, But marine scientist Jennifer Jacquet ofthe University of British Columbia in Canada and digi- {al artist Dave Beck's illustration uses this absurd, grotesque image to make their point: Overfishing and climate change have signif- icant consequences for marine ecosystems. As the numbers of larger fish dwindle and ocean temperatures rise, the sea becomes more and mote ideal for the floating creatures, Jacquet say. “Jacquet’s adviser, Daniel Pauly, frst dreamt up the jellyfish burger as an absurd metaphor for where overfishing was leading us. To illustrate the idea, Jacquet contacted Beck, whom she's known since the fifth grace. Beck set up a fake burger shoot with ‘buns, tomatoes, and lettuce from the market. He then digitally added the computer-gencrated jellyfish, adjusting it for just the right amount of rubbery gooeyness. “I wanted it to be both funny and scary.” Beck says. "My ultimate goal isto reel viewers in so that they're no longer interested in the image, but they're now thinking about the larger issue" Along with the burger, Jacquet has also imagined a series of follow-up illustrations to create a possible exhibit of seafood of the future, including krill chowder and squid and chips. Back to the Future Mario De Stefano, Antonia Auletta, and Carla Langella, The Second University of Naples Nature has been building microscopic cellular solar panels for ‘almost 200 million years. So lets follow her lead, says marine biologist Mario De Stefano of the Second University of Naples in Italy. De Stefano and his collaborators have been studying dia- toms, microscopic algae, and they believe the organisms’ cellu- lar structures could inspire the design of solar panels, This illus- tration demonsirates the principles of biomimeticism, which involves looking “to natural organisms to see our future,” De Stefano says. Inthe foreground, a scanning electron microscope image shows the blue fans of diatom colonies from the species Liemophora flabellata that have attached themselves to a sand ‘grain with afong, gelatinous anchor, called peduncle. Each cell isa lat wedge with a glasslike wall shaped to maximize its sur- face area and absorb sunlight more efficiently for photosynthesis. Behind the sand grain, the team presents computer drawings of their bio-inspired solar panels, which would sland 3 meters tall with a span of $0 meters. De Stelano and his collaborators have started building these panels and believe that hey could be used ta create solar-powered street amps. 948 19 FEBRUARY 2010 VOL327 SCIENCE wuwssciencemag.orgInteractive Media First-Place Winner Genomics Digital Lab: Cell Biology Jeremy Friedberg and Andrea Bielecki, Spongelab Interactive Video games allow players (0 rock 0 ‘touchdown passes like Peyton Manning. In this interactive media entry, they can turn sunlight into electrons and convert sugar into cnorgy likea plant cel, Jeremy Friedberg and his team at Spongclab Interactive in Toronto, Canada, designed this Web-based game to teach high school students about the intricate eyeles and pathways that keop the cell alive by generating and burning encrgy Ate start of each game, the camera zooms into the cross section of a leaf and focuses on like Jimi Hendrix or hurl slowing cell. Then the students must pl the role ofthat cell, building and maint points. Inone game, players have to man age glycolysis, the pathway that breaks down the s slucase, by shooting enzymes at molecules marching on tracks to convert them into new chemicals thatthe cell can use. The final games focus on transcribing genes and synthesizing proteins processes on which cells spend ‘much of their energy. Similar to Guitar Fiero, players transeribing a gene must hit the appropriate RNA base letter as the DNA template serolls down the screen, Too ‘many mistakes and the cell becomes sick, ning its eyeles and pathways Games can be important educational tools that go beyond rote memorization, Freidberg says. “I want to know if my students ean think critically and be ereative and figure out ways through problems,” he says. “That's ‘what games ean do: They can create scenarios that make students problom-solve.” ‘The judges “were impressed with the incredible attention (o detail and accurate repres. tio structures... a8 well asthe use of story ines to draw in andl sustain the users’ attention panel of judges member Tierney Thys. “My only wish is that Thad more time to play wonusciencemag.org SCIENCE VOL327 19 FEBRUARY 2010 949Noninteractive Media First-Place Winners The Epigenetics of Identical Twins Harmony Starr and Molly Malone, University of Utah As children, identical twins are indistinguishable. But as they reach adulthood, physical differences develop: One twin may go gray ear- lioror the other's facial features may scem more youthful. This short video from the Genetic Science Learning Center atthe University of Utah explains that one reason for this phenomenon is epigenctics, the chemical changes cells make to chromosomes, ‘TheA,T, C,and G letters ofour genetic code are familiar enough. But enzymes write another message onto the nucleotides of our DNA and our chromesome's proteins to control how our genes turn fon and off Harmony Starrand Molly Malone tell the story of how two twins” epigenctic codes change with their different environments and life choices. The video stars with tubes of cotton batting clipped onto 1a clothesline to depict the chromosomes the twins inert from their parents. Red+topped sewing pins pushed into cach tube represent the chemical tags of each chromosome’s epigenetic code. As the twins grow ftom infant to adult— visualized by a scroll unraveling from a rolling pin hands place objects that symbolize environmental dif: ferences next to clothes baskets filled with each twins” chromosome 19 FEBRUARY 2010 VOL327 950 tubes, With each new factor, such as an ashtray’ for smoking ora jump rope for exercise, the hands push more pins into the white tubes. By the end of the video, corresponding chromosomes from each twin hhave visibly diferent pin paterns dovting them, ‘This low-tech approach to explaining a technical topic was inten tional: "Because there is so much use of computer graphics [in tence videos], we hoped the style oF this piece would catch peoples attention with its simplicity and quirkiness,” says the center's direc- tor, Louisa Stark. The judges agreed, “It was a delightful way to present an extraordinarily detailed scientific concept that is acces- sible and entertaining to an audience.” says panel of judges mem- ber Alisa Zapp Machalek, “You could tell that the producers had a tremendous amount of fun conceiving and making it” says judge Thomas Lucas. SCIENCE wi sciencemag orgFollow the Money: Human Mobility and Effective Communities Christian Thiemann and Daniel Grady, Northwestern University Ever wonder where your dollar bills travel after you plop them down for a cup of coffee? The Web site Where's George? allows you to do just that: Record your bills serial number and then track its journeys as other peo- ple spend it across the country. But i's more than just a game, Because every time dollar is spent in a new place, it means someone moved it there. Christian Thiemann and Daniel Grady of Northwestern Universit in Evanston, Illinois, have been using the Web sites data to tudy how people move within the United States, “They produced this video to explain their project and snimate the results. Tiny bills stretch out from county to county on a map of the contiguous United States. Some places, such as Los Angeles, California, have many bills passing through it from across the nation, while oth- ers, such as Anderson County in Tennessee Grady's hhome-have just a few that mainly cycle locally. From this travel data, the team ran computer algorithms to find ‘what they call effective communities within the United States. People tend to travel more within these invisi- ble boundaries than outside them. In the video, coun- ties flash and wigele as the computer algorithm tries to decide which counties belong in the right community. “Normally, ... voujust push a button and wait for 2hours and thenall you get are numbers, which is really boring; Thiemann says. “But [the animation] makes the process visible and shows you how interesting it can be.” Grady hopes the video will stimulate other people to think up new ideas about what to do with these data, They've already started using it to study how diseases, such as HIN1, spread, and linguisties professors want to compare these travel boundaries to dialeet boundaries, Panel of judges member Corinne Sandone thought the video had a whimsical, quirky appeal, like that of independent films. “I liked the extreme yeekiness of it and how it made me want to watch it again to absorb it all” says judge Thomas Lucas, “It was so rich.” wonusciencemag.org SCIENCE voL3z7 19 FEBRUARY 2010 951Noninteractive Media Honorable Mentions Decision Support System for Tsunami Early Warning Gregor Hochleitner, Christian Gredel, and Nils Spanvasser, German Aerospace Center (OLR) After the 2004 Indian Ocean earthquake triggered a devastating tsu- nami that killed more than 200,000 people, Germany and Indonesia designed a new system to warn people when a tidal wave is about to strike. Nils Sparwasser and his collaborators at the German Aero- space Center produced this video to explain how the new German Indonesian Tsunami Early Warning System combines data from underwater probes, orbiting global positioning system satellites, and floating buoys to better detect a com ing tidal wave, Starting with a hypothetical earthquake off the coast of Sumata, the video's animations show hhow the different components relay their information oa central decision-making center and how computers there then predict which areas should be evacuated. “We ‘wanted to explain thatthe new system works with much ‘more information [than previous systems],” Sparwasser says. “So if it generates a warning, they have a really good reason to evacuate.” Inside the Braii Unraveling the Mystery of Alzheimer’s Disease Stacy Jannis, William Dempsey, and Rebekah Fredenburg, dennis Productions Ina brain riddled with Alzheimers disease, protein tangles grow and connections between nerve cells shrivel. This video by Stacy Jannis| and her ‘eam at Jannis Productions in Silver Spring, Maryland, ani mates these microscopic damages to explain how the disease starts, ‘The team produced the movie for the National Institute on Aging to life (2)~ 1 hour) in eget to GL-eUre3 (N- terminal Gly) and CK-e-Ura (Lys a position 2 which were geval Fg 1, Band, fig SOB and fi. $3, A and D). Other nonbasic residues at Position 2 also yiekld shortlived CZ-<"-Uns (2= rp, Gin, Gly. ole) (Fg. 1B ad fig. $3, Several other XL-eUra repress (X - Met Ser, Val la or The) were ako shortsved in viv, like CL-Uin3 and in contrast long-lived MK Lin Lys at poston 2) MRL (Ang at Poston 2), GL-¢*-Ura (Nermiral Gi). and PL Un (N-terminal Pro (Fig, 1 Ato Ds fig 82, ‘A and By and fy $3, B, C, and E). We sho performed S-pulse chases (8, 19) with Le ‘Ura and ME-e*Ura3 venus CK-eUra and MK-e*Ura3 (fig. 4, A tC), The techniques of CHX chases and S-pulse chases are comple= mentary, becans the former method monitors the degradation of all molecules of a spefic ron, whereas the later assay measures the degrada tion of newly formed (pulse-labeled) molecules. puke chases confirma the instability of XL FU (X=Cys or Met) and stability of XK-c™ Uns (X= Cys or Met) (Fig. L.A and C: fig 82, and B; and fig. S4, A tC), The degradation oF MLe*Ura3 was proteasome dependent, brause the MGI32 prozasome inhibitor significantly ine creased the level of the normally sbr-ived ML U8 but not of the longived MK-2Un, whose levels were high bo in the presence and sbsence of MGI32 (Fig. 1G). The Doal0 ubiquitin ligase recognizes acetylated N-terminal residues. To scarch fora ‘Ub figase o igases that meat the degradation of XL-c*-Ura3 (X = Met, Alt, Val, Ser, Thr, oF ), we expressed CL-e-Ural in. cerevisice ‘mutans that lacked specific F3 or F2 enzymes (fig SAD), CLe*Uia3 bocame long-ived in the sbsence of Doa0 (Fig. TE and fig. $4, D and F) Moreover, ther shor-ved XL-¢*-Uras proteins (2—Met, Ser, or Vl) were also stable indo? cells (Fig. 1F and fig, 84, Fad G). Doatd is a transmembrane E3 Ub ligase that functions with the Tbe6 Ube F2s and resides in he endoplasmic tsticuum (ER) and inner nuclear membrane (ISM) (22-29, To ales the above rests, we focused (on MATA2, a physiological substrate of Dea 0. ‘The 24KD) MATA? contains more than one degradation signal and hasan in vivo halite of 5 10 10 min (13, 25, 26), MATa ropes ta sctnton of apecifc goes in ers, whereas in se dpb the MATo2-MATa conmiex repress haploidspcife wenes (27, 28). The 67-residve [Naerminal region of MATa, termed Det, has been shown to harbor DoalO-dependet degron 974 22-24), MAT absent fcr databases of Ne acetylated proteins (5,6), possibly because of shart in vivo hale, We expresso fulblength MAT@2 in doat0S ubetS yeast and analyzed purified MAT2 axing liga chromatography tandem mass spectrometry (LC-MSIMS) The scsuls Gg, SSA) nated vieully comple Nt scefyltion of MATs2 (oo MAT? tat lacked Neavevlation could be detested) in agreemest with Neaeeslaion of ober pons ening the Nerina Metso ($7), Similar LC-MSMS ofthe Doal Otani, purified ML-<*-Uia3 (ig $5, and C) inated the Nescetlaion ofthis ssportes in agreement wih Neaceyatin of ober pyobinsconaining the Nein MetLeu (5-7) We alo obseved Nealon of a Deg eanng report was pried fom E of a inc ated with Scena exact (ig. SOA). Tn ation, we produced an anbody, ee ani MATAD, that recognized the Neaceyated, Necominal sequence of MATa? (Fig. 2C) and was speifi forthe acetylated, hewn (A) tagged, MATa2-erived, MNa2-o a3 eprter, denned MNU2 Fig. 2, A and B) Antic MiATud and antl (he ater anibody recognized both Neacetyatd and unacciylated assess? 8 Ss MN. MNa2 MNa2 > doat0a nat3A Coeteemeal,..cte [Ra tbatin Pee eee ee wee e— Socom ee| TI sas a S epi ino. FPP © EE gtopw eon & Mepioom XZ-0K 3-11) « . Actmarmounc- © © rcxzeKoi-@ 9808 eee 1234 12.3456 7089 anti“ WATa2 antibody F100] 80 DD xXa22 _Kenspat® al 2 ©. Fr ae ; PIPPOP SS io # g- -Doato 2 12345678 0 chase i Fig. 2, Doa10 as an Nerecogin, (A) Extracts from wild-type, doct0, and nat34 5. cereisioe that expressed XZ-o2"*7- 2. Form < 2, (7) does no exhibit pronounced peak, and RAT) decreases monotonically with decreasing temperature without showing the uptum. These resulls indicate that the magnetic order is suppressed when the Celn; ayer thickness de creases, and is vanishing ner m = 2. This conclusion is renfored by the results shown in Fig. 4A, where 7 decreases neatly lineaiy with wonusciencemag.org SCIENCE VOL 327 Lim and goes to 2520 in the vicinity of ‘Therefore. it s natural to conclude thatthe en- hance aniferomagnete actuation associated with rwo-dimersovaliy ae responsible for de- siroying the magnetic ord ‘The vanishing magnetic order implies that 2 quantum phase warstion occurs close to = 2.Close to 4 QCP, the Feisty stongly deviates ftom the Fei liquid behavior p(7) py + AT™ with = 2, where is the Feri liquid coettcient. tn Fig, 4B, the exponent is mapped in the T versus In disgram. The Tadependence is observed st low temper atures for m > 2. In contrast, for m = 2. a marked deviation from such behavior i ob served (Fig): A Tinea dependence with = 1.01 + 002 is seen below 05 Toy which coresponds to the Fermi temperature of heavy fermions, At even lower temperatures, below ~I40 mK. (7) shows a faserhanlinear de- crease, which i suppressed by small magnetic fields (Fig. 4C, upper ins. We eoaelude that, form =2, the lowered dimensionality suppresses the magnetic order dowa to Ty ~ 140 mi, which can be further suppressed by « smal field (17). The Tinear behavior is consistent, with seatering by the 2D antieromagntic focations (22) that are cnnanced by the QP This isin const tothe 3D case, where a~ Sis, expected (22), and indoad @ ~ 1.6 i observed near he pressure-induced QCP in he bulk Celns (4.15. Even for w > 2, where 7° behavior is b= served the ini slope of p~ pas Tapreuhes 0 K becomsslngr With desing sand is enhanced toward m = 2 Fig 4), Because 4 is ‘itd to he Sommerfeld coccint of specific heat y 38 4 ay. with cose the universal value of 10° fem (Kroll) (4, 27) the Inne 4 imma nds the enbaned quasi parcle mas. A enperaturs below 1 K the ‘exisivity of Lal is och smaller than that of superltices, and temperature dependence i nope, Ths the observed lang 4 wale ine dicate that he temperature dependent par of the resistivity is governed by the 2D heavy fermion Taye Ta fet they vale estimated fom i eshanged fom the bulk Celay value of -120, ‘nl (K mol) ane reaches ~350 m'(K° mal) far m3, hich conesponds to quasiparticle mas, 8 least several hundred times larger than the fice lec mass. Beceune the Fincarhehivior observed for m= 2 suggest fur enhancement of Ais tempting w ascii esl with éivering quasipaicle mass because ofthe quan- tum facwation enhanced upon approaching & QCP. For m= 1 the determination of 4 6 arm binons because of the weak tempratre depen- dence ofp. The diverging behavior of year QCP is als repre in bilayer films of He sys- tems (2, ‘Quantum erica i epoted to he ston ly modified by maznetic fields in strongly conelatel electron systems (2 9 23, 24) The quantum criticality for m = 2 is removed by 19 FEBRUARY 2010 REPORTS i strong magnetic fields, and the Fermicliguid properties with a= 2 are recovered (Fig. 4C. lower inset). Simultaneously, the 4 value i quickly suppressed with increasing: magnetic field 1 (Fig. 44). Thus, both temperature and field dependencies of the wansport properties provide strong support for the presence ofthe ‘quantum phase transition in the vicinity of 2, which is tuned by the dimensionality pa- rameter. The successful growth of epitaxial Ce- based superlaties promises w provide a setting Jn which to explore the fundamental physics of strongly comelated election systems such asthe 2D Kondo latice and, potentially, 2D super- ‘conductivity near a QCP. References and Notes {ce Heson, Be Rod rode 0 Hey Feros ‘Canondoe Ua. Pes, cane, 2003 P-Garwar, 5, Sth Nt Ps. 4 186 008 3. Sic, Quantun Pee Tnston Cm Ui Pres. abe, 2000 4. metodo Hath, Hai, Nave 398, 47 5.0 Gen, 6.1. Cae ate, AB Mewes L-AMeosh, Py ey. 864 168435 2000, 6 eae a eg Pps. ete 9, 122507 (2007 7. Meg a Ps er. eB 986 (2000 1 C Few et a Cnt 33, (337 20, 9H Sha RSet, Haring, ¥ Ont Pye Sao. 75,1103 rs. 10. Seta 1.08, Hon, Yu Py Sx Jon 178308 008 12 6 Zheng ea Phys A et 86,4664 20%) 12M ean, Ny 8 Coon, J Sav, See uy, bse G00? 131M meee, 5. Shap, Pi, 8 22,6379 14 0D. Mathur ea, Nae 398,39 998. 1. G tobe 0. Bhat, FC Ganels 6. Lap 1, Reo Pie. 8 5, 02875 2003 16 Samal ea, f Pp Soe Jon 73,167 1, See supporting mae oe cece One 1. scrap, selon, Tash Tach 2 9s Soe pn 88,1541 (1988. 19, Woke a Card. lt, 25,3741 202), 20, Y.nalajna el Py. Soon. 76, 028709 ‘200m. 2. Monta, ep. Pag. Py 7026501 (208 BET Met Uda As hy 49, 595 200, 2S A Banc R Moro vei, Paps, 1 Sor, fe Le. 99, 27001 (003, 24 T Shae te Pe Ho ee USA 205 720 @008. tesa, Pye Re B48, 9292 1992) 26 We tank ay Kaka, a, yee, nd kanal cst, Ts wa a sip by (Garhi fr Set esearch QBKENHD a Japa Soc forthe Praton of Scene rd by 9 "The es Geran of Pps, Sn or ie and Engen” om ie Myo Eaton, Ce Sars See ana Tey Supporting Ontine Materat Srmactnenoy epiarell2 759689800 figs St 055 15 Oxoer 208; cepted 7 Joray 2010 sa 1zexcence 183376 983The Silicate-Mediated Formose Reaction: Bottom-Up Synthesis of Sugar Silicates Joseph B. Lamber,* Senthil A. Gurusamy-Thangavelu,t Kuangbiao Mat Understanding the mechanism of sugar formation and stabilization ts important for constraining theories on the abiotic origin of complex blomalecules. although previous studies have produced sugars from small molecules through the formose and related reactions, the product mixtures are complex and unstable, We have demonstrated that simple two- and three Se tom Gia stacey temamorelecvty sudie, 8 Gf ° 2% 2 0 2 a, daseeomer rato x er er) irae OO, ALLO, Ah temied by apc ON WS HOW NY 8 Sow 8 ons Etre ea 8 e 4 8 © w ysis using commercially X28 ke er able ira calimns aa, ° ; auinotne siuacture of » varity of important, A Ay 32 see Biologically active compounds (27-23) cad > a ing mteline (11, Fig. 20,1 matraly oc ye Bite curring nonpeptide natural product that hasbeen 3 Ere is pane ‘denied as brykinin BI and B2 receptor Nasa 6 mal anlagorist (24, The enantioselective cate “48-06 Be Yiesateie Povarov method provides an eflcint enantiose- ZI -20°S-20°¢ Hy Assay Jectve oe to his natal prt andi analogs 2 Oe te eee sy Ga, rst» nee ome 7 os "The cooperative activity of an acid and 8 Y=H,3CL38r 35.0Me, 4,40) 48%, ¢Me, Mo, 3,5¢e; ZaHsCoMe, 4h 46), 25M ¢Me 8 1a (4 mot %) [i sdetzethy cooky tune, sks, BOY e Seta eiihes ce > wot " Fig. 2. (A and B) Asymmetric Povarov reactions catayaed by YaINBSA with enamide 5 and enecarbamate 7 as the nucleophilic reacting partners. Molecular sieves (5 A) serve to sequester water introduced withthe hygroscopic NBSA reagent thereby preventing competing imine hydrobss pathways. See tables S2 and 53 for yields and selectivities obtained wih each subsvate. (C) Matineline (22), 2 naturl-produc ini concentation Isp = tr of bradykinin B1 and B2 G protein-coupled receptors (median inhibitory 4 and 0.25 jh, respective). A previous study demonstrated that a racer form of ester 10b could be converted to (:)12 after epimeization of the corresponding aldehyde, 50 the enantioselective syrthess of 10b described here constitutes a formal enantioselective symesis of rmartneline 25) Although moderate dissereoseletivtes favor- ing the exo diastercomer were obtained inthis 880 Beu/Benta~ 1 0 42:1), tis product was also generated in high enantiomeric excess (ee) (90 10 98% ee) and could be isolated in diastereomercally pure form in useful yiekds (4S to 73%). In gener, imines derived from efecton-deficent aldehytles—especially imines wonusciencemag.org SCIENCE VOL 327 derived from glyoxylate esters underwent reac- tion more rapidly in the Povarow reaction, but uniformly high enanioseletvities were obusined despite these reactivity differences Povarov reasons between glyoxslate imines 9a oF 9b with 2.3 | i i : : Fig. 3. (A) Formation of a complex between wea 12 and iminium sutonate 2eHOTE. HNMR chemi shifts of the formy| proton of 2a as ree and ures-bound ‘als (B) Geometry and energyminimzed structures of 2aeHOTHea calculated at the BBLYP-31GIe) level of densty funcional theay. Ar = 3S4CFCH. Selected bond cistances are shown in angstroms. () Kinetic parameters of racemic and enatioslectve Povaroy reaction of 2a and 3 cocataljaed by 1a and HOT (D) 988 19 FEBRUARY 2010 VOL 327 Pot of inal rate of the Povaroy reaction versus (2a) at three diferent corcetrtions of HOT, [2a] = 02 oF 04 I, [3] = 16 M. The blac cures represent eastsquares fs othe rate aw derive fom the kinetic scheme depicted §n(©. E) Pltof ee of 4c vesus ala tee different concentrations of HOTT. (F Pot of intial ate of the racic Povaray reaction cocatayaed by achiral urea 112 and HOTE vers [12] with HOTF = 0.80 mi [2a] = 02 1, (3] = 1.6 M. SCIENCE wwwsciencemag.argiminium ion, Computational analysis (27) of the temary 2a+HOT Pla complex suggests the basis for the observed effect Four energetic minima of comparable stibility were identified (Fig. 3B), with the Laetriflate complex ating asa dual H= bond acceptor through the tiflate and sulting mide groups, and the iminium ion acing as a dual H-bond dooce through the iminium nitrogen and form protons (23) ‘The editioal stabilizing interactions involv ing the catalystulfinamide group and iminium jon formyl proton group have 2 propounced effect on the rate ofthe Povarov reaction, Kinetic analysis of the reaction between 2a and 3 that was cocatalyzod by HOTE and chiral sulfnami- dourea 1a under homogencous conditions re- vealed tat 1a induces a substantial decrease i reaction rat: (Fig. 3D) In contrat, the simple chiral urea 12 has a slight accelerating effect (Fig. 3F). Inthe presence of ta, the reaction rate can be expressed by a wo-term rate law in which the pathway catalyzed by HIOTTalone dominates at low [la], and a HOTE a cocatalyzed pathway dominates at high [1a] (Ea. 2). binding con slant of K'=9000-£ 2000 Mfr the interaction beeen 1a and 2aeHOTF can be determined from the kinetic data (Fig. 3D and fig. S11), This relatively high value stands in sharp contrast to the much weaker binding between chiral H-bond donors and neural subsites (2% 3), but is come Fig. 4. (0) Posible A mechanisms and rote liming steps inthe asymmetic Poway fe dction. 2, an electon- donating oF thang subsivent @) Kinetic isotopic pines {> distinguish beeen diferent possible rate Uestng eps (© Geome- tyand energie lomestenesgytransiton stucure for cycoate- tion caelated at the BSLIPESIG(d) lev of dersiyfrctonal theoy. Selected bond dares St are shown In aogarons fs, 3545). (D) Alierate view of the stucurein (highligh ing the sbiliing === ineaction between the anlgoural ec Pw dergoing cycloaddition. (to © Analogous view of the transition structure leading to the minor en- antomer of product. This tration structure Lacks stabiling —r interac: HH aa mol), CFSSOSH (9 ol), tohiene, 55°C sistent with binding constants determined bex ‘ween tetalelammonium salts and ureas and thioureas (57,32). Rate = dls)/ae = iinesHHOTH|[3) + AnynliminesHOTT4a](3] (2) Here, Kagyy is the second-order rate constant forthe enantioselective eyeloaddition between 2ueHOTFta and 3 ‘The observation of tight binding between room tiflate 2aoHHOTY and sulinaidn urea Ta serves to explain how high enantioslee- tivity is obiained during the formation of 4a, even uucder conditions where the HOTEeatalyzed race mic pathway i several times more rapid than the cnamticselestve palway (Fig. 3. and E), The ‘slum constant for complex formation i suf ficiently high that virually no fee iminium ion exists, and the reaction is thus chanced through the asymenstic pathway. The enantioselectivityin the formation of yi increased futher to syne thetcally usefal levels by carrying out tbe reaction at lower temperatures (tat is, 55°C, 97% e). To glean insight into the basis for stereo Induction in the Povarow reaction by sulfina- midoures catelyst 1a, we carried out x fall experimental and computational snalysis of the stepwise JoF;80. cydonadton athe 0.92 #003 REPORTS i mechanism of addition of dihydrofuran 3 tothe protoiminiu sulfate 2HOTPIa, Ia principle, any of several elementary steps inthis process may represent the rate and enantioseleetivity= determining event (Fig. 4A, steps a to d). The absence ofa primary kinetic isotope effect on the fonbo hydrovens of the aniline group of 2 in- dicates tht rearomatzaion (stp 4) is kinetically rapid (Fig, 4B), A positive Hammet correlation was obtained in the analysis of the effect of snilino substituents on the rate of teaction (p = 1.96 + 0.06, fig. S17). This result indicates that cither the fist step of a stepwise process (sep) ‘ora concerted cycloaddition (step c) may be rate- limiting, bu itis inconsistent with the eyeization step of a stepwise process (step b) representing the slow step. Definitive distinction between steps a and c is more challenging (33), but the kinetic isotope fect data suggest that paral ‘chybridization ofthe onho-carbon of dhe aniline jeeurs inthe rate-limiting step 34), and this is Indicative of a concerted, absit highly asyn- chronous [42] eyclosddition. This conclusion vas supported by a computational analysis ofthe reaction leading tay, which predicts that the lowest-energy pathway involves an endothermic celeaddition and a comparatively rapid depo ‘onation/earomatizaton scp (fg. $19), ‘The catalyst-bound iminium ions depicted in Fig. 3B cach have one face exposed to solvent 3 4 Hay re-aromatizaton A” eu CY Bsa! . Aw \ap le gl 2 h + CF,S0H+ ta ‘tons andi distavored relate tothe structure in (0) by 2.3 kcalmal in calaations using the BSLYP/6-31G(4) method, and by 3.6 to 3.9 kcalmat using MOS 26-3144 p) or MP2/6-316() single-point calculations, See table S17 for further detalls wonusciencemag.org SCIENCE VOL 327 19 FEBRUARY 2010and the other x face shielded by the catalyst, and ae all expected to be energetically accessible under the reaction conditions. Computational analyses using ether density fimesional theory ‘or ab initio methods (Fig. 4, C to E) predict that the enantosslectiviy-determining cycloaddition occurs preferentially with complex I, leding to the experimentally observed (R)-cnantiomer of product 4tqqe, The lowestenerey cyclo- addition transition structure displays iminium N-H-Osctnamite el formyl CH Orstire hydrogen bods (Fig. 4C), and is predicted have >13-kealimol lower energy than aliemae tives arising from complexes IT to TV. which is consistent with the experimental data, The basis for enantioselectivity may be ascribed to a stabilizing -n imeraction between the (CF3}eCAHN component of the catalyst and the cationic aniline moiety of the substrate, This interaction is evident in transition stuctores leading 0 the major enantiomer of 43eu, (Fig 4D), but it is absent in transition structures leading to the minor enantiomer (Fig. 4E), Enantioselective catalysis by 1a of a stong Bronsted acid-catalyzed Povarov reaction thus involves tight binding to a highly reactive cationic ‘mtermeatiat through multiple, specific boning imeractions, and these noncovalent interac= tions ace maintained in the subsequent stereo= determining eyclouddition even, One of the four enerpetically accessible ground-stite complexes lundergoes reaction with the nucleophile prfire ential, illustrating the ability of bifunctional catalyst Lato control precisely the outcome of this reaction though noneavalent interactions slone, Given the known ability of urea and thiourea derivatives 10 bind a wide range of anions, the strategy demonstrated here is appx cable, in principle, to cationic intermediates with 1 varity of counterion structures. References and Notes 2. Wenn at Ed ro 3, 11968. 2. T. flys. Ykta, Koh, agen Che, Ig £43, 546 2000) waa x. Strachan, fm Chom. Se. 426.804 Goo ‘Ama Cem ey. 107, 5784 200 Nala, Maman, A. Cham. So 428 9626 2006. 1 tang, 1 Mo «Maa, Aabe Kb, 1 he Ceo. Se 130, 1838 2008 isha foes, Yara, Aa Chen. fetamamnta Raat Age hom It 6 4924 2005, 9. TW Me © is, Sb, Og ett 40,153 008 to, Rs. Rowen When, coy Le 2,87 coo 131. Stach HALO, Angew. Chem nF Eng 8, 20 0915, 12, LT Ran, FS. hi, 6. Palen ace, Jen Cher So 129, 13804 2007. 13, 22mg P&S, Cr Soe Ret. 38, 12872009 1M, AG Oo £8 Jane, ew e407, 57 2007 45, VL, keane Teton 65,2721 G00), 16 Hea 5 Kaa eae et 37, 357998, 171, yaa, Horta, K Fb, jm, en, Se sag, D070 @006 2, Lu, Dagus, 6 aso, Fels. 2h, te chr So 1, 4598 (209. 19, hers nd meets ae late 3 ppg 20, Lan EN Ipcaa, Age: Chm na 86,3315, e007 | Me. Cem, 35, 19541952 100-Million-Year Dynasty of Giant Planktivorous Bony Fishes in the Mesozoic Seas Matt Friedman," Kenshu Shimada,2* Larry D. Martin,* Michael]. Everhart? Jeff Liston? Anthony Maltese,* Michael Triebold® Large-bodied suspension feeders (plantivores), which include the most massive animals to have ever lived, ae conspicuously absent from Mesozoic marine environments. The only clear representatives of this trophic guild in the Mesozoic have been an enigmatic and apparently short-ved Jurassic group of extinct pachycormid fishes. Here, we report several new examples of these giant bony fishes from Asia, Europe, and North America. These fosils provide the fst detailed anatomical information ‘on this poorly understood clade and extend its range from the lower Middle Jurasic to the end of| the Cretaceous, showing that this group persisted for more than 100 milion years. Modem large-bodied,planktivorous vertebrates diversified ater the extinction of pachycormid atthe Cretaceous Paleogene boundary, whichis consistent with an opportunistic refling of vacated ecospace he largest veriebrates—fosil oF ving — I ‘are marine suspension feeders. Modem clades adopting this ecological stateey diversified in the Paleogene (6 t0 23 million years ago) (7-3) and ince baleen whales and Tour independent lineages of carilaginous shes (sharks and rays) (4) In striking contrast tothe 990 may of giant suspension Teds found in Cen 2oie marine environments, this il has appeared to be absent during most of the Mesozoic, an iterval tha 6 marked by the ecologial ascend ance of modern plankton groups (5, 6). Possible candidates have been proposal (7,8), but the clearest examples of Targe-bodied planktivores in 22 RA. te FD. Sno, in Si A tsi Gm, Comm, Cam Mh 621 099, A Tataman Tana Ma, SBS an Che Soc 323, 601 (2003 24 CAL eng eal Cem Se 197 6682 (995, 25. Cua US. Heng, Dla ead Let 3, 105 00a, 26 Hua Oa EU Wart, NC, An (hen oe 129, 12727 499, al Ft eo, Gauson 3, Reson E01 usin, Mating 304 26 GR Ea RF Chi F Bite, OSC. Ya, 8.0. Stasi, Can | Chem 74, $91 0996: 29, Pata Me cabin An, Che Se 428, 10012 ‘aon, 50. PA Save, A. ition, Ott. 4 217 (002, HL. LU Soul Pk, Gl, Wes ha, Aen agar hemi RSC Pasig, Canben, UE 2009. 22 Tol MH iJ Ch SoC 396, 2072 asm 33.1 Goede, KN oak 70. Chm. 57, 3031992 1 Nts Py Onan Chery, ee nk, 195), pp. 296301, 55 1s wi appre by MM as GH3216 an 50 e721 aed by lini apo mh DreheFaneston 9 he keno Cea Sow rt Rte 09S), he Are rama Sect gh the iin , Sil Foal erat of arpa Aang are alae Fee {8 chage tom te Candie Crysallaphie Dain Conte, 2 Supporting Ontne Materat scenenag anaes Tu327 59689860. Hert nd thar Totes St S17 Fleeces 5 oxober 209; sept 13 ora 2040 the Mesozoic seas have been a handfil of bony fishes confined toa brie 20-milion-ycar window during the Jurassic (Callovian-Tithonian, 165 ‘145 million years ago) and known almost ex- clusively from European depesits (9-12). These cnigmatic taxa belong to the extinct family $Pachycormidae (the daguer symbol indicates extinct groups), a stemcleost clade that is otherwise composed of pelage predators con- vergent upon unas and billshes (0). Giant ‘achycormids include the largest bony fish of all time (the ~9 m fLeedsichthys) (9, 13), but their short stratigraphic range had implied that they were an inconsequential component of °oepetmet of Each Science, Urvesiy of Onr, Parks Font, Ono OL JPR UX “Endre Sexe Poem tnd Deprmentof Bal Scere, DePaul Unverty 225 Neth Con emia, Chea, sO814) Usk ‘Seine Maem of Natal Hay, fat Hay State Un vesiy, 000 Sten ike Hay FS 740, USA Mah Hitey Museum ard Bidversty Research Cet, Unies cf Kans, 1345 Jaya Bout, anerce KS 68048, Us "Dion of Eloy and Beluionay Bio, Fal 1 Bimal and Lie Scenes, Unversty of Gogo, Uses ene, sgn G12 660, UK "td Pas doy ad Ray’ Mout Dewsau Resource Cer, 201 Sout avn set Worn Pak, CO 8085, USK “To whom conespondence shoul be alesse. E-nat inaigeath nasa 19 FEBRUARY 2010 VOL327 SCIENCE www.sciencemag.orgMesozoic marine ecosystems and a einor— sand ultimately unsuecessfil-experiment in sus- pension feeding at lange body sizes. re, we report newly recognized remains of suspersionfeeding fpachycormids ranging from the lower Midlle urasse tothe Upper Cresccous and discuss the implications of our discoveries for “Mesoaie marine ecosystems, With the exception of one recently discovered specimen, these fossils were housed in museum collet in which they were either unstdied or had boon described but incomeclly Mentfied in the 1th century. Further preparation and examination, combined with an exsting systematic framework (10, 12), led us to revise previous intepreiatons ofthis materia ‘The fossils reported here include & gencri= cally indeterminate cranium ffom the lower Midale Jurassic (Bajocian, 172 10 168 millon years ago) Infeior Ooite of Dorset, UK (Fig): the nearly complete skull of #Rhinconihilys taylor gen. et sp. nox. (14) fom the lower Up- per Cretaceous (Cenomanian, 100 o 94 million years ago) Lower Chalk of Kent, UK (Fig. 1) a Similar fish ffom the Upper Cretaceous (Ceno= ‘manian) Middle Yeo Group of Hokkaido, Japan (5); and_ multiple specimens of tBon- nerihthys gladius wen. now (16) fom mid-upper Upper Cretaceous (Coniacian-Maastrichtian, 89 10 66 milion years ago) deposits of the Westen Interior Seaway and Coastal Pain inthe United States (Fig. 2) (05. 17.19). These specimens reveal the anatomy of suspension-feeding.fpachyeormids, previously known exclusively fom poosly” understood Junasic fossils Material of {Leesihulys is crushed, fragmented, and disartculated (9, 1), tnd detailed stricture is obscure in rare atioue lated examples of Asthonocormus (20) and ‘WMartilchihys (12), We base our description on ‘Bonnerichehys (Fig. 2) The soout is covered by & median rosto- decmethmoid, which is a #pachycorid syne Pomorphy (10, 15, 19), Two ral projections slong the orl margin a unknown in other forms, but their position suggest that they might be co= cossifed premnillae. Thickened ridges trace the Iara margins ofthe rostudermethmoid, rk the positon ofthe supaotial canal, and imply that the rostexermethmoid of }Bownericahys also incorporates the nasals. The rectangular frontal bears ridge om its visceral surfice, marking the course ofthe suprabial ersory carl. Te hooke shaped dermosphenotic ies lateral to the font, defines the dorsal margin of the obit and bears the anastomosis between the infrarbital and o> sensory canals. A trindite caal-bearing osifea- tion is identified as a dernonteraic. There is no indication of sume contacts betwen the dermes- phenoie, dermepterotic, and frond, or beeen the fontal and the rostradermethmoid, The condition in #Bonnerichhys is derived rave allotherfpachycormids, n which dermal bores of the sll roof are tightly inked AS in many suspension-feding fishes (4), the dentary ofthe lower jaw and the maxilla of wonusciencemag.org SCIENCE VOL 327 the upper jaw are clongate and edentulous, Absence of dention even extends to the preatc- ular whic lines the inner surface ofthe mani, and the parasphenokd, which forms much of the roof of the onl chamber, The parasphenoid is pierce by paired foramina forthe imtemal carotid teri, a derived feature of teleosts (15). “Members of the operculogular series recon cred for tBonnerichtlys include the opercle, subopetel, and gular plate: The hyoit arch is represented by liye, imperforate hyomanlibe Ine with well-developed opercular processes and rod-shaped ceratohyals. Gillarch remains ine clude hypo- and inffapharyngobranchials with complex articular surfaces, plus long and deeply grooved cerato- and cpibranchials, Enlanged gil rakes bearing long, needle-like projections are found in fsthenocormus, tLeedsichilys, and +Rhinconichhys (9, £0, 12,15). These probable sspension-foeding structures have not yet been recovered for *Bonneriohhys Typical of thachyeormids, the pectoral fins ar seythe-shaped. The leading edge of the fin is fused, with an iregular margin that contrasts with the precisely patterned serions in some Tom REPORTS i species of $Protoyphyraena (10, 17, 19, 20) Distally, te fn-ays bifurcate in the “Y” pater saractrstic of fpachycomids (10, 19). The pectoral endoskeleton of FBomnerichihys agrecs closely with those of *Orthocarmus (20) and $Proiosphyraena (20). The second radial is "C-shaped, makes (wo independent articula- tions with the primary shoulder girdle, and is tralled by a series of paddle shaped radials (15), ‘The sul of ¢Bomericuhys specimen KUVP £60692 measures roughly | m from the tip of the snout to the rear of the pectoral wtdle (Fig. 20), Tsometic scaling relative to articulated fosis of {Asthenocormus and *Morilichthys (12) sua- gests a total length of approximately 4 m fr this specimen, Other less complete remains of ‘ Bonnericthys (associated skeleton Univesity fof Nebraska State Mascum UNSM 88507 and Pectoral fins and girdles KUVP 465) are over 20% larger than equivalent elements of KUVP £60692, indicating thatthe genus i likely to have reached at least Sm in length. This is smaller than revised length estimates for FLeusichays (13) bul approaches the size of some moder plankin= forous chondsichthyans (4,2). eu ent Fig. 1. Nery recoorized fsls of giant Mesomic supersioneeding bony fishes. (A) fincoictys tor gen ts. nov, BAN 233, rom the ues Upper Cretaceous Cenomanin) Lowe Chal of Ker, UK in ight ard etter views. (8 Indeterminate edentulous fpachycomil, BH P4146, fam the Loner Mil Jurassic aja Irae Ooi of Dost, UK in doa and veal view arterir oward ‘te op) atx hasbeen dgtaly masked so sto enhance conta and preserved bone that weld have been visble extemal in the verval and dorsal views fs shaded in terttve recnsructon based on ‘Wanticys ad YPoctycormus Scale tas apy only to fo. boc, baccptat bs, branchial ras: hy, cebrondials dh, anterior crtbya: de, det, dot, demopeso; et extopesyi frontal gu, median guar hy, hypobyl: ym, hyomandbuia; mp, metaptacd; mx, masta, ps0, parosperoi: é,rsradeemetmoit: ard st cleo ng. Paired bones ae tse a it (oe 19 FEBRUARY 2010 991992 Fig. 2. {Bonnerchtys glodius gen. nov, a giant suspersior-feeing bony fh from the Upper Cretaceous ofthe United States. (A) Newrocranium and prasphenoid in venta view. (B) Guta platen vetal view. (Q Craniat and pectoral skeleton, shown in rghtlateral view. Bones that were recnstucted from other species are shown Jn gay. (D) Hypural plate in right Latera view, Scale bar in(© applies to (At (0). Ato (0 show specimen KUVP £60692; (D) shows specimen FHSIA (temberg Museum, Hays, Kansas) VE-17428,(E) Tertatve reconstruction in vertval (op) and Lateral (bottom) views, inating Ue positon of bones shonn in (A) to (0). ant, antrbtal ar, articular cle, cleithrum; dsp, dermosphenotc; ect, fectopterygoid; thym, hyomandibular facet of neuro: cranium; op, oper: p§, pectoral fr; qu, quaate; 5, scapulocoracoid; and sop, subopercle. Other abbrevia tions ae asin Fig. 1. Fig. 3. stratoraphic diti- bution of giant -tpachyorid fishes and modern plank- tivorous whales and chon drichthyans (cons of tving ‘groups are not shown to Seale, Previous exeurerces of suspersior-feeding tpachye carmids were confined to 3 shor interval inthe Jurassic Ghown as a thin line rep- resenting “tLeedsichthys, {ortiichetys, and tasthe- rocormus), but occurences reported hee Shown as thick lines) expand this group's stratigraphic range to ap- proximately 100 millon years, Convincing records of modem large-bodied.plank- tivorous vertebrates: only appear in the Paleogene, after the extinction of giant ‘tpachycormids 19 FEBRUARY 2010 VOL 327 A ! A. clastic analysis provides rabust support for the monophyly of a clade comprising ‘Bonnericuhys plus other large edentulous ‘pachycormids and reinforces the interpretation of pachycormids as stem teleosts (15). This subset of Fpachycomids is «unique example of suspension feeding at messive body sizes within the teleost total group (23), which is a diverse radiation that otherwise contains a bewildering say of bodypans and whose living rgmesen= tives are wxonamie dominants in marine verte brate faunas (22). With lengths between $ and 9m, tBonerichihys and }Leedsichthys are ‘comparable in size with some modem suspension- foading sharks (4, 22) Even the more modestly Sized {Martlichiiys und ‘Asthenocormus are ecological giants in comparison with extant plunktivorous teleosts. At over 2m, the ‘fpachycormid genera are an onder of magnitude Jonger—and by extension several orders of ‘maygitade more massive—than living teleost suspension feeders, most of which are on the oer of 10 em long (23) The only moderately Jange planktivorous actinopterygian (the more extensive bony fish clade that contains teleosts) is the padlefsh Poh edon. This surgeon relative is confined tothe freshwaters of North Ameria nd rarely exceeds 2 m in length despite « greatly congated rostrum (24) SCIENCE wwwsciencemag.argThe discovery of carly Middle Jurassic and Late Cretaceous *pachycocids with anatomical features. that are consistent with suspension feeding (4) alters the picture of the evolution of this ecologial guild in the Mesozoic and aiferward, Ooeans during moch of this interval have been viewed as devoid of large-bodied suspension feeders (25), but we now recognize that fpachycomids occupied this ecological role for much of the Mesozoic (Fig. 3), Marine replles diversified prolifically during this go= logical interval, attaining massive. sizes and evolving specializations attributed 1 suction and ram feeding (26), but ther is no clear evi- dence tha they ever adopted panktivony. This observation, coupled with the perceived absence of large-bodied planktivores during most of the Mesazoic, led to suggestions that anatomical constraints prevented these othervise diverse ‘marine amniote clades from exploiting suspen sion feng (25). One findings suggest that marine replies might have been excluded fiom this trophic stategy by incumbent fpachycormids The fist sil occurenoes of modern large bodied suspension feeders are confined to the Cenozoic: manta rays and whale sharks inthe late Paleocene (J), basking sharks in the mid- Bocene (2), and plankion-feeding whales near the Eocene-Oligocene boundary (3). The only example with a possible Mesozoic record is the megamouth shark Megachasna, but there isa 7S. millin-ycar intel between a few isolated Late Cretaceous teeth and the next oldest oocurence, hich dats othe Ite Oligocene- carly Miocene (27), The radiation of largehodiad suspensione feeding chondrchihyans and whales inthe Paleo- gene follows the disappearance off Bonnerihilys and many other large-bodied marine teleosts (28, 29) during the end-Cretaceous extintion, sugaesting that familiar modem groups of plank tivores diversified into the ecospace vacated by siant fpachycommids, References and Notes 1 Wappen, Ct}. Bake Fs es 2 1s (Chagnon Ln, 1999p. 55-40. AL ne AS Ragu Ane st 10,88 G98 EW. Hagel Ba St 273,25 206), SL sande, Waser he Sil 3 Jfanuen bf Eos (na cheage es, (heap 1983) pp 37-212 SN Toman Be Une So tnd 73, 19 (200. 2.6 fake of, Scene 305,354 208, 1 Bota Mor: Ques Ms 49, 523 G08. nd Poesy, 6 ra P-ibal,-V. ss, Es elas esi Mine, 2000, 9. DIN, Man Kai, eo Pleo ‘ava 10. F Lares, hes, sues risen, Geoninoen, Neerans (1992 1, | Uae neo Fees 9st, Polearnnoneas aa Rader, 6 uaa, AT, (erage le en, 208, 12. on, neo Fees 4— Haman on Dreyer, sae, AH Wan, de, eag Or Fiich Pe, cer, 008, pp 285-98 wonusciencemag.org SCIENCE VOL 327 22, lta, Lob Ach Nat Mt 31, 236 2008, 1H, Eamon: The gene name 3 romano an Umpire by Hate Rynenonenye) Sper Oscyes ay, 1860, Atop ova 1891 Test Mil, 18 Gast de Pa 1996) aches Nona, 195; {icant get 9. oH: BN {he Haralson, Londen 238, rey ‘esa pecs Edeulns payor {ram ae mers af tgp m hang exepsenly slong pose races fe demopaas ad 2 font ea of oman rant he Leaty empress iy a ge over Cn ae ism Cerna, Ket 15, user sd me ae able 3 pate 16, Eyal The gee rae bana te Seve aly, Scat Cy, asa ich hs made may pra finer nthe Mca Forman, nang KOUP ‘Gray Kee Mat Hy Rew, tee, a 60692 Sena One He as Gene de Ps 990 aha Weootar, 1895 Banners no gen aed ‘enenchny: cau Cope, 1870, comb. rv aye AH ean se of Rata Hy, ‘ew ot FE 1889, incl petal. Doge of ‘era pce! Ertan psyco erg Tesch dep at oe Geral irs oh stu usec, an rio rages pea fs tneqary cela oalty and ye: ye om Nitra Foran te Geen, mam Cope, USK 17.1.0 Stews, Gels, Pooley ond Soenopy of etn Kea ME aon Et ays Ste Ure Mj S988, pp 80-84 REPORTS i 1, 0. Pam 8 Scones, WB. Glagher Spee Pap Geo Sein #27, 99 200. 19, A) Aaonring te, Hest Clipe, nv Landon, ann, UF 1570 Aalestogapia sary, Lanson, UK, 192) 21 HLL sen, Fost srt, 1872, 22 1am, Fes of he Wn Me, Hobs, 2) B,J A frestinan, 0, LG ook, Re Fi Bk Fh 2, 403 2002, Nat deri ard hot of exo aon Mit, Boca, 199, 2, Rab CM ans in Arco Moe ps, eM alo €L Meal essences, Sam Deg Ch 1997p 5-6, 26, CU Nal, sae, Vee Ponta 24 636 2, shina, verb. eo. 27,512 (20. 26. Gann aot and Bloc! Ee of mpect Fenn tes, © Kare foe. Goae Ves Bern, 201), p. 1-158 29, Ueno, Na He Sl USA. 206, 5218 530 IF nas ede an neon Pecton Agency Schnet here es elle ar 7916730 Wethan 1 Ujeno dH. Manabe fr poeence. (afore japanese pach, 5. trea or ‘acing spinon greprton, eho ‘carton {Bone dM. Bre, Supporting Ontne Materiat mcenenag ag erieTa2 759689500 Figs Stasis Petree 1 Novant 2008; ace 25 eu 2020 Climate, Critters, and Cetaceans: Cenozoic Drivers of the Evolution of Modern Whales Felix G. Marx? and Mark D. Unen® Modem cetaceans, a poster child of evolution, play an important role in the ocean ecosystem as apex Predators and nutentcstributors, aswel 2s evolutionary “stepping stones” forthe deep sea biota. Recent 10", hypergeomeni tes) and exons codependent on these factors were Independently validated by RT-PCR assays (fig S13, B to D. MRGISPTB codependence was ore pronounced for exons tha display relaive- |y moderate changes upon PTE depletion as com pared to strongly PTR-dependent exons (Fig. SA and fig $13). Funhermore, MRGISIPTE code- pendent exons contained, on average, weaker PTB- binding sites than did strongly PTEdependent exons ig. ST3A) (24). These resus sugeest that histone modifiations preferentially modulate the splicing of alternative exons that are weakly regulated by PTB. ‘Our rsals demonstrate are for histone moxl- ifcations in AS contol. We propose the exsience of adupir systems consisting of histone modif- cations, chromatin binding protein that reads the 999histone marks, and an interacting splicing reg. ubator (Fig, SD). Such complexes are a means for epigenetic information to be transmit wo the pre= mRNA processing machinery and probably act by favoring the reratment of specific spicing re ulators to the pre-mRNA, thus defining splicing coutcome, We show here that fora subset of PTE dependent genes, this adaptor system consists of H-K36me3, its binding protein MRG1S, and the splicing regulator PTB. Is tempting to spec- ulate that other combinations of adaptor sys- tems exist hat act on other types of alternatively spliced exons. Physical intrtion betwoen sev eral chromatinassovated proteins and splicing components has besn reported (9, £4). Our ests rein ine with recent indirect evidence based on genome-wide mapping of histone modifications for a role for chromatin structure and histone ‘modifications in exon definition and altemative splice ste selection (5,13, 15-19) Although our observations argue for & direct linkbesween histone maifiations and he spicing machinery, histone marks may also affet splice site choice indirectly, Extensive evidence dem onstrates a re for RNA polymerase clang tion rate or higher-onder chromatin structure in splicing outcome, and itis likely that histone radiations ct in concert with these mecha nisms (6-8, 10-12). Based on our findings, we propose chat the epigenetic memory contained in histone modification patterns isnot only used to determine the level of activity ofa gene but also transmis infomation to establish, propagate, and regulate AS patterns daring physiological pro- cesses such as development and differentiation, References and Notes 4. fo. Bereove C226 37 (2006 2A} Mati Ck, C8 Sm, Aa. er. a el ot 6,386 05, 2, Marg el, Noe 27, 56 2008) 4.65 mang 1 Caper Nt Re Geet 8,749 (2007, SALAS rl, Rat St. a i 6,717 09). batch Yarn € aca Not Sc Ml Bi 1H dea et ol Ma, Cel 2, $25 2002, A orb Seta Nt, Pde] Muna, Nom, 2425, 1288 200 9. RL: Lomi el Ml Ce 3,450 209) 120 | Mute Ce 237, 708 2000, EG. hoses, Sort Famer, . Bete, mete, i, Chem 277, #8110 G00, 12, 8.0. Rots Dion, MA Goan, | Bot chen 279, 29075 2000, 19, LE. Ser, Maka, Flick M.A 14 RSs 3a. Ml el 28, 665 (2007. 15, ers, Eat, ads Cases, J: Kemer ene Re 39,3732 209 6, Bevo ey cnet 3.376 208 26,990 2000, 18 Nps © Men, RA Podge, 8. Bae, Mol Ce 36,245 2009. 2, Hoe ea, Hat Suc Mt Bol 26, 996 200 20. RP. Cases E Magne, M.A, Gace Bance, a Ge. Bi. 20,7388 200). 21. CC Mana, 1H Sata 8 abet). Magra, LP Castns a Cal 3,591 2008 Regulation of Cellular Metabolism by Protein Lysine Acetylation Shimin Zhao,*# Wei Xu Wenging Jang," Wei Yu,"# Yan Lin, Tengfei Zhang,” Jun Yao,? 1 Zhou,* Yaxve Zeng," Hong Ui,” Yxue LL ong Shi,¢ Wenn An,” Susan M, Hancock,” Fuchu He? Lunxiu Qi,? Jason Chin,” Pengyuan Yang? Xian Chen,2* Qunying Lei,*=* Yue Xiong,"t Kun-tiang Guan™=*?} Protein sine acetylation has emerged as a key postranslational modification in cellular regulation, in paricular through the medifcation of histones and nuclear transcription regulators. We show that Uysine acetyatin i a prevalent modification in enzymes that catalyze intermediate metabotism. Virtually every enzyme in glycols, gluconeogenesis, the tricarboxyc acid (TCA) cele, the urea cc, fatty acd metabotism, and glycogen metabotsm was found to e acetylated in human iver tissue. The Concentration of metabolic ful, suchas glucose, amino acids, and fatty acids, influenced the acetylation stots of metabolic enzymes. Acetylation activated enoyl-coengyme A hycratase3-hydronyacyt-coenzyme A dehydrogenase in faty ack oxidation and malate dehydrogenase inthe TCA cycle, inhibied argininosucinate lyase in the urea cle, and destabiized phosphoenolpyuvate carboxykinase in ‘luconeogenesis. Our study reveals that acetylation plays a major role in metabolic regulation, regulation of transeripion in the nucleus (), at much less is knowin about non- ruclear protein acetylation and its ole in cellu- lar regulation, To investigate non-nuclear protein scetylation, we sepated human Tver issues into nuclear, mitochondrial, and cytosolic fractions Proteins in cytosolic and mitochondrial factions were digested wih uypsin and acetylated peptides wore purified with an antihody to scetliysine Pps sceryation has a key role in the (ig. S1). The purified peptides were analyzed by tandem Figuid ehromatography-tandem mass spectrometry (LCILC-MSMS). From three in- dependent experiments, we identified more than 1300 acetylated peptides, which matched to 1047 Aistinet humen proteins (table $1), inching 703, proteins nat previously reported to be acetylated, ‘A previous report identified 198 acetylated pro= teins from mouse liver (2), and 135 (70%) of these wore alo present in our data St (Fig. 18), 22), Wager, MA Go lars, Ml Cl 0, 962 2 Sptinan onan CW Sth a C27 «20 25 Pahang et, Mee A Re. 34,6521 26 0. Fan et ol Hot Cal 26, 929 20H 27 Q.Fan,O- Sha | eB] Fey, B I encom, Net Govt a, 13 260. 26D Raye a Net. Bena. 2,66 2009. 29, Me han | Paton, 0. Sank, 8. Seal Set, Dv Bek, M, Gea Slane, ar Hania eens PS fo econ aro fr atone Mgt fori ac: aa Moi ‘eae on atta ard Tonge ral cor. THs reac as sare by ferro Aaah gam he otal Cn ote (Ge Cac Read A gn tam end (Goer ee of Cana, Gada nso lh Rewaeh art OF 6731) an Grae Cas ‘Soir tig Ontra Cerais Ireie) 9 BE RUMI pont OL AOE (QU OPS, es vec Hea Aon Yan CSS a Ganpce MUA seepage date a mate be (Gn preston Oras ese ra. SEI373, Supporting Ontne Materat tomacencenag argent ulacerce 1124208001 Hert nd tase Fie Stia S12 ene 4 Neb 2009; acpi 17 Deceber 2008 Paes are 4 Fry 201° Ide natn cng th pape. Indicating that our proteomic analysis reached high degree of coverage. Choudhary eta, very recently reported the ideniication of 1750 acete ylaed proteins from a human leukemia cel Line (3) but only 240 ofthese were present in our data set Fig. LA). Comparison of these three acet- vylome data sets indicats thatthe spectrum of ‘acetylated proteins is highly conserved in the liver between mouse and human, buts very dif ferent between liver and leukemia cll ‘We compared the aceiylated proteins with the ‘otal liver proteome and discovered that enzymes that participate in intermediate metabolism were preferentially acetylated (Fig, 1B) Indesd, almost "soa of ie Sees, aon une, shat 2003, (Gna. ear and ella ab ada Usesy Sangha 20032, china. “ent of Proteomic, ste et fiemetea Scenes fen Unie. shaghs 20022, (ina. “Deparment of Bers and Bogs, Une toe Comprtenive Conc ene, Uses of Nath retin Chapel HL NC 27589, USA. Bisted Pros Hossa, Fedo Unity Sanghi 2003, Chis. "Be Inoats Cate, ey abt Systeme lly, nha Instes for Bulag Scenes, Chie Academy oS ces, Shnghl 200031, China. "Medial sear Cutch ‘Ghote of Mier Slgy, Ws Ron, Contigo Bz (Uk Depart of Binal Chemis, Fun Uri ‘erty, Shang 20032, ie, "Depart of Pema ay a Mores Canes Center, Unversty of Cai, Sn Deg a Jl, CR 92083. USA ‘these autos contd equally 1 is work {a whom corespondence stl be added. E-mait Yesngemane eds C0; kaguangued ede LG) 19 FEBRUARY 2010 VOL327 SCIENCE www.sciencemag.orgevery enzyme in ghcolysis, gluconeogenesis, the TCA cycle, the rea eye fat acid metabolism, tnd glycogen metabolism was acetylated (Fi 1, C to Gy. The high ovcurence of metabolic n= ‘zymes identified in our MS analysis is pparenily not due to the abundance ofthese procins, be cause only a few rbosomal proteins (7 of approx imately 80 cytosolic ribosomal proteins) were acetvlated (table $1). These results indicate a previously unrecognized and potentially exten sive role of acetylation in regulation of celular tnstaboism, Therefore, we investigated the effect of acetylation on representative enzymes fom four metabolic pathways. Enoyt-coenyme A hydatise’}ydroxyacy cocnzyme A (EHHADH EC code 11.135) cat alyzes two steps in fatty aid oxidation (Fig. 1E) 4, 5), and its deficiency causes abnormal faty ‘cid metabolism (6). We identified four acety- ln psn sides (Ly, ys! ys and ys) in EHHADHT (able $2), immanopre= cipitation of ectopically expressed FLAG-tagged ENHADH and Wester bloting with antibody ‘tw acerllysine confimed that EHHADIT was in- Fig, 4. Acetation of liver metabolic = names (9) Comparson of tree acee SHY 5 lation proteomic stuces: this study and Pawar Faas G, 3.) Preferential aceon of n- 5 ef Kinley aa ‘Coarse ca] es exact test of comparing acetated tee = protest er pois shows that y fomnogruga. | 240*10 acetylation ts much more prevalent i Km era, Co a ST intermediary metabolic enzymes. (C to G) Acetyated metabolic enzymes iden- ‘ied by proteomic survey are marked in red, See supporting onine material for key to abbreviation. F wonusciencemag.org SCIENCE VOL 327 deed acetylated. Is acetylation was enhanced by 80% after treament of eels with wichostatin A [TSA, an inhibitor of histone deaetylse (HDAC) and] and nicoxinamide (NAM, an inhibitor of the SIRT family deacetlases) (Fig. 2 and fig, 2A), To quantify the sceiylaton of EFIHADH, \we used isobaric tags for relative and absolule quantiation (TRAQ) MS of immunoprecipitated EHHADH. TSA and NAM teatment increased ys" acetylation fom 43.5% to 62% and Lys scetylation fron 468% w 77.8% (Fig, 2B), espec= tively Consistently, dhe comesponding unacctylated Depts were deceased by TSA and NAM teste ‘ment. These resuls show that a substatil portion cof EHHADH is acetylated and that EHFADE scetyaton can be dynamically regulate in vivo. ‘To detemnine the elect of acetylation on en= zymatic activity, we weated cultured Chang hue ‘maa liver cells With TSA and NAM and detected doubling of endogenous EHHADH activity Fig, 2C). Similar observations were also made with ectopically expressed ENITIADI in TEK2937 cells (Fig. 2C, right panel). Acetylation of an ENHADIT'® mutant, which had the four pute REPORTS i tive acetylation lysine resides replaced by gitar mine, was decreased (fig. S2C) and its activity vas no longer regulated by TSA and NAM (Fig, 2C), Adkiton of fat acids to the culture medium increased acetylation ae activity of EHHADHL by factors of 1.7 and 1.3, respectively (Fig, 2D snd fig, 2D). Thus, acetylation of EHHADH can be regulated by extracellular fues; this finding supports a physiological ole oF acetylation in the regulation of EHHADH and fat acid metabolism, All seven enzymes in the TCA eyele were ssccylted Fig. [Cand able SI, ineluding malate eydrogenase (MDH; EC code 11.1.3) in which four acetylated lysines were identified (Lys!™, Lys, Lys", and Lys" ables $2 and $3) Eciopcally expressed MDI was acetylated and ss acetylation was increased by a factor of 24 in cells treated with TSA and NAM (Fig, 2F and fig. S2E), To quantify MDI acetylation, we per- formed Fourier transform ion cyclotron resonance (FTICR) MS. This approach identified the wn- modified fulldength MDI and two addtional peaks, each with a mass increment of 42.01 dal- ‘ons, corresponding to mono- and diacetylation sahon ante act Urea cyale hase ch SS ak 25> yy Say sok ——~. veatemaeycak Sema eae Sir abe se eck Gece sospae — Gunstsrospa Ix choosen oe nesSemn 19 FEBRUARY 2010 1001: REP 1002 A racennaon - 4 4 B wanetsa == 4 [ein papian | elt pop atundanca Co ® afin eee ee Ack [ME] [LetoK"Wnsopveran| es-22 | coo-e7 ° 2) [iinsoovecan sescaa [mae onac MiTSVLER CASK west mast 8) irsviex Beet | eesat 10 Zia AGeHWADH Fon @ 50 100200 = —smC« Angatant ASL" was refractory to inhibition by TSA and NAM (Fg, 3D) o¢ ectvation by amino acids (Fig 36). The ASL mutation didnot alter global protein structure, a8 determined by limited proteolysis and circular dichroism analyses (fg. 83, D and B), ‘Therefore, extra amino aids appear 10 activate ASL by dereasing acetylation of Lys? ‘The urea cycle is coupled with the TCA eyele because fumarate generated fom the rea cycle cam be fed ino the TCA eye or energy proue= tion or gluconeogenesis (8). We therefore deer- mined the effect of glucose on ASL aetvity and actyation, Ghucose incre acetylation of AS by a factor of 2.7 (Fig. 3F and fig, S3F) and decreased activity of AST. by 80% (Fig. 36). Tn vito inubation of ASL. immunopartid from 19 FEBRUARY 2010 VOL327 SCIENCE wwwsciencemag.orgFig. 3. Inactivation of A ASFA + ASL by acefation, (ANAM OTEA = and 8 ASL acetylation. - re FLAG-tagged ASL or oan ASL vas ove pressed in HEK293T fells, immuroprecin- tated, and probed with antibedy to aceysine 9 oF to acenttys™™ ®. (© inhibition of ° 8 Aeatve ASL Activity) a8 ASacaten yor B MAG = mm _w Ss ‘tra amino acids. ASL 7 =. Sikcon = oe | was. immunoprecii- e ae 200] ted fon toneced odo (EE HEK2931 cells, which were treated with varios ain acid con centatios. (0) Inst tin of ASL in espns {DNAN ard TSA Wld- ‘ype and mutant ASC ASLFLAG + + + H K2BER (132% of Asovousoun 0 4 . wipe seg yo we teins were expressed in = roxas. Seewereran Stee « aa ere sed th NAIM 5 ask moo # and BA as nda BSS enc HO heiviy of immuno: i precipitated ASL was normalized to total protein. (E) Requirement of Lys" acetyation for ASL activation by amino cells incubated in medium containing various amino acid concentrations i ove fof PR] ees a acs. Wil-ype and mutant ASL proteins were overexpressed in HEK293T F and G) Effects of glucose on acetylation and activity of ASL. ASL was similar to Fig. 2. A Ewuera = tke Cyeebeximiseimn) 9060120 180 «0-30 6120 10 =| eae CGuecrownt 6359 8D Aen ai — Fasunas 4 4 4 4 - CGueose(ma) 0 as 125 25 25 ® OFLAG =e oo = @ oFLAG overexpressed in HEX293T cells that were treated with various concentrations of ‘glucose. Acetylation and activity of immunoprecptated ASL were determined. (H) Activation of ASL by in vitro deacetylation. Invitro deacetylation was Fig. 4. Destabilization of PEPCKE by acetyia- ‘ion, 0 Glucose induces J=P=POK pePCKA acetylation, (@) Amino acids. decease PEPCKL acetylation. (C) Jevencer Glucose induces deple tion in oF PEPCK pro- J=setn tein. Endogenous PEPCK levis were detected vith /=PSPR 3 PEPCKL antibody. (D) lesein TSA and NAM reduce PEPCK protein abun- dance. (E) Glucose destabiies PEPCK Cj- lobeximie vas added ‘coro (nt) 9 a1_a2 TBS a Joraa at time zero to block translation in HEK293, Jem cells. PEPCKA protein abundance was deter- mined by Westem blot- —- = SS eee deacetylases. destabil- a Ee — ore te dpe but D ect _cungstircat FAC EPR |<< nin Nat the aetation- umes =| ei art PEPCKL < ——| == wimusciencemagrg SCIENCE VOL327 19 FEBRUARY 2010 1003‘mammalian cells with CobE deacetylase increased ASL activity (Fig. 3ED, supporting a direct role of sceilaton in ASL inactivation. The dual regue lation of ASL by both amino acids and glucose indicates that acetylation may have an important role in the coordination of metabolic pathways, Inthe presence of sufficient glucose, amino acid catabolism for energy production and gluconso= genesis would be inhibited. Inthe presence of shundant amino acids ar low gluco, cll woul swt to using amino acids for energy pruction with enhanced urea cycle activity, Cells may use sceiylation 40 coordinate multiple pathways in onder to achieve these metabolic adaptations Phosphoenlpyruvate carboxykinase I (PEPCK BC code 41.132) isa key regulatory enzyme in sluconeogenesis (Fig, 1D) (9, 1D. Thee acetate lysine residues were identified in PEPCK by MS analysis (Lys, Lys", and Lys™: table $2). Acet- ylation of PEPCK | was enhanced in cels rated ‘with high concentrations of plucose (Fig. 4A) but was dss by alton of emino acid in gucose- fice medium (Fig 4B), These resus suguest 2 po- ‘ential mechanism by which cells could regulate shiconcogencsi through regulating acetylation of PEPCK1 in response to the availability of extracellular face Inscarching for an eff of acetylation on the regulation of PEPCK, we noticed that levels of endogenous PEPCK protein were decreased by high glucose (Fig. 4C). Furthermore, treatment With TSA and NAM eatsed a 702% reduction in the amount of PEPCK protein in both HEK203T snd Chang liver eels (Fig. 4D). PEPCK vas sax ble in ces n ghicose-fe medium but unstable in high-locose medium (Fig. AE), When lls ‘were ated with TSA and NAM, PEPCK was unsable even in glueosefiee meium or in the ppesenoe of amin acids These sus indice da Sexton muy regulte the sabiiy of PEPCK We replaced the the putative acctyltion lysine scsiduts by anne (PEPCK) or glutamine (PEPCK:®) io abolish of mimic aetylason, respectively. The PEPCK? mutant was more stl tan the wid ype, whe the PEPCK I mutant remained unstable (Fig. 4). Moreover, treatment of els with TSA and NAM filed to destabilize the PEPCK*® ant The importance of lysine acetylation inthe regulation of chromatin dyramies and gene ex pression is ll apresiated. Ourstety and others ested te cope of ell elation by sine acct Ylaion to an extent comparable to that of other ‘uso postanslatonal modifications such as phos phorylation and ubiqitination. We how tha most, fntrmediate mtsblc enzymes are acetylated and that acylation can diel alert the enzyme ac- tivity oF salty, We found that acylation of metabolic enzymes changed in response 10 the sro of exter ier sail, po viding evidence fr a physiological role of y= amie acylation in metabolic regulation. The mechanism of acetylation in regulating mtbo- lism may be conserved during evolution. Many ictabolic enzymcs in Escherichia cal at ate ‘lated, hough the functional importance of these actylations has not boon investigated (1). We propose hat hsneaetlation is an evolutionarily conserved mchanisn valve in egulation of ‘etal in response t nutrient aay and cellular metabolic status. Acetylation may play a key role in the coordination of different Imctabolic pathways in response to extracelular conditions References and Notes Arf Nano € So, el Cal 3,249 2008, E Ghauhay et, scence 335, 36 (00 Site tl. Coco 233,297 20, #8 Wats a Jn. es 83,77. 989, 1. Tein 1, Hu, Mu 28, 68 (2000. 8 ELL Hangin ML Hagin, Beran, Piel. 72,49 0992 9 etary fhe, Ci. on. Reg 28, 101 10, Ste Mel el ok 20, 6808 2000, 11 J Thong ML Cl Pro 8,25 2009, alate ps gow is say and. ado oe ‘eiing the mans Supper y the 985 pray ‘om the Ginese Miya uation, sate ke erlopnentpeas af bs each China {caren 20098918401 an 2006880670), te ‘anal gh tery rsa ard delet (own of China ant 2064402408), Chee Anna Sere Foundation gant 30606212 an 30671255, sangha! a ise reach oj ‘gars oseates, orueo, oesaost0, srs rns ROACH, ICRI, 08 HESSD IKLG XC, a8 Supporting Ontne Materat sienehag ogre Tul32759¢010000C8 Hert nd tase Totes St 53 Fleeces 27 uy 208; aap 7 ory 2050 Acetylation of Metabolic Enzymes Coordinates Carbon Source Utilization and Metabolic Flux Cijun Wang," Yakun Zhang.? Chen Yang,” Hui Xiong," Yan Lin Jun Yao," Hong Li Lu Xie, Wei Zhao,? Yufeng Yao,” Zhi-Bin Ning,” Rong Zeng,? Yue Xiong,** Kun-Liang Guan,*” Shimin Zhao," Guo-Ping Zhao™**** Lysine acetylation regulates many eukaryotic celular processes, but is function in prokaryotes i largely Lnkrown. We demonstrated that central metabolism enzymes in Salmonelia were acetyiated extensively and diferentaly in response to different carbon sources, concomitantly with changes in cell growth and metabolic fx, The relative activites of key enzymes controling the direction of alycolysis versus gluconeogenesis and the branching between citrate cycle and glyonyate bypass were al. regulated by acetylation. This modulation is mainly controlled by a pair of lysine acetytransterase and deacetylase, whose expressions are coordinated with grouth status. Reversible acetylation of metabolic enaymes ensure that cells respond environmental changes va promptly sensing callular energy status and flexibly altering reaction rates or directions. represents a metabolic regulatory mechanism conserved from bacteria to mammals oicin Iysine acetylation regulates wide ange of cellu fancions in eukaryotes, especially transcriptional contol in. the rueleus (J, 2) Tealso plays an extensive role in regula of metabolic enzymes though vari- fous mechanisms in human Tver (3). In prokary= ‘te sch a Sahnanella enterica, reversible sine acetylation is known to regulate the activity of sanetyl- coenzyme A (CoA) synthetase (4). To de- temnine how lysine acetlation globally regulates he metabolism in prokaryotes, we deteminod Uae overall acetylation status ofS, enterica proteins under either fermentable glucose based glycolysis or under oxidative cite-baed gluconeogenesis By immunopuriicaion of acctylate peptides with antibody to acetyllysine ad peptide identification "sate Key Laban of ent Engnetng Deparmert a Uicesidegy, Soe of Ue scenes and ste of flamedeal cence en Unies. Shang 20032 (ina MOST Shanghh Laborer of Deze ané Heath Caenomic, Cine atonal Homan Genome. Centr at Shon, Stang 201203, Chin Key Labay Sats isogenic Cees at Latrty & Sens Bley nue of Plant Pyslogy ad Ey, Songhai or Blog Scenes, ries Academy ‘fScenes, Shanna 200032, cia ae Cl egy Ladnntay tt of irs inc aden Unies Shani 200032, China aoa of Hon Boca Pathog, epatment of Medal Moeslgy od Prag, rte of Me Scenes, Sangh To Unley Sa Madi Shenghal 200025, China "Depatment of Soden and Bop nd Lnebege Cerreersive Crs Crt, Uist af Nat Crating st (Gpet Hit Chope il NC 2750, USA. “Deptt Prmclay and Moors Cancer Cee, Unies of Calflora Sn Dio, La Ja, CR 92095, USA "Department ‘f Meloy an Ka Shing tte Heth Sees. The Ces Uns 0 Hog Kong. ce a aes esa ht, Mew Tete, Heng Kong SR, Chia “To whom conespondence shoul be adtessed. Era hana abe 52) gpuhang acer GP) 19 FEBRUARY 2010 VOL327 SCIENCE www.sciencemag.orgFig. 1. Acchaion of Bi canal metabacenaynes soc Sr 0 ene © hy Came in'S eerie. (2) Goble ir Sma] ee] ere xetlaton of S entco Pe eolam ome’ | Wwe | pure tngynes of central me ve | ae tabolim, Clored boses zou | so reper: bot gente OP atom ee om [ag (ne snd grey sa th roe ore ee drown irsie to inate me se | one seen He estimated by courting coe | 2 [ah ‘number of positive hits Ms vou | oe | ah from mS analyses. Com- ones se | puto wre nate fe anthems ew, oe ae wg ls rm See cor [ee SETS with ether lacs red cee iene 3 : Bo) or cna ate) os a jan 2 the sle carbon source, Papen iy Pree ato ro veel ana for WT cals low) en ine and Spat (gay, bath ERP ad ca oan on 18. Blak bores ea 18. inde that aceyated ms a pepe was not detected = = B)SUACqunticaionat on a rave action. Act = = a Maton of WT cals grown eS eau 7 wth glucose Wn) OF Si au in ee al argon at i with glucose pty) ad 20pm sos. bid seat gam wey | as = 2 et core (cobBy.) or crate (ob) were quatied by SWAC. Relative acetylation i presented wih the top mean value ( 3) of each pair set as 1 arbitrarily. ND indicates not detectable. Full names of abbreviation use inthis figure are available in supporting online material SOM) tx. A B 5.0 mM Glucose 5.0 mM Citrate a 2.0% Glucose or Citrate ge 3 oe 16 Bool = gu 3 at Z 3 ° 02 ae 02 8 cal : co a acme Cc D cmoneagp — eee Tae 2% L SacBee i 7 aac aaa nes Veen ! Weert citrate 0.2020.02 0.142003 0.310.068 oh Nie ong, Se es sce ee Fig. 2. Growth phenotypes of pot ard Acobe (A) WT ac), pore), cobs (ea, of pate green) sans were grown in rnin medium with indicated concetrations of glucose (et) or citate (igh .D. indicates optical density. (B) Gronth cuves of WTS. enterica in minimal medium cortaiing lucoe (red crate hue with (4) or thot (@) NAD. (Cin vivo metabolic ux profiles in S enterica during growth on glucove or crate. Invacelluar flux etiburion was wonusciencemag.org SCIENCE VOL 327 determined by C labeling and GANS analysis. rows indicate the dtecton of et ies, and thei its are scaled othe flux vats. (0) Altered metabolic Mn. rato of Spat and AcobB Flux profiles through dyes (represented by Ve juconeogenesis Yn), dhorate bypass acy) ard TOA ux (cy) Were ‘quanttated and used for caelating Vaga¥nus and Van fx Ft, Data shown are mean values of hee independent measurements th SD. 19 FEBRUARY 2010 10051006 by mass spectrometry analyses, we identified a ‘otal of 235 acetylated peptides that matched to 191 proicins in S. enterica, About 50% of the sceylated proteins participate i muliple bolic pathways (ables Stand S2), and about 91? of the enzymes of central metabolism were sceylated (Fig. LA). Stable isotope labeling with amino acids in cell culture (SILAC) quantitative analysis showed that, among 15 enzymes that had altered acetylation in response to diferent carbon sources, all showed greater acetylation in cells grown in glucose than in cells grown in citrate (Fig 1B), consistent with our estimations based on numbers of repetitive detection of acet- late fragments (Fig, 1A) ‘The abundant acetylation of metabolic en= ‘zymes anel the concerted chinges in acetylar tion dependent on carbon source indicate that acetylation may mediate adaptation to various carbon sources in S. enterica, which has only fone major bacterial protein acetyluansferase, Fig. 3. Regulation of centrat metabolic enzymes by acetyla- tin, (A) Aceyation of metabotic ienaymes expressed in Arab ard ‘pat sais. Hiagged. Gaps, AceA, or Ace proteins were ovr expressed in the WT, Acobé, and Apat strains and purified to homogeneity. Equal amounts of each protein were used and acetylation 3s determined. SDS PAGE indicates SDS poly: acrylamide gel electrophoresis. (B and ©) GapA, by Patmediated acetylation and CobB-mediated A Exoression Sra GGapA, AceA, and AceK proteins were purified from WT S. enterica and sub- jsted to in vitro acetylation by Pat or deacetylation by CobB. (8) Reciprocal Tegulation of alycolytc and gluconeogenic activities of GapA by Pat and CobB. (C) Reciprocal regulation of AceA. activity and AceK-conteolledICDH activities by Pat and CobB in vitro. Eror har indicate SD of three measurement, A Pat, and one nicotinamide adenine dinucleotide (NAD?) dependent deacetylase, CabB (4, 5) ig, S). Acetylation of central metabolic enzymes in 8 par null mutant (pat) was reduced, whereas acetylation of these enzymes was elevated in a ob ull roast (AeobB) compared with that of the wildtype stain when grown in glucose (Fig. 1B). When grown in cit, the acetylation of metabolic enzymes in AcobB cells was also increased over the wildtype cells (Fig. 1B). On the other hand, there was no change in acety= lation of ribosomal peotzins in either AcobB or ‘Apat cells (Fig, 1B), supporting the notion that Put and CobB are the major enzymes responsi- ble fo the reversible acctlaton of central meta bolic enzymes in S, enterica A physiological roke of enzyme acetylation in mediating cellular adaptation to different metabolic fuels was suggested by the different sgramth properties of wikktype, Apa, o Acobit Strains grown on diferent carbon sources (Fig, 2). The AcohB cells (with increased acetylation) frew faster than wild-type cell in minimal glu- ‘se medium but grew slower than wile ols ‘in minimal citrate medion (Fig. 2A), whereas the Apat cells (with decreased acetylation) had the opposite growth propstis (Fig. 2A). The ‘Spa/AcobB double mutant bebaved sully 9 the Apat stain, consistent with the notion that the CobB deacetylase would bave no effect on target proteins if they were not acetylated by at (Fig. 2A). Treatment of wild-ype ces with nicotinamide (NAM), an inhibitor for CobB (6), like cob mutation, suppressed eel growth in the eitrate-containing minimal medium but had litle effect on cell growth in minimal me= ium containing glucose (Fig, 2B), consistent ‘with the fact that metabolic enzyme weeylation is higher in S. enterica when cells grow on gh cose (Fig. 1). ‘We tested the regulatory role of Pat and CobPa in coondinating carbon source adaptation by mea nwt pat sea 2 ons o8S Sse esess AceA, and AceK activites deacetylation, Hi-tagged Cooma a otha! ED oueaed roca Lg" tg fag Stoney, Ions Sg" FSS femora, ———— | oe ees | em at oo | ee bisa tton rested ook a Not. COBe, Pat eeibe wats wees Fig. 4. Ditleertialtran- serpin of pt and cob in response to metabolic status and various ci bon sources, 5. enterica cell were incubated in 1B and then mashed and ‘wansered into minimal ‘medium containing 50 mi glucose or citrate. Cells were sampled at indicated growth phases WA) Growth curve (a) £ 3 4 isteg pt and cob® mRNA amounts normalized against 165 RNA (b and ratio of ‘Bat mRNAcobB mRNA (ed). () Changes of Pat and CobB protein concentrators at ciflerent grow phases in glucose- or citate-conaining medium, 19 FEBRUARY 2010 VOL327 SCIENCE wwwsciencemag.argsuring in vivo metabolic fx profiles with °C labeled ghicose or crate a the cer followed by gas chromatography-mass spectrometry (GC MS) aulsis (7, 8). The overall metabolic fax profes nS. enterica showed distinet pats ding rowsh on gcose or cite Fig 2C). The slvorsite bypus as ava, ad the caboa flow favored glusoncogensis when cell were sown on ciate. We also qunitavely compared the fos profes ough peal (ag io ncogeness (aah catbongic ack (TCA) exete (nc at alvoxslate bypass (7.0 oF the wiklaype, Apt std Aco sins wo ase the relation benveen metabolic Hx profiles and acetylation status, When cells were gown in he presence of gos, the Acobd san had 2.07- fold peter lyolysislusonagenesis Hux ratio than the Apr stain hal (Fig, 2D), whereas he alycolyssthconeogenesis us rato ofthe wide ‘ype strain a 47% lower han that ofthe Acai stain but 40.7% higher tan that of the Apar stain ig. 2D) When ells were grown in the presence of erate, the Apt tin hal 221-0 seater plyowyate bypass/TCA fx ratio than that of the AcobB strain (Fig. 2D), whereas de lvowsateTCA tux ato ofthe wildeype stain was 88% lower than th of the Apt sein bu 43% higher than that ofthe Acoba! strain Fi 2D), These results and the eet of gcoxe on acetylation of matabole enzymes (ig. 1 indica that carbon source associated acevlation may affect the rate atv of metabolic enzymes an ths module metabolic fx pois. Dice evidence of acetlation mediate ulation fr cena meubole enzymes was dive fiom biochemical studies with thee exzsmes (able Sir the gleeralichyde phosphate de- Iydrogense (Gap), which channels svcoisis o gluconeogenesis fun bition a well 6 the icieae Iyase (ACEA) and the inca detyérogsase UCDIN kanasephospatse (ACK, both of which control te ux distibution of isocitate between the eneey-geerating TCA ele and the gloeoncopenesirequiing glo sate pathway when only an oxidative carbon Source (such as cite) is availble Y) Ant= scatyysine immunoblot showed that all ees enzymes had increased acetylation inthe Acobe stain (ig, 3A)- Acelaion of these thee ene zymes dereasd to vations degrees inthe pat Stain. The remeinng acetylation detect in the ‘pat sian may refs the prsence of other aetyansferass (ig. $1). All these enzymes were more hewily acetal in ells roan in minimal media with glocose than those grow with ciate (jg, $4), These obsenations con- firmed hat Capa, AceA, and AccK ae under regulation of Pat nd Cob. To demonstrate a direc effect of acetylation on enzyme activity, we puri recombinant Pat snd CobB proc and tested Gap, AceK, and ‘Ace eyesin vi ig, $2) Tnreasing Gap acevlton by Pat aceyase weament increased is plycolysisaetivity by 100%, whereas is a= tivity in promoting ehiconeogenesis was inhib wonusciencemag.org SCIENCE VOL 327 ited by more than 309% (Fig. 38), supporting the notion that acetylation stimulates glycolysis but Inhibits gluconcogenesis, Consistently, in vitro deacetylation of GapA by CobB caused a 30% Increase in gluconeogenic activity bu a 27% de~ crease in glycolytic activity (Fig. 3B). The ine crease of AceA acetylation by in vito treatment with Pat Fig. 30) or aceylation-mimicked mutae tion (fg. $3) led to 30% and 20% loss in AceA~ spifc activity, respectively In vito deacetylation of AceA by CobB, on the other ane, led & 24% inetease in AceA specific activity (Fig. 30. ‘The activity of AceK was measured by its bilsy to inactivate is native substrate, CDH. which cata- Iyzes the conversion of boca o ket, Invitro acetylation of AceK by Pat led to 260% reduction ofits ability to inactivate ICDIT (by phosphorylating IDF), whereas in vitro deacet- ‘lation of AceK by CobiB increased AceK s abil- lay to inactivate CDH (Fig, 3C), suggesting that acetylation may activate the phosphatase atviy (of AceK toward ICDIT, consistent with the fact that both acetylation and the acetylation mimetic ‘mutation of AceK activated the phosphorylated ICDH (figs. $4 and SS). Together, these results demonstrate that activities of GapA, AceA, and Ace enzymes are regulated through acylation, ‘which in turn may direct the fax diretion toward alyeolysis or gluconeogenesis or the distibu- tion of isocitrte between the TCA eyele and alyoxylate bypass. We also investigated the expression ofp and cobB genes in cols grown on diferent carbon sources. Realtime reverse transcription poly ‘merase chain reaction (PCR) analysis, using 165 ribosomal RNA (FRNA) as the intemal control, reve that both par and cob genes were more sctively transcribed during log phase growth than ther growth phases when cells were grown in the presence of ether lacose or citrate (Fig. 1A), Accordingly, tbe concentrations of Pat and CobB proteins were increased (Fig. S6 and Fig, 4B), Dring the stationary phase, cols that grew in the presence of either glucose or citrate had sime ilar ais of pat mRNAJeab! mRNA. However, ‘when rich medium incubated cells were tats- {ered to minimal medium containing plucos scetlation peaked at mid-log phase (Fig. 4A), wheres if they were transfered t0 minimal me= dium containing citrate, acetylation decreased in both prelog and early log phases before it resumed 1 basal level in late Jog ail staonary phases (Fig. 4A) The lower amount of celular acetylation was apparently needed for efficient utilization of citrate, These results indicate that the expression (and probably totl cellular ac+ tivities) of Pat and CobB are regulated diffi ently im response to diferent carbon sources, although its underlying mechanism is yet to be revealed, The central metabolic pathways constitute the backbone of cell metabolism. Such patsiays use various carbon sources to provide building blocks, cofactors, and eneray for cell growth. For calls to respond flexibly and promptly to avail- REPORTS i abiliy of environmental carbon sources, the dic rections andlor rates of the metabolic reactions need to be modulated concomitantly and gb ally. Transription regulations merely adjust the mount of metabolic enzymes in unidirectional ‘manner, whereas allosteric effects only mode late the catalti activities of individual enzymes responsible for specific reactions of metabolic pathways (10). We have found a potential rea- latory circuit coneinating the carbon flaw of S conterica central metabolism by reversible sete lation, Acetylation uses acetyl-CoA and NAD’, ‘0 molecules that directly involved both in me tabolism and energy sensing, as substrates, pro- vides unparallel advantages in sensing cellular nergy status, and thus could fit the role for slobal metabolic modulation. The identifica tion of extensively acetylated metabolic enzymes in both human and prokaryotes and reports of broad metabolic regulatory soles of this made iMfeation (/-F6) indicate that reversible lysine acetylation may represent an evolutionarily con= served mechanism of metabolic regulation in both eukaryotes and prokaryotes, References and Notes X fang € Sa. Orage 26, 5310 2007. 5: thee, scence 327,109 G00) Nef Sra ee, RC, Bote, 1. Exabnte Sener, Serce 298, 2390 2002, Wf Sura C Beer semeet) Mel Bol 30, 1005 2003 Ch Bitar, M.A HY Cen, ate tees, Asie Bl Chem. 277, {5099 (2000, (thio C Yang Too, Man. K tino Taare 383, 7053 2003 1 Zaoan Fer, U, Sauer, NC Boigermees 6 209 2005) 9K Corns dany, Ro: era, $2,127 ‘asp, 10. FC Nelda, sehr a end area (Ait Fess, Washing, Oe. 2 1996. 11 | Dhange ey UL Cl Pres 8,215 200 12 € chou et, See 325, 834 200) pbs frog 1 aly 2009 (126m 9590, 13 VV Line Ce 136, 1073 2009. 1M, 0,8 eect, MG, R Califa Mat. Gert 32,06 ao02. 15. Landy al, Po Na end SL USA 97, 8807 Goo, 16.9. Halls, See, 1. Den, Fe Ao. Si Usa 303 10230 ote Wek members of Fusin Mela al oy [Gorter inputs ratghut ewer. Lag anal Urwesty, Gea, pared the Sere (tee for ead. The nk spre by sate fey aecloomert payors abo rseath& Cra aRDHCAPINAD 4 MUKCHEDON esl Mh ‘elo rest ad Seep poten China 1 cna 20530602), and shang hey Base ese pre, china aIC1209500, ‘Supporting Ontne Materat omscieneag ogearerttul327 58681004002 Mette snd tas SO Tet Figs st ose 27 uly 208; ace 7 Jory 2000 19 FEBRUARY 2010Significant Acidification in Major 1008 Chinese Croplands Guo, X. J. Liu," ¥. Zhang,? LH. Kw. LL Shen W. x. Han.* W. F. Zhang,* P. Christie“? T. Goulding,” P.M. Vitousek,* FS. Zhang} Soll acidification isa major prablem in sols of intensive Chinese agricultural systems. We used two nationwide surveys, pared comparisons in numerous individual sites, and several long-term ‘monitoring-field data sets to evaluate changes in soil acidity. Soil pH declined significantly (© < 0.001) from the 19805 to the 2000s in the major Chinese crop-production areas. Processes related to nitrogen cycing released 20 to 221 bilomoles of hydrogen fon (H") per hectare per yea, and base cations uptake contributed a further 15 to 20 kilomoles of H* per hectare per year to soil acidification in four widespread cropping systems. In comparison, acid depostion (0.4 to 2.0 kilomoles of H* per hectare per year) made a small contribution tothe acidification of agricultural soll across China. ciiction can arth gsc Anessa aber as bo (eo) Powter sas Se ve bse haga iy ye poe clan (monty by a tnd mat and Sie cana {fl mea fon een cabo tc (GOs) he onan coe et inate bdo nee acon ‘htop ois ar ech (an Sols ae sony bute yon Samp tion y eahery te nis the eg) yn th timinan (AD an on Sk ly ey ‘caloge of Rous and Erinn ences, Cina fair Unies lig 10193, hina. "Agr Evora ‘nay, ard Breas stu Bal 59 SPC UC ‘Derm ot Sal Scere Rete Rech, Harpe, Het ALS 2, UK “eprint ol, Sani Uns, Str cA005, USK Tres authors contd quay 1 this work {To whom coreipondence shuld be address Ema Pungl@auede, slowly under natural conditions over hundreds to rillions of years. OK soils and soils in high= rainfall regions tend toward greater acidity (8), Naturally acid soils occupy approximately 30% of the work’ icefce land and are commonly associated with phosphorus (P) deficieney, AL toxicity, and reduced biodiversity and produce tivity Chinese agriculture has intensified greatly since the early 1980s on a Timited land area with lange inputs of chemical fertilizers and other resources, Gin production and fertilizer nitro- gen (N) consumption reacted 502 milion and 5326 milion tons nationally in 2007, respectively, Jnereses of $4 and 191% 8 compared ith 19ST (U0), High levels ofW feilizaton can deve soil acidification both drelly and indirectly (11-13), and the rates of N applied in some regions are extraordinarily high (14) as compared with those lof North America and Europe (15). These have degraded soils and environmental quality in the ‘Nonh China Plain (/6) and in the Tabu Lake region in south China (/4). Here, we investigate whether they also cause significant soil acidife cation ata national scale. {A national soil survey was conducted during the ely 1980s, and pH was determine in all ‘opsils that were sampled (7). Far comparison, we collected al published dats (/3) on tpsoil pH from 2000 to 2008 and compiled to (unpaired) datasets (1980s versus 20005) on the basis of six soil soups according to geography and use, with ‘xo subgroups per soil group: cereal craps and cash crops (bles SI and $2 and fig SI) (23). In Cina, both systems receive very high nutrient inputs as compared with those of other agricul= ‘ural systems worldwide (8), especially the cash crops (such as greenhouse vegetable system which have developed rapidly since the 1980s a9), ‘The results reveal significant acidifeation of all topsols (average pH declines for the soil groups of 0.13 19 0.80) except in the highest-pFT soils, which represent only a small percentage of Chinese culivated soils (able S1). In all other scl groups, aviication has been greater in cash crop systems (pH decreased by 0.30 0.80) than under cereals (0.13 to 0.76) (Table 1), These are substantial changes. The pH scale is logarithmic and a pil decrease of 0.30, comesponds 40 & doubling in hydrogen ion (FT) activity Soils in group I [for example, leached red soils (Arg-Udic Ferrasls) and yellow soils Ali- PPeriulic Argosols}] are the most acidic in south China and have acidified further since the 1980s, with pH declines of 0.23 and 0,30 (P < 0.001) in cea and cash crop systems, espectvely Table 1) Although nec pH decreases for group T sails were somal s compared wit these of other groups, the ‘impact may be more pronounced because these soils are approaching pH valves at which poten- Wally toxic metals such as Al ancl manganese (Mn) coal be mobilized (20, 20, ‘Atthe other extreme, soils i group V [ein ly fuvovaquie soils (Qohri-dquie Cambosols), ‘which are widely distibuted in north China] are Table 1. Topsoil pH changes in major Chinese croplands between the 1980s and 2000s. The soil groups are defined in (23). NS, not significant; pH range is an average (5 to 95 percentile) 19805 20005 sa yon Sane eae ped Tae ele aE ' agate ares OF 7 aneay 27 " 157 gancson 8 garry “Ot 1 asecray 0254 " a7 sont azrstog O78 aan 80 w seas 97 agents) “O22F Barta ORE v gana garam “AR 5 gaetay O58 “ gave gave 9 gaan “Cerwin cop Gh ce whet ma nd aon Wignpa a os(ch vegeatien a Hee od tal P< OOTR 19 FEBRUARY 2010 VOL327 SCIENCE www.sciencemag.orgconsidered resistant to acidification because of thei relatively high CaCO, coatent (5 0 108%) However, they have also acidified significantly (P< 0001), with ptt decreases averazing 0.27 tnd 0.58 under cereals and cash crops, respec= tively. The pl decin in grou Vis smal Sompared wid hose of groups I and TV (Table 1) (13) but probably resulted in substantial loss of CaCO, Therefore, soil plT decline might be expected to soceerae in the future. The broad-scale comparative results are supported by data ffom 154 agricultural fields, in which srl pared data were available from the same sites in the 1980s and the 2000s (13), Fig. 1, Topsail pH changes fom 154900 pated sta oer 38 ssn seven Chinese provinces bencen the 1980r an the 800 2000s The Ure and square thn the bor rpresert the medan and mean = rool talus of al data he bot and top = : cages ofthe box greet 75 and 78 Be goa percentiles of all data, respectively; and the batam and top ba represent Sand gg 9 pene, respecte rived £ tests show that these topsoils were significantly (P < 0,001) acidified, with an average pH decline of 0.50 (Fig. 1). Topsil pH in 53.2% of the sites decreased by over 050, 182% by 0.30 to 050, and 18 9% by 0 4 0.30; only 9.9% the sites increased in pH, The pated national data stongly support widely occuring soil acidification in Chinese cropland We also summarized information from 10 long-term monitoring field. (LTME) sites in which soil pH was measured regularly aver an 8+ to 25-year period (13). Decreases in pl were substamial, from 0.45 vo 2.20 (Fig. 2). We found signifleant soil acidification cccurred only in Fig. 2. Long-term changes in opi pin some tpi- cal Chinese sos Grou oi @ Groups WV and V recived conventional rates Of NPK ferlizers for 8 to 25 years. Soil groups are described in (13). Data are means » SD. ¥ sa : SYPEE crm = crown a= —— Group 1-2} —2— Group IV-2 Sen ae Srp i ~ Soh Sowa Seem Seva a Cropping year (yt) Cropping year (yr) Fig. 2 don gto min (asin fo geal Coe soon vccie Sere Wil eave aes anal Saou Tacha see mel gentase ies vegetables. N cycling, S uptake, and P up- 220} }2Cs uphake take denote the H produced by W ccting and Sand P uptake processes. BCs uptake Indicates H released by BCs uptake, Net production isthe algebraic sum of H re- sulting from N cing and P, 5, and BCS Uptake, Data are means ¢ $0. 8 & 8 H production fx (kmol ha yr") wonusciencemag.org SCIENCE VOL 327 HEBNatHprosucton rw BR Cropping system REPORTS i NPK (conventional frilization) plas (P < (0.001), wheres sol pl didnot show obvious change in CK (no fetlization) and Fallow (no Fetlization and no crop) plots (i. $2) (3). Further analysis shows that eset soil acide ification in China has resulted mainly from high fertilizer inputs and the uptake and removal of base cations (BCS) by plants. Inthe tree major Chinese double-eropping. cereal systems (wheat-maze, rice-wheat and cee) annual N fertlize aes ar usually above S00 kg Nha ! wilh N use efficiencies of only 301 50% (Cable $3), Calculations based on inpus and outputs of ammonium and nitrate N indicate that N loading contributes 20 to 33 kmol HT ha! year of proton generation in these systems (Fig. 3 and table $3). Greenhouse vegetable sys tems, the major ash ropa, recive even higher N fertilizer inputs: in Shandong province, N fer- tz rates above 4000 kgN ha! year" are com> ‘mon with N ue efficiencies below 10% (22,23) ‘Unde thismanagement, about 20 kml [ha year" of poteatial acidity accumulates in each hectare of soil (Fig. 3 andl table $3). The proton generation related to N eyeling 20 o 291 kmol TW ha’! year!) in China is extremely high as compared with values (1410 11.5 kimol H” ha" year!) at ower N ferlizer ats in other regions 8. lant upke of BCs together with emoval of economic yields and crop residues fom lds nother driver of soil acdication because the net removal of excess cations over anions leaves behind equivalent 1” released wo the soil. At current ertlization levels, approximately 25 tons of dry biomass (rain and stalk) (able $4) is harvested annually in the three doubk-eropping systems, lading to an estimated I" production rateby BCs upiake of 15 920 kro Tha year! (Fig. 3 wed ble $3) In greenhouse vegesle sys ‘ems the importance of BCs uptake varics greatly with plant species and ick! but overall appears similar tothe cereal systems, Increasing N fertile applications hes been & major management technique diving high crop Yields, which in tum increase the removal of BCs. These ficiors combined have prouced potential acidity equivalent to 30 to 50 kmol IT ha ' year in double-cropping cereal systems snd “230 kot Hh! year in greenhouse vegetable systems (Fig 3). Although acid dep- ston is an important regional environmental problem in China, stonaly affected ares with 3 precipitation pl¥ of 4.00 w §.60 (25) receive 0.4 ‘0 20 kamol FT" ha year, much of which i buifered by other depsitional or sil processes. Overall, sntiropogenic sification driven by N fertilization isat leat 10 to 100 times greater than that associ with wid ran (13, Fig. 3)-Tnosher regions, serous acidification was found in the longer (NTT):SO,-reated Park Grass soils at Rothamsed in England when no lime was ap plied to buffer soil acidity (26), and Wallace (27) reported soi aeidiction from routine feiiza- tom practices fo erp prosiveton. 19 FEBRUARY 2010 1009Anthopogenicailifition of Chinese agr-_ References and Notes cultual sols wl bedificao corrects fongas + € Duss, hats Pot Py, 27, 335 excessive levels of N fertizaton conte, OS Gouin and Aris found that ach SO Kg 7S ahaa See a8, ha! of added ammonium-N generates ~+ km 4, 1.C Om et af, Natere 437, 684 (2005), HE ya indsouins S00kgCaCOs he 5B le ae 98 tn year' to neutralize in their field conditions, ¢ 1-M. NAPSpencer et al Nature 454, 96 (2108) Similar thortical eacultion show that each kg ©, ACSW Cae, Gero en, 322 of applied NITeN leached as NOyN' demands 4, Hfvton Us. dt Mot S741 72 ky of CHCOs to neutalize the acidity A) enero (29,30) Adding appropriate amounts 7 HEA an. WT, Gas Sate of lime in China would be arduous; intensive yp, hina dyin Hearook Chins Aagiclal Ps, dublecropping systems tat genente 30 4 50 Sj. 982-2008, lkmol 1 ha ' year ' would teretically require 1. 4 oa A Dai, Ranlethes, Aon Si 1 1737 @00?. 1.51025 tons CaCOs ha year Yo counteract 4) SOP soil acidification—and greenhouse vegetable ons Rom ae systems would equ ea Ue this ae. aa nd eats el pig ‘Ovense of N fein contibee subi» mina ne. ally to reional sist inChina Since 16 2M ol Pe RO dS, US 36,204 Sab coppeiionbsinsese vip yaw apn ann eg tress N fz sotsumpton (i Seat Gove eng en Decreasing N ue cfcieny (Oi. $5) inicaes fr Slo td Fos msl B20, UatimrefeiliesNisbeingltiotheenvione EL tment (27), casing futher nogaive evans M+ EEL Lu 5. 2an Po, Orie Fe ‘mental impacts. Optimal nutrent-management 9, $3 Suney Ofc of Cita, Chino So Specs, vs 110 statgies an signlicanly reduce N feline” £cheeve Pes Sy 190, fk without creasing copys (24, 32,44), MB. Vina eta eee 34 1319 20M, ‘with multiple benefits to agriculture and the en- 3 Centra uenente teopuetSpetat ‘vironment (15), including the slowing of dangerous ‘Changes end ConteolingCourtermeasues of tes of anropegenie ain. Fenton Soi Beacon nH Re So tego ot based on comprehensive, knowledge-based N Seu Crna Goence Pes Ben, 2002, Mangement psc hes become one of he, ATO sean tno urgent eqiemens for ssainable agel- 7 G ee E Hon As ture in China and in other rapidly developing 2, regions worldwide, TH, By. 8 Chi JL, Ae rot Fron 1, 70205) B81 CL fou Ft, 2. Do. 5. Zhan, nian Fost 385,437 2000. 24 fis Frac, yak, Sarna Pont So 346,261 208). 25, tne ovelaig200 gh 2009061 {eons 125e6 ie 200, 26 Cate WT Geo, C8. Ma A Js, fi} Sot Se, $0,401 993 2B, A Wille, Commun, So Si lant Aa. 25, 87 990, 29. 8 Upon Fern, a. Cores a dpi. Su ley ego) 67209 acy rin 2008) 50, Wt Pos, wn seonaors uF ‘eel 9813L hem 1998, 3 G Xe Xing, ZL Zh, Blogecherny 57158, 405 068. 58.6 H. Worg. 0. € har, Wit). Buch, Arc Sj 94, 1 (2007 24, We Mon) Nag dt cal, ag ee fetes cect ard. San) en pole Soil rami ate MF se Fran pps ‘er hs war was pred by he ches atonal Base ase Progr (CUSCB100 308 2422206, te rei Gn Gra rom SEC (20321031, a he Sel Fd Ages Peteion 290803030, aw et Agacans 74,92 ‘Supporting Ontne Materat Fie Stas Tate St Se 28 Seger 2009; accepted 1 Janay 2010, Paes one 1 en 200, 0126 scence 1182510 Peptidomimetic Antibiotics Target Outer-Membrane Biogenesis in Pseudomonas aeruginosa Nityakalyani Srinivas,” Peter Jetter,* Bernhard J. Ueberbacher,* Martina Werneburg, Katja Zerbe,? Jessica Steinmann,’ Benjamin Van der Meijden,' Francesca Bernardi ‘Alexander Lederer,” Ricardo L. A. Dias? Pauline E. Misson,* Heiko Henze,” Jirg Zumbrunn,? Frank 0. Gombert,? Daniel Obrecht,? Peter Hunziker,’ Stefan Schauer,’ Urs Ziegler," Andces Kach,* Leo Eber,” Kathrin Riedel,* Steven J. DeMarco,”* John A. Robinson” Antibiotics with new mechanisms of action are urgently required to combat the groning health ‘threat posed by resistant pathogenic microorganisms. We synthesized a family of peptidomimetic antibiotics based on the antimicrobial peptide protegrin |. Several rounds of optimization gave @ lead compound that was active in the nanomolar range against Gram-negative Pseudomonas spp, bout was largely inactive against other Gram-negative and Gram-positive bacteria, Biochemical, and genetic studies showed that the peptidomimetics had a non-membrane-{tic mechanism of action and identified a homolog of the B-barel protein LptD (Imp/OstA), which functions in uter-membrane biogenesis, as 2 cellular target. The peptidomimetc showed potent antimicrobial activity ina mouse septicemia infection model. Drug-esstant strains of Pseudomonas are a serious health problem, so this family of antibiotics may have important therapeutic applications. aturally ocesting peptides and proteins ‘make invresting stating points for the design and synthesis of biologically ac~ tive peptidomimetics. We previously synthesized Tibrries. of ehuitpin-shaped_peptidomimeties (1, 2) based on the membranoltichostetense 1010 peptide proterin I (PGI) (3), These mimetics contain loop sequences related to that in PG-L but linked to a p-prolins-t-profine template, ‘hich helps to stabilize P-hsipin conformations within the macroeyele (4, 5) (Fig, 1A), One se- ‘quence variant, L8-1, hd. broed-spoctram anti- microbial activity ke that of PGel, but with a reused hemolytic activity on human red blood calls (2). To optimize this lead, we performed ‘teraive eyeles of peptidomimetie Hbmary syn- thesis and sereening for improved antimicrobial ‘activity. The optimal it from each brary was used a8 a staring point for the synthesis and testing of variations ina subsequent bear, This ssructre-etvity til led sequently mimetics L1945, 126-19, and L27-I1 (Fig. 1) L271 Scprsty Oeprtmct, Univesity of Zach, Wiebe srsvase 190, 8057 Zach, Swizuland “Pavan RG Hegeaheinemetiveg 125, "4123 lich, Satetand Funcional Genoms Ceres 2h, Winterreise 190, 057 Zach Stal “Centr tor Mey nd Image Pasty Urvasty of Zach, Witethuestasse 150.8057 Zurich, sited. "Deartnent of Mri ay, Unveiy of 2c, Whereas 190, 8057 ae ‘To whom conependece shuld te ase. € mai rabrmengociurhch JAR), stevedenarco@yepeccon 510) 19 FEBRUARY 2010 VOL327 SCIENCE www.sciencemag.orgpossessed an interesting spectrum of animicrobie al activity, including minimal inhibitory concen- trations (MICs) in the nanomolar range against many Pseudomonas aeruginosa (PA) strains and totier Paewiomonas spp: the mimetic is only weakly ative or inactive against other Gram positive and Gram-negative bacteria [Table | (6) Tn contrast PG displays broad-spectrum antimi= crobial activity against Gram-positive and Gram negative bacteria the law micromolar range?) The enantiomer (ior image) form of L27-1] is essentially inactive (MIC > 32 py'ml) against PA, unlike the two enantiomers of PG-I (8), suggesting that the antibacterial target of the tie chiral and not dhe achiral ipid chains ofthe cell membrane tangled by PGet (9-12), Further effors focused on optimizing. plasma stability ane drug-lke properties. The compounds POL7001 snd POLIO displayed much ime proved plasma half-lives while retaining a potent and selective action against PAs the MIC’ cover- ing 90% of move than 100 74 clinical isolates pA Rpt ears” cain — =a = Pa! 123 412 18 24 time (n) =ee =H, . 0 @ 0 te oy ~~ aoanien om Fig. 1. (A Structures ofPG-1and peptiomimetics. Dashed tines indicate sufi bridges in PGA. The single eter code is used for amino acids (30), except as shown, and X is L-2.4-diaminobutyrc acid, "P= prline, and" is proline, PAL‘ contains -phatPro in place of. (B) The Kneis of bacterial cell death at 37°C i shown for PA PAOS in MH broth after treatment with the indicated antibiotics at 4% IMIG, or for the contol (no cua). The remaining colony-forming units (CFU) are shown asa function of time after expsure tothe antibiotic. (Pa membrane ineaity was measured by fluorescence spectroscopy. PA PAO cells in MH broth were treated wth SYTOX and each of the antibiotics (added at time 1= 0) at a concentration of 5 jf. The fluorescence charge is shown versus time Table 1, Antimicrobial activities of the peptidomimetics The MIC values were determined by the ‘microdilution method in MH broth in the presence of 0.02% bovine serum albumin (BSA). The MIC values are typically four to eight times as high when the assays are performed in cation-adjusted IMA (UHI) broth in the presence of BSA. ND, not determined. Strains: Pseudomonas ceruginose, ‘Acinetobacter baumanai, Klebsiella pneumoniae, Stenotrophomonas mettophilia, Escherichia col, Enterococcus faeces, Staphylocaceus aureus. ATCC, American Type Culture Collection; OSM, Deutsche Sammlung von Mikroorganismen. MIC (nant) stain tea 1945 2619 (27-11 POL7O01 _—POL7080 Paevuginoso ATCC 27853. «8 «1020008 (OL P-ceruginas AOL 3 2 003008 0.008.004 A boumannii 053008 =—~ND ND 36864 36a K-pneumonige ATCC13883 ND ND 3643643 368 S. maltophitio ATCC13637 «ND ND 364 3646 36a col anc 25922 8B 3 6h 3 E faecalis DSM12956 «ND ND 6h 36 26 S.reus ATCC 292138 864k 264 wonusciencemag.org SCIENCE VOL 327 REPORTS i text (MIC) were 0.13 and 0.25 g/ml, re= spectively, fr these two mimetics. These isos ‘were of European an Nar American origin and most were resistant t© one or more csses of clinically wed antibiotics (6). ‘We next focused on identifying the mecha ism of action ofthis family of peptidomimetcs. ‘Theirmod of action against PA at concentrations close to the MIC was clearly different from the rapid membranolyti ations of both PG-(7) and polymynin B, a eationic maeroeyelc peptide anti- biotic of bacleral origin (73). Whereas PGI polymysin a4 MIC caused rapid ell Isis, he killing caused by the pepidomimetics was much slower (Fig. 1B). The ability of the pepidom- Imetes to permeabiize the PA cell membrane was also tested withthe fuoresent nucleic acid stain SYTOX (14). No fluorescence increase was apparent over 120 min when P4 cells were exposed to SYTOX and 126419, 127-11, of POLTOOL, compared with 2 rapid Moorescence inrease upon exposure 19 PG or polymyxin B [Fig 1C (6)). A dirt interaction between the Peptidominctic antibiotics and ipopelysachae Fide (LPS) was examined with adansy-polymyin displacement binding assay (5. This revealed an interaction between LPS and L27-11 in the low micromolar range {median inhibitory con- centration (IC) 08 uM. whieh was not en sntoxelectve (ICzq 1-1 pM forthe enantiomer of L27-11), suggesting that LPS is not the pri- mary site of antimicrobial ation. Also, 726-19, LZ7-11, and POL7O01 caused no notable Isis, of human red blood cells at concentrations up to 100 ug/ml ‘We tested whether the peptidomimetics have any effeeton protsin or mle acd biosynthesis in Pd by examining te knetes of incorporation of adolabeled precursors into macromolecules ‘No nouleinfuence was detected (6), suggesting that inhibition of protein, DNA, or RNA bio symesis are not the primary modes of action. Similar attempts to monitor effects on cel wall biosyethess by radiolabeled Nexceylgacosamine were frustrated by low lees of corporation of this precursor into cell wall biepolymrs {AS an altemative approach wo investigate the mechanism of action, forward gencti sreen was established w characterize the genetic basis for resistance tothe antibiotics. Spontaneous re- sistant mutants of PA PAO! could be selected on the anibiotc POLT080 at $x MIC, at an es timated frequency of < 1 10. Three mutans (PAIN) showed MICS toward POL7OR0 and 26-19 of >32 wim but were more sensitive toward POLT001 (MIC 8 nef, vers 10.06 ual for wikdsype 24 PADI), No other changes in growth ate were observed. Compared ‘o wildaype 24 PAO!, the mutants showed only ‘minor changes insensitivity toward several oer antibiotics (able 2). To identify the causative mutation), we constructed three plasmid Fibra ries fiom restrictionigested fragments of PA PAO" enomic DNA and transformed wikl- "ype PA PADI wit selection for growth on seat 19 FEBRUARY 2010 10111012 conttning earbenicillin and POLTO80. Resistant clones were isolated from all three libares. Plasmid DNA isolated fom 12 clones contained 3 common 3.440 overlapping DNA. fragment Inucleotides (nt) 652,070 wo 687452 in the senome quence (/6) of PX PAO] containing, vo contiguous open reading ames ented homologs of ut and ast (also called inp and nore recently JprD (used below)] (17). The Saekb DNA fragment conferred the resistant phenotype on 4 PAOI, wheres a smaller fay ment containing only surd had mo effect on resistance. The urd gene possessed the expected wild-ype nucleotide sequence, whereas the xD homolog from Pa PAOI™™ (here calle pr) contained & mation, consisting of a sin 18— base pur tandem duplication of at 628 to 645, coctesponding to a tandem duplication of resi= dues 210 to 215 withthe sequence LRDKGM (6, with 90 other changes in the eating oF up stream promoter sequences, The dprDI gene ap- ars to act as & dominant resistance marker in the wiklaype P4 PAOK background. This docs not appear to be a gene dosage effet, because ingoduction ofthe wikliype allele on plasmid VLT3I (18) does not infuence antibiotic sen sitivity for PA PAOL or PAO, Moreover neither wild-type PA PAOI, containing plasmid bore copies of IpD7, nor PAOIF™, contain ing copies of pu, has sere sensitivity to most. of the other antibiotics tested (Table 2). This argues against a general change in the perme ability ofthe outer membrane caused by this ‘mutation, TEpiD is an outermembrane protcin widely distibuted in Granv-uezative bacteria that func tio inthe assembly of LPSin the outer leaflet te outer membrane (17, 19-21). LpD isan es- sential owabundance entersmerirans pre in Escherichia coli (19, depletion of which causes stress in outer-membrane biogenesis (21, 22). TxD in PA PAOM is predicted to contin a C= terminal barrel domain (epproximately residues 300 to 924) embealded in the outer membrane and an Neterminal domain (approximately resi- dues 34 80M) (19), which may reside in the periplasm andor parly plug the Bare! Fig 2A, The Brbarel sequence is highly conserved in TptD homologs in Gram-negative bacteria (17, although the N-terminal domain is more ‘arabe in leneth, comprising about 300 residues in Pd but only aout 180 resides in Ecol K12 (6). The size difference in the periplasmic doris of LpiD in Feo and P4 the location within this domain ofthe xD mutton, and the hey fanetion of LpxD in outer~membrane bio genesis suggest that Lx) may be @ primary target ofthe peptdomimetic antbiotics, ‘Photoafinity labeling experiments (25) were pecformad to detemine whether the antbotcs bind to pe in itt cells. For this ask, am ane slog (PAL-=1) was prouced hat contains 8-4 diazarinylproline (C-photo-neoline) in place of pine, a wel bitin ty at postion 1 (MIC OfPAL-1 aasinst 4 PAOT=005 im Fi. 1A) ‘Table 2. Antibacterial activities (MICs in MHI broth) of various antibiotics tonard wild-type PA PAOL 30") and resistant mutant PAOL""),as wel as toward bacterial cells containing pasmid-bore copes of wld-ype fd or the esstance gene (ptD1) slated from PAO". The plasmid vector used for these experiments (pVLT31) has no effect on resistance to the antibiotics shown, when introduced into PA R02" or PAOY Antibiotic pro" pan Pot7o80 0.96 oa 619 on 366 Gentamicin os. 1 Tobramycin 025 025 Croftoacin os, 0.06 Colsin 1 os (6), Photoaffinty labeling with PAL-1 consist- ently revealed a major photolabeled protein with an apparent mass by SDS-polyacrylamide get electrophoresis of 100 kD, close otha expected for PA LpiD (calculated molecular weight 100,751) (Fig. 2B). When the photoalfinity labeling was repcated in the presence ofa 100% excess of L2T-II. the labeled band disappeared from the blot, demonstrating competition berwoon 127-11 and PAL-1 for binding to LptD, The entity of the photolabeled protein was proven tobe LpxD (PA0S9S)by two-dimensional (2D) gels and in-gel protease digestion LC-EST- “MS-MS (liquid chromatography-clectrospray fonization-tandem mass spectrometry) analysis and by immusobloting with polyclonal antibodies raised against a synthetic C-temninal peptide fragment of LptD (6). When the photolabeling experiment was repeated with the resistant PAOI'** mutant, photolabeling of LptD was not detected, indicating that the priein was no longer able to bind the anibioie with compare ble affinity the finction of Lye is imped upon bind ing to the peptidomimetic, then some effect on fouleramembrine struct and biogenesis should become apparent (19, 24), When grown in “Muellr{Tinton (MIT broth for several hours in the presence of groninhibitory amounts of LIP-I1, PA cells became more sensitive to de- tergents (Triton X-L00 and SDS) and to various antibiotics, including teraeyeline and rifampicin (6), PA PAOI cells wrown in MH roth with 27-11 or POL?OO! were also examined in thin sections by transmission electron microscopy’ (TEN), This revealed internal accumulation of rmembrane-like material in many apparently in- tact calls Fig, 2C), by two different nation methods, an effect not seen in cells grown withe fut the antibiotic. Similae accumlations have buen observed in E colt cells depleted of fx (C1, 22) and in other bacteria exposed 10 anti- microbial peptides (25) Some cells (10%) grown inthis way formed filaments comprising multiple concatenated cells Fig. 2D), suggesting an ime ppirment in cell division, which was nx observed ‘with amreated PA PAOL cells of with L27-11 IC (ug/t prow" pai"? pag ipt01 -ptd sipr01 4 oa oa 16 368 368 os. 1 1 oz 025 025 1 oaz oz os. as 025 treated PAOL cells, Cells grown in the pre cence of growth-inhbitory amounts of T27 could be uniformly stained with the membrane ye 3S-dipropylthiaeurboeyanine [diSCx(S)) (ig, 2E), whereas unteatsd P PAOL or L27- 1-weated PAI" cells exposed to this dye did ot Auoresce. Thus, the action of 127. during growth impairs the outer- membrane pet- meabilty baie. Interference with the function of LpxD may also allow entry of phospholipids into the outer leaflet of the outer membrane, In E, coli and Solmonella spp. this is known to activate the fouter-membeane enzyme PagP, which modifies LPS by convering the bexs-acy form of lipid A Jno the hept-acy form by transferring a palmitate rou from outlet phospholipicls to lipid A (26, 27). The same effect has been observed in [peDvdepleted E.coli 21), Pd can also modify its lipid A by addition of a C16 fatty acid, to gen- tate from the normal pent-acylated form (mo lecular weight 447) 4 hexa-acylated derivative (molecular weight 1686) (28). This palmitate- ‘containing hexaracylated form as been observed ‘in lipid A feo PA clnial isolates from patients with cystic fibrosis (29), although it normally absent ffom laborstory-adapted strains (Such as PAOI) 28). ‘We first confirmed thatthe lipid A prepared ‘om LPS isolated fom PA PADI grown aoemal- Jy in MIT broth was indeed the expected pene acylated form {negative-mode matrx-asised laser desorption ionization (MALDI)-MS mass! charge rao (m2) 1446 [MELT }. However when cells were grown in MH beth with L27-IL, afer 5 hours the only lipid A detecuble by MALDI- MS was the exs-acyated form (observed mass 1684 [MI] ) containing « palmitoy! re ‘due (6). A further indication of » peruroed rrcmbrane stricture eae from fractionation of ‘membrane extracts from cells by sucrose density tulracentrifgation. Fractions containing LPS and LptD appeared at higher density in the gradient when they came from L27-I1-culivated cells 18 compared to untested cells (6), a result sim= ‘lato that reported for fd depleted F cot el (19,20, 19 FEBRUARY 2010 VOL327 SCIENCE www.sciencemag.orgA LptD (imp/OstA) rite te F___wtpaot+ Fuot + antibiotic + dye PATS + Fluot Fig. 2. (A) The predicted prbarel domain of LptD (924 rescues) is shown integrated inthe outer membrane, (B) Photoatfnty labeling of membrane proteins in PA PAO. PAL-L (1 jgimi) was incubated with A cells at 37°C ad iradiated with ultraviolet light. Membrane proteins were extracted, separated from cytoplasmic proteins by ulracentifugation, and subjected to gel electrophoress Biotinylated proteins were detected by a steptavidin-based chemiluminescence detection system after blotting to 2 polwinyidene difluoride membrane, with (100x excess, lane B) and without (lane A) competing 127-11 during photobss (6). (C to E) Etfecs on PA clls after gromth for to 3 hours at 37°C in MA broth either with 127-14 (+ antibiotic or without antibiotic no antibiotic); (CTEM showing accumulation of extra membrane-tke material within cryo-fned PA PAOL cells (bar, OL uml; (D) fermation of filaments comprising multiple concatenated PA PAOS cells; (E) staining PA PAOL wit the rmembrane-sersiive dye diSC3(5) and washing, () Staining and light microscope images of wild-type (wt) PA PADS (lefD and resistant mutant PAOI"™" (igh), after groin the presence of Flt and washing, ‘A fluorescently labeled derivative Fhio-1 (Fig, 1A) of L27-11 showed an MIC against PA PAO! of 0.1 ug/ml, suggesting that it might Inbel potential antibioe binding sites. PA PAO cells in MIT broth were incubated with Fhuo-l (S jim) for 1 hour at 37°C, washed, and ex- mined by fuorescence microscopy: The reste ing Muorescence staining was not inform over the cell surface, but rather appeared to be con= centred in spots (Fig. 2F), suggesting a lo- calized binding site. A similar labeling was not fobserved when the 7 POT mutant was Stained inthis way. ‘The ability of the antibiotics to provide pro twetion aginst a lethal PA infeton in a whole snimal was also tested, Foe this, the in vivo ef= wonusciencemag.org SCIENCE VOL 327 ficacy of POLTO01 and POLT060 was evaluated jing mouse septicemia medel at doses of 10, 3,1 103, and O.1 mg per kilogram of body weight given subcutaneously a | and 5 hours after bac~ eral inoculation with either PA ATCC 9027 o ATCC 27853. Both antibiotics demonstrated sub- tanta ectivty against both stains with calene lated median effective doses (EDaq vals) n the range 0.25 to 0155 meg, as compared to gen- umicin (sed asa posive conta, which showed an EDs of 3.1 and 29 mele, respectively. ‘inst these sts ‘The mechanism of sotion of the peptidem- metic antibiotics reported here Tikely includes perturbation ofthe ertical LPS transport function ‘fF LptD, The in vive activity reported here raises REPO! the prospect that his family of antibiotics may be ‘useful in a clinical setting 1 combat nosocomial infections and lung infections in patients with cexsiefbrosis, where multiple drugresistant PA strains area serious health problem. Reterences and Notes 2. Juin ea Boe Me Chem, 13,2055 ons 2.5. Shanta eto, enBiochom 3, 126 2. YN hoya a, FERS Ut 327, 238 992 4 Lag. Mai arpa 0, Cea A ese, 5. eA ebm, Gera, F. Gombe, K Moe, 0 bre, Drop Ds ody 13,94 (2000) 5. ah Satya nits Ars Cate. 2, 38 G97. 8. ¥. Gh, NN. Dh, RL abet Inc. oman. 66, 686 G99, 9.1.) ate ee ogy 85,2368 (2008, 10, Ou ile Pr, Nat Bed Sci, UISA, 200, 02 Gon, 12 Ranta, Sachem 45, 26100) 12, Man, lie fe 96 295 0992, 1, 8 Lh Pace Sev, irs, ee Eon, deri 63, 2421 0590) ‘henatier 30,923 (386 11. WLP. Bos, V.Raber, |. Tommasien, Ara Rew. Microbial. 3, 3912000 Gere 123,17 990 19, Wun 1 | Sia, al Mab 45,1209 20 Tr Mace. Ce 224,235 ne, BLT. Wuet es Pac Na Aa Gone. 2. Speanso ay | oto, 19,8460 (208. 2. Darmins & D. Peis Tend Ban! 18, ‘2000, 24 BA Sampson, Misa S.A Boor, Genet 422, 492 25. CL eich 0, Moles, 1. Beige, KE None Jaimie tone Creat 4%, 2686 2000, 26, Cm eo Cat 95,189 0990, 27 W. hve | Bl Chem. 278, 4966 2000, be RCs oJ at 188, 191 200 39K ie Scene 286, 1561 (199). 5a. Sle eter tvs fr the ana a8 ress ae as alls; ye, he Ks 3, We han Aer fr ec prt Th dy 9 Servs by ia om the Ss atl Soe lnovaton ar the Epson Unc 7th Fame Fogare ot NAGATA Fels pert ae WO! 2007079605, WO20107997, aa WD 2008" (1850.18 ett fF Hs, Poets sbi 07207 79605, WO2007019597, ‘W02001019503) te ome yy Unies ot Zureh ndPepor A usa m0, a175¢ Supporting Ontne Materat nomena ngeearter 327 596820100¢2 Aerts ud aaa Figs stiasiz "abe St 87 Fenn 1 ober 2009; cepted 19 Jy 2010 sane 1182748 19 FEBRUARY 2010 1013NMR Structure Determination for Larger Proteins Using Backbone-Only Data Srivatsan Raman, Oliver F. Lange,"* Paolo Rossi” Michael Tyk," Xu Wang? James Rami Goohua Liu Theresa A. Ramelot Alexander Eltsky,” Thomas Szyperski, Michael A. Kenmedy,* James Prestegard,? Gaetano T. Montetione,” David Baker™*{ Conventional protein structure deter ton from nuclear magnetic resonance data relies heavily ‘an side-chain praton-to-protan distances, The necessary side-chain resonance assignment, however, is labor intensive and prone to etro, Here we show that stuctures can be accurately determined without nuclear magnetic resonance (NMR) information on the side chains for proteins up to 25 kilodaltons by incorporating backbone chemical shifts, residual dipolar «couplings, and a proton distances into the Rosetta protein structure madeling methodology ‘These data, which are too sparse for conventional methods, serve only to guide conformational search toward the lowest-energy conformations in the folding landscape; the details of the Computed models are determined by the physical chemistry implicit in the Rosetta all-atom energy function. The new method is nat hindered by the deuteration required to suppress nuclear relaxation processes for prot ins greater than 15 kilodaltons and should enable routine NMR structure determination for larger proteins he fin spin prin sacred "Tne ctr mis (NM) i chal asaprment ti batitone omen conta othe be gunn asignnon of ie ein, i roses iw mapa cuble and ingly atta (15, Gta backbone ssl infomation Somplementing he eal unr inbrmaton proved by Backbone chemical sip iments (6, 7) can be obtained from 1-H" nuclear Oven opecscopy (NOESY) ri tal dplr coupling (RDO) and ob. 10) ‘spect For ng prin denealon ber comes esse oem ieee spin ‘nation popes ring fen thigh tional contin tins (1,12) ut emo ing pcos ss chines laiprnge NOESY ination fom side chins, eae free thely poate sdeshain moles (0). The ticly in ctemiing acute acs wih no lied eden infest baukck tha trey prevent eine ap ean otNM larger 18 KD) oes (2). Tre we show tat sce of pron up to 200 ress (23 KD) can be dcr Aoepatment af Sechemy, University of Washington Sestle, WA 98195, USA. ‘Deparment of Malecuar Bet tg and Bachem, Cente fr AivacedBtcology fd edicne, and Notas! Stuchiral Genomic Con toro, Rtgs Unie, Pacey, 06858, USA, ‘Compe Cayce Reach Cente, Urey of Go sh, Ars, CO 30602, USK “Deane of Chey rd Bock and Nothess Seal Grn Carson, Miami Unvesty, Qxtord OH 45056, USA “Department of Chemis, Ste Unive of Men York at ut, Stl, 114260, USL. nar Hugh Mal nt HH Seat, WA 96195, UR, ‘Tras authors conte equally tts work {Pec ares Deparment af Canes, Fanard Ma {BI Séhol, Beton, 02115, USA. {lo whom correspondence shuld be adds. Ema fabotergs washington nbs 19 FEBRUARY 2010 VOL 327 ith the use of information fom backbone (1, N.C*,C%, C) NMR data by taking advantage of the conformational sampling and allstom energy function in the Rosetastrosture prediction met= ‘dology (15), which, for small protins in favor= able cases, can produce atomic accuracy models stating from sequence information alone (16,17) Structure prediction in Roscta procesds in «wo steps: (i) a loweresoltion exploration phase using Monte Carlo fragment assembly and a coarse-grained enengy function, and (i) a com Putstioally expensive refinement phase that ‘eles between combinatorial side-chain optic zation and gradient-based minimization of all torsional degre o feel ina physically rele Fig. 1. Impact of Roc A ata on conformational search Lies depict VSD histograms of stucures selected inthe lowest 10th pecensle of conse gained energy for ers Bes generated wih the tee of Coot tad) oF CS ROCRoseta re. (A) | BRLOBA,B) DRI, Rena, and iD) siRISC. “PS ‘ng, angstrom. c a> Ware ASD tonne (Ag) Inti lleatom forcefild (16). The primary obstacle to Rosetta structure prediction from amino acid seuenee information alone is conformational san pling: nave stuctures almost always have lower energies than non-native conformations, but they se very seldom sampled in unbiased trajectories, Incorporating NMR chemica-shift information Jn the selection of the fragments used in the exploration phase [chemical shift (CS) Roseta} (U8, 19) provides a robust aproach to determin- ing accurate strctures of small (<100-rsidue) proteins using only backbone and C®chemical- shift dat, Foe larger (12-ED) proteins, the pe formance of CS-Rosetis very trget-dependent Structures sulicienily clase to the naive struc- ture for the energy 19 drop substantially may be generated rarely or not ata, We investigated whether RDC data, which provide long-range information on the ofienta- tions between bond vectors, can guide the low resolution search closer wo the native stacture and overcome the sampling problem for larger (100 to 200 residue) protsins. For every atempted Monte Carlo move, the aligameat tensor is ale culated by singular value decomposition (2 and the decision to acceptor reject the confor- ‘mation is biased by the change in the agreement berween the back-alculsed and experimental couplings (21). Incorporation of RDCS dramati- cally improved convergence on the correct struc- ture in a benchmark of 11a, 8, and wB proteins ranging in size from 62 to 166 residues (Fig. 1. ‘Table and fig SI). As indicated in Table 1, CS- RDC-Rosetta consistently generates accurate models for proteins up to 120 resid and, in favorable eases, for ager proteins. Tor proteins with more than 120 residues, conformational sampling becomes limiting, even fo the CS-RDC-Rosetta protocol, and the low= SCIENCE wwwsciencemag.argenergy ensemble is not always close to the native structure. To further foeus sampling, we devel- ‘oped an iterative refinement provecol that in- corporates assigned backbone 1-H” nuclear (Overhauser effets (NOES) in ation to back- bone RBCs, AS in the previously: desenibed “rebuild and refine” protocol, a pool of diverse low-energy conformations is maintained, and the highest-eneray structures in the pool are peri- cdically replaced with offspring (22). The new protocol, a genetic algorithm, generates hybid cone formations by recombining firs sheet pairings and, subsequently, fagments of the lowseneray structures (17), To further enbance sampling, ta- _eetories ae seeded with conformations harvested from previous trajectories that led 1 low-enerey conformations (23) ‘The improvement in the model population With increasing generations inthe iterative pro- tocol is illustrated in Fig. 2 for the 200-residue ALG protein using experimentally determined chemical shif, RDC, and assigned backbone amide HH™ NOE data (24). The C™ root mean square deviation (RMSD) to the native structure and the energy improve from generation t gene eration, and after several rounds, discrimination toward lower RMSD structures is apparent (Fig, 2A, light blue to yellow). Affe high-esolution refinement (Fig. 2A, orange w re), the lowest energy structures ane closet the native structure, The final low-energy structural ensemble (Fi, 2B) recapitulates the unusual topology in the previously determined NMR structure (24) (Fig, 2D) to within 3.44 RMSD (Table 1). The Rosetta ensemble fits independent RDC data, as well as the NMR structure, and the backbone variation in the ensemble is comelted with back bone dynamies as probed by the RI relaxation rate Table 2, Accuracy of models generated with backbone-only NMR data, Number of REPORTS i (ig. 20), The iterative CS-RDC-NOF-Rosetta ‘models of ALG thus appear to be comparable in quality to the previously published structure «hat required substantial effoxt, including prepara tion of selectively methy- and aomatc-enatst samples (24). ‘The teative CS-RDC-NOE proocol was ested father on 12 proteins ranging in size fom 1200 266 residues (Table | and fig, $3). For al proteins except 1g68, a considerable part ofthe structure converges (Table I), Backbone H™-H NOE data ‘were required for convergence of 22% Lil arf, 2m2, and Isua but not for Spat, Isp, 11, and 553, The RMSDs to the nave structures over the converged regions range om 1.74.3 A. with the exceptions of Isua and 1121. For 21, high aceuracy (1.6 A) was reached for a 92-esidue subset (ig, $3), Sidechain accurecy was genet ally quite high inthe converged regions (fig. $5) Nedian residues Aion estan Depth of energy change Protein native umber of RMSO to coras converged resulting from name" Poem tne arene ensemble energy incuson of converged in toneged——Lonestenergy Sale ent econ of computed regiont (A) models pier structure onterae snka37 2s ab eur 26 954 3251 as 0 2 a» vars a5 9 674 “03 DvRitsc# Det 3 66 1 s00 243 “07 wins 23d a» sara za aa 3801 az acti034 et 3 snes 3a a3 -27 a3 simuisce 2k A 20095 1 861 3 078s maeaisne 2k 8 0296 2 ei -39 06 armas 20m ab 10482 24 668 =29 2958 serzsere 25 a nams 1 yaa “571 “ts 1553 2s ab wwsns 52 seuss 4 25 ane 215 » aera 26 133 -216 35 seo are 2x8 an 12258 30 v0 375 aes esa 2iye at wns as 767 316 “93 kay" et am swore oa 766 235 3588 R53 ks a raan36 1 852 “385 a34 amaze har 3 a501a8 2a 793 “516 = kor 1b 8 ssuantt 2s naa =333 285 kay sinh 2tt 8 ssuasstt v ony 1003 05 kroy" ane ab ass 3 29 “75 240 kay sent ab asst 30 ne 388 “30 key 150 a sorte 3 708 -99 a4 ARF 2st a» aseize 25 m2 -286 “81 ray 22i a 9s 18 7 465 26 ALGis 2x ab aouassit 3a #7 a8 =28 kar 3a a 263173 62 so “35 “265 key 168 ab 219 32 wats 353 351 “Ts cote are iad lr roe sce aban ih corvnecral Raa NEG and POR co ah esr prob Te eis show he pHs ere eed he (SC Rs rua and We enaig wine tate SOC Roses POUCA. Ho the Habe pt reise cole omega ey ae mans the Lge Sl ‘ebm tht sprigs win 4A Fr porate pot thereat sled with te Fone sty, 27 ado he cower Meee, re id ‘hone es sre DS) the mage raf Ge primp min, 2,3, 2 hy wgety 29 show the mda of CTS care camped oe pi fie 0 tftne Deve enrgy mice suger tien he meson eng fn are ey mabe dn 10 leery md at ey tot FGDs trey motel omeseaeg wns, eens vee te redion ergy of 1 west energy mde ln th ROC mer MDE dats een cesgy te 1 es ery. Feo mde et energy on. ney ap camped ih Aci als encod TAY #0 ne. “Paral ly pete da ner ee ables). AL Farell pores Lawwrtedan WM NOE dare comtamt af A 071 #8 estou mh dca coreg cue) Hiatal 18,53, sd 176 ede ‘Sree mda mean BSD 9 523, nd 89 A for 2 an rege). Hea ol abo ron, wonusciencemag.org SCIENCE VOL 327. 19 FEBRUARY 2010 10151016 We catrid outa blind test of the new meth- ventional NMR structures were detemmined. For vergence was not observed, and blind stucture ds on five data sets generated in the Northeast four of the prowein, the CS-RDC protocol con- determination was instead carried out with the Structural Genomics (NESG) Center before con- verge (Fig. 3, Ato D), whereas fora fifth, con- iterative CS-RDC-NOE protocol (Pig. 38). In Fig, 2, Determination of ALG ste ture fom backbone NR data ith Reta. (A) RNSDs and energies of Stucues generate in ates of 2000 rng the erative rte ah er tration of stares (or code Blue tore coesponds to numberof gen trattonbased on information rom previous rs 17, Song convergence Seach already inthe computational tes expense, low-resolution mode The last genetatons (orange 0 ed increase bath he precon ad air ray ofthe ensemble by refi the Strdectres hin the Roz alt om nergy. The RNSD ts computed ver the residues for which convergence shin 3 rot mean sore fata: ors NSA) wos reached nthe 50 Uavestenergy Rosetta models es dues 5 070,81 t6 139, 151 w 180. {B) Encemble of 20 lowest-energy | Roseta suc fbelow te in A) \) Regions wth mare than 3A RUSE are depicted in gray. (C) Comparison of the RMSF at each residue in the low- . o_o ad ners Roza ervemole to MR RL ‘mater ‘edaation rte Ged, relsation rates blak, RSF in Rost ensemble) The relanaton data werent sed in he structure cleatin. Regions variable in the low-energy stuctresenibitinrased dynamics in solution: tes ata wee rot wed nthe structure calculation. (0) NI saluin ensemble based on sidechain NOES (POI: 2) oe we tet yz Fig. 3. Bind predictions wt the C-RDC Rosetta and iterative C- E 2 ROC Rosetta protocols (Left side ofeach panel Superposition of the 10 lowest-energy predicted stucturs (ed) over the experimentally solved ensemble of NNR srucurs (ue). Right side ofeach panel) Magrified view of the cre side chains Rosetta models in (A) to (D) were deterned wth CS:RDC- Rosetta, and in () with iterative CS-ROC Rosetta. (A) BAD, (@ DARIISG (C) MaRZ1AA, (D) SARIS, and (E) ALT. 19 FEBRUARY 2010 VOL327 SCIENCE wwwsciencemag.aorgall five cases (Table 1), the resuliing Roseta- determined structure is very similar t0 the con ventionally determined NM solution structure cover both the backbone (Fig. 3, lft side of each pane!) and the core side chains (Fig. 3 right sid= ‘of each panel), which is notable because no experimental side-chain information is used in the Roseta protocol the details of core packing ae determined by the Rosetta allatom enerey function (magnified views of core side chains are shown forall ofthe remaining targets in fig. $6) Thus, our methodology is able to generate sccurae structures of potcins up to 25D from sparse NMR data without side chain assignments. To be use in practic, iis important that there be a means of assessing the rlibility of the computed models. Crossvalidation with inde= pendently collected data are an excellent way t0 do this, but truly independent data: may not al- ways be availabe, and ifthe available data are already sparse, it may not be posible to remove a subset for independent validation ‘Our approach to structure validation is based fon the interplay between the two contributing sources of structural information: (i) the detailed physical chemistry implicit in the Roseta alle stom energy function and (i) the experimental NMI data As illustrated in Fig 4, the allstom energy landscape (back) is rugged with many local minima, making optimization difficult. The experimental bias based an backbone NMR data (fed, though smoother, is degenerate and lacks A ‘A610u9 © auors oo 5 maa) mss A) Fig. 4. Effect of incorporation of experimental data on energy minimization. (A) The Rosetta all-atom energy tlack fine) has many local minima, making minimization dict, but the global minimum generally close to the naive structure (N). The experimental bas (red line), though smoother, has de- ‘generacis and lacks resolution because the data ae sparse, Local minima ofthe all-atom energy and the texperimertal bias ate uncorelated far anay from the native stucture but caincde clase tothe native structure Acocingly, far from the labal minimum, including the experimental data during optimization usually results in higher energies (arrow 3), wereas close tothe native stcture (),incloding the data results in lower energies (arrow 2). (B) Lines represent the lowest energes sampled by structure at various USD after optimization inthe absence (black line) or presence (red ine) of experimental data. Gener- ally, the all-atom energy and experimental data are in concordance for conformations close to the native protein structure but not for conformations fr fom the native structure ths concordance condition ‘met, the experimental data can guide sampling toward the global minimum close to the naive structure (arrow 2), and ths, constrained optimization can result in lower energy confrmatians than unconstrained optimization, whereas biased optimization is les effective than unconstrained optimization distant from the native structure leading to higher energies arrow 3). (Cand D) In contrast, all-atom energy and RNSD of inal Rosetta ensemble fom iterative refinement, wth an without experimental data, are shown. Lines represent the median ofthe 10 lowest-energy models per RMSD bin. (C) 121, an unsuccessful calculation. Biased optimization with RDC data (ed) yields similar energies as unbiased optimization (hack there isa large remaining energy gap tothe native suructure(bive dot). (0) Alg23, a succesful calculation ised optimization with the experimental data (re) resuts in lower energies than unbiased optimi- zation (blac. wonusciencemag.org SCIENCE VOL 327 resolution. Because the constrained minimization ‘ofa function will almost always result in higher function values than unconstrained minimization, NMR dta-constrained optimization, in general, shoukl result in higher-energy’ structures than bias-fce optimization (arow Tin Fig. 4). This scenario may bold for trditonal structure deter- ‘mination in which the search is almost completely «riven by the experimental dats, However if the to sources of information are in concordance, the bias from the experimental data ean have 40 favorable effets (Fig. 4B), Firs, optimization far from the native minimum is impeded, resulting ‘in an upward shift ofthe energy of non-native struts (arow 1), and second, optimization near ‘he native minimum is improved as the data guide the search toward the global minimum (Fig. 4, sand B, sow 2), ‘Bete optimization in the presence of exper- ‘mental data (Fig, 4B) is unlikely to occur if here sno sampling near the coneet structure, as data and the energy function will almost never ine ‘dependently favor the same incorrect structure, Hence, we propose the following thre ertria fo evaluating the reliability ofa calelated trac ‘ure (Table 1, columns 6 to 8) Fist the calculation should converge: The lowestenergy conforma tions should be very similar to each other aver a large fraction ofthe structure. For both the CS-RDC-Roseta and the iterative protocol, whenever the caleulation converged for more than 60% ofthe structure, the RMSD to native over this region was less than 4 A (Table 1, column 6), Second, the converged. structures should clearly be lower in energy than all nificarly iferat (RMSD > 7 A) structures: this was tne for nearly all of our test cases (Table 1, column 7), Third, the stuctres generated with experimental data should be at lest as low jo nongy a those generated without experimental ta; for none oF the succesful calculations does the energy increase significantly when the exe perimental data are included in the optimization (Table 1, column 8). For larger proteins (120 residues), dhe data in ict guide the trajectories to Jowerseneny strictures than those obtained by unconstrained opsimization (Fig. 4D and Tube column 8). As argued above, this is @ strong in- dicator tha the corect stuctere has been Found When all three criteria were satisfied for the 20 proteins in ur test se, the low-energy’ n= semble resembles the independently determined structures. Importantly, the clear structure caleur Tavon failure, 121, which converged to a wrong conformation with an RMSD of 9:4 A to the native ils the third exteron: The enery is higher rather than loser when the experimental dat are ‘inclded in the optimization (Fig. 4C and Table 1, column 8}. Because we had only ne such failure, we simulated aditional failures by de- leting all neaenative smctares from the model populations and computed the three metrics de- scribed above for these “ike” minim, (able ST) (22), For almost all of the proteins, these con- stmctodpathologieal cases again fail the chit 19 FEBRUARY 2010 1017i REPORTS 1018 criterion; They have higher energies in pesimentally biased optimization For the proteins n our set in the ~80-KD ‘molecular-weight range, the computed stuctares are not completely converged and have large dis- condexed regions. This i clearly a sampling prob> lem because te native structure has lower eneray (Fig 4C and fig. $3); even withthe NMR data as 1 ghide, Rosetta trajectories fl to sample very case to the native sae. Increased convergence om the low-energy native stite can be achieved either by collecting and using additonal experi= mental data (Lilb_2 in fig, $3) or by improved sampling. Though at present the former isthe ‘move reliable solution, the later will probably become increasingly competitive asthe cost of computing decreases and conformational search algorithms improve, ‘We have shown that accurate structures ca be computed fora wide range of protcins using bbackbone-only NMR dats. These results suggest ‘change inthe traditional NOE-constaint-based approach to NMR structure determination (Fig, 4), In the new approach, the bottlenecks of side-chain chemical-shift assignment and NOESY assignment are eliminated, and instead, more back- bone informaiton iscolectad: ROCs in one o mone ‘media and a small number of unambiguous H HY constraints from thee- of four-dimensional experiments, which estict posible strand reise tes. Advantages ofthe approach ae that 'FL!N- based NOE and RDC cts quality is relatively tunaifected in slower tumbling, lamer proteins and thatthe analysis of resonance and NOESY peale assignments ean be dane in lingely automated fashion with fever opportunities fr croc, The approach is compatible with deuteration neces sary for proteins greater than 15 KD and, for larger proteins, can be extended to include methyl NOES on selectively protonated samples, ‘The method should also enable a more complete structural chanscterization of transially pope Inte states (25) for which the availble data ae generally quite sparse References and Notes 4,0. dimmeman eal Mo Ba 269, 592 2. C Bone, Pita, ls, Watch, 1 comput Chm. 18,139 1999, 2, WCC Baan) Hr HL Mose, GT. None, (hem fer 108, 35412000, 41S ang Beds, oo. A 30,22 5. Wes WA Wet, A. Baan H.R. ba 1-L Note. anfometes 25, 2085 (2009. 6 Beane a, tee A es 37 Me Sere fe W670 20 1. Yh, Fagin G Coles 4420 (208, 8G foreas, FD, A. Bs, Methods Eat 398, 91 C Latin, Pag Pri, tan ans 29,5782 008 10, 14. Pete Bugs, A Khare, Chem. er abs, 3819 Ib 11 S Gres A 80 J ial ANE 3, 627 (1993. 1 O.M teaser FM ars, Becher 27,142 ‘a3ea. 13, KL Gove Re, LE. oy, Bei 36 2349 4997 14, 6 Magne J Biora AWK 3, 375 0998) 15. R Dan. Bake es. Bichon 77, 363 16, Prey LS Mla, Ele Sc 309, 1668 ears. 1, lst and ead ae able af spain tier an Scene Oni 10, Al sal, CM. Osi, ent 19. Shen eto, Pee. Natl. Acad. Sc USA. 105, 4585 201 Aone, Ae. Fb), rsena Alger 138, 338 0999) 2, Ck Bob Bak, J Am Chem, Sor 128, 2723 e002. 228 Qanet ot, Nowe 450, 259 (2007, BT Bart 0. Bet, Pres 73, 958 (2008 24 Mang Tasers Phang, 8 Inge, I. sear toc 46, 965 (2000, 5 lant ne Limits of Predictability in Human Mobility CChaoming Song," Zehui Qu," Nicholas Blumm,*? Albert-Lés2l6 Barabsi*=* A range of applications, from predicting the spread of human and electronic viruses to city planning and resource management in mobile communications, depend on our ability to foresee the whereabouts and mobility of individuals, raising a fundamental question: To what dearee is human behavior predictable? Here we explore the limits of predictability in human dynamics by studying the mobility patterns of anonymized mobile phone users. By measuring the entropy of each individua’s trajectory, we find a 93% potential predictability in user mobility across the whole user base. Despite the significant differences inthe travel patterns, we find a remarkable lack of variability in predictability, which is largely independent of the distance users cover on 2 regular basis, ren it comes to the emerging fie of Ihuman dynamics, there is a funda- ‘mental gp besicen our inition al the current modeling. paradigms. Indeed, al- though we rarely perceive any’ of our actions to bee random, fram the perspective of an outside lobserver who i unaware oF our metivations and schedule, our activity pattem can easily appear 25, Han eg, UM. Horo, Ps Chem 8 26 OK Ser T onebne Pie 58, 672 2008 26 A teria, ele As fe 32,1370 203) Iovate a Novel Campana mga oF Tear ad pine Aa for psig es oe Bae ‘Goa? svprompse a the fame enh {apa Fst arto Rsetaghone arian for thor geemau etn cpg gone Yo Mang and Tang et otbton dat cia sues wig sae NOE contacts wth (tse 5. oral He ean. boo calecion 1 Rc ot, A Leak br pod the Cotlbyanhy {nd RNR Sem ROE refine’ pat a he NESS Cantor ae oar uote NR ety Shut a fated metots depen, OL PR GM, and D8, cess esta dered Sd we ibe CS ROC Host reat OFL desgd Sd ested he tare CS-ROCNOE asta prea Us semepes th xm einarent pseet ‘OF a BB. Sesed ane prom rect Ie ory tae str at KW ate 1 aad ‘He ALG semi: 1A GL, TR, AL UK ang 18 oe ns NI St; and SOF, (Ci. ane. te the mano Te vx ‘Spped by iHome Fmt See Fog {OU NI ge T6222 (0), te HA (G1o74958 (GTA, ard he Resear Resse (yon RRCOSSL (} PWT al aS ey Welcome Posoatal Felbwsip ROC ané Prana Retain Eoanceert ita dead he PH Baia Bok OB) mth scion abe Supporting Ontne Materat scenemag ngeareTuleoce12636290CL Herat ond etase fie St tos Totes 5183 Felneoes 21 oer 208; azepled 14 amy 2010, Fis one ata 201, Ince the narnabon in cing th pee. random and unpredictable. Therefore, curent models of human activity are fundamentally stochastic (J) fom Erang’s formula (2) used in telephony to Lévyawalk models describing hue ‘man mobility (3-7) and their applications in viral dynamics (8-10), queuing models capturing bu- ‘man communication pattems (1/13), and mod els capturing body balancing (14) o¢ pane (15) ‘Yet the probabilistic nature ofthe existing mode eling framework raises fundamental questions What isthe role of randomness in human bee hevior and to what degree are individual human ‘actions predictable? Our goal here is to quantify "cetera Net Ree, Depetmentscl Psi, Biko, and. Comper Scavea Noten Une ston MA 0215, USA “Deparment of Mite, Hanae ea See en enter Cae Ses doy, ana Faber Caceres, MA 02115, USA. Sl of Comper Scene and Erne, Uniesty of Bec Sclne a Tele of Cha, Cheng 610054, Cie “To whom conepondence shoul be adtessd. E-rat alb@rev 19 FEBRUARY 2010 VOL327 SCIENCE www.sciencemag.orgREPORTS i the inerplay between the regular and thus pre- mobile phone tower, with about a km? recep- location, chus capturing the fll spatiotemporal dictable ad the random and thus unforeseeable, ton area on average epresenting the uncertainty order present in a person's mobil parte. To be probing through human mobility the fundamen- in our bility to determine the user's where- specific if 7) = {Xi, 5." Kz} denies the tal Limits that characterize the predictability of abouts), and links represent the observed move- sequence of towers at which user i was observed human dynamics. iments between thse, The uneven node sizes, _ateach consecutive hourly interval, the ent 'At presen, the most det infoematon on conesponding to the percemage of time the user §, 8 given by —SreP( 7} lous PCT) whe human mobility acwoss a large segment of te spent in the visinity of the paticuar tower, PI7') isthe probability of finding a partiulse Population is elected by mobile phone carrer indicate that individuals tend to spend most of time-ordered subsequence 7 inthe trajectory (4, 16-21). Mobile caries record the closest ther time in a few selected locations. Finally, 7; [(22) section $4]. Naturally, for each user, mobile ter each time the user uses his or her each mobility network has an essoiael dyname —_S, < 10 km, 11" is largely independent of rp, satu ratingat 1" =0.93.(D) Thetracon oftimeauser —& Spends inthe top most ised locators, the re: (kam) sulting measure representing an upper bound of precicabittyI1™. Thus fr =1 we can pric corecty ‘the user’ action when he of she found atthe most likey locaton ("home"), whereas for 2wean predict corecty the use’ whereabouts hen he sin one of his top two locations home” o ofc’) Inset) appears to grow approximately logarithmically wth, Ao cw Time sm) Fig. 3. (A) The houly regue larity Ra) over 2 weekciong time petiod, measuring the fraction of instances when the wer is found in his of her mest visited location dut- ‘ng the corresponding hour- long period. (B) The average umber of visited locations / (0 during each hourty time frame within a week, revealing that high regularity A) correlates with small WO, (C) The averaged AUR versus the adius of gyration (), showing that the users with large have high elatve regularity. prodicubiliy 11 < HI™(SN), where 1 sven by S= TIM) +1 TI) las with the binary entropy function HT™) TI fogs(ft™) (1-11) log. (t — 1. For a scr with FP" = 0.2, this mans that at feast 80% ofthe time the individual chooses his location ina manne that appears to be random, an only inthe remaining 20% ofthe time can We hope to predict his or her whereabouts. In ther terms, no attr how good our predictive algorithm, wwe cannot predit with ever than 20% accuracy the future whereabouts of & ser wih IT" = 02, Thorfore TT™ repre Sent the fundamental iit for each individl's predictability. ‘We determined 11™ separately foreach user dn the database. To our surprise, we found that PXUI™) does not fllow the fasted dist tion sugested by the travel distances, but i rarowy peaked near TI™* = 0:93 (Fig. 2B) ‘This highiv bounded distribution indicates that, csp the apparent randomness of the indivi: ‘ak actors, historia! record ofthe daly mobility pate of the users ides an unex pectlly high degree of potential predictably We have also determined the maximal predict. sbiliy TP and he random predictably TI exacted fom Sand." As Fig, 28 shows, the result strikingly diferent PTT) is tremely widely dsiibuted and posked at 11 ~ (03, wih indicates tha, i we rely nly on he hetragencous spatial dtrbution, the prec sbily aeross the whole population is insignt- dean and varies widely ffom person to person. Sei, (TT) has ap at TP! =0, hie suggests ot only that T™™ and 11 ae net fective a pritive tools, but aso that a se nificant share of preci is encoded in he temporal arr othe visitation pater ow can we conc he wide varity in the observed wave dtances, as captucd by the fated PU) withthe highly bounded predict ability ebserved across the user population? To rawr this, we measured the dependency of 11 on 7, ae found that, for 7 > 10 km, pre= dictbilty becomes largely independent fr Saturatng at "= 093 (Fig. 20), There Fig, 2C explins the failure of our eater by pothesis: Indvidal with , = 100 kr, covering hundreds of slometers ona rl basis, est, as predictable as these whose life is constrained (-km neighborhood, saturation that lis behind the high predictability ebserved aross the whole usr base ‘To determine bow mach of our preicaily ‘sreally ote in he visiation pattems ofthe top locations, we calculated the probability 1 tha, 4 given mement, the user sin ene ofthe top rmost vised locations, where m= 2 typically capnures home and wok. Thus 1 represents an ‘pper bound for TI, 25, even if oar proitive algorh is 100% aceurte, it cam foresee the Fire locaton only when the seri found in one ofthe top laations monitored by heal grt, [AS Fig. 2D shows, the top two locations (r= 2) » 19 FEBRUARY 2010 VOL327 SCIENCE www.sciencemag.aorgoffer only 260% overall pic, Gradually adding mote locations ineeases Tin), but we neg several dozen stint locations to converge toll = 1 (Fig, 2D. ise To understand the crigno the observed high potential prediciliy, we segmented each week {nto 24 #7 = 168 hourly intervals, and within cach hour, we identi for each usr the most, vied location Fg. 3, A and B). For example, if between 8 and 9 amon Monday, ser was found 10 ies at tower I, ewie a wower 2, and once at wer 3, we assured that he ost key location during this hou wil be tower 1. Nex, We measured each use’ regularity, R defined as the probability of finding the user in his mes. sited location ding that hour. reresents 3 lower bound for pedictbity Ta itignores the temporal corclatons in usr mobil, We found that actss the whole user base, R= 07, which roca that on average, 20% ofthe tie i most, sted location coincides wit the user's atl location, The ptt is ie dependent: During the nigh, when mont people tal be eably at home, peaks at ~ 09, but between noos and 1pm. and between 6 and 7 pam, has clear tninima, coresponding to tansion periods (vel w lune oe home). Indeed if we measure the total numberof stint lations NU) a user visited each hour (Fig. 3B), we find that momen of low regularity R comespond 1 sigieant increase in MO a signature of high mobility. and wen & peaks there ea drop in NO. IF the users were to move randomly between thc 'Toctions, then 2%! — iN, whic i 1/22 0.016, an order of magptude smaller than the observed = 0.7, This gap once asin indicates thatthe high regularity chrsctoiing each user's mobiliy represents significant departure fom the expectation tha hey wil be random. In Fig 3C, we plot he lative regulary RRC asa faction of rn observing a cleat increasing tendency. That is, counerinutivey the relative requaniy of uses who tavel he sos (ive high r,s higher han he elaine regularity ofthe move homebound individuals, “To explore whether demographic Factor. inuence the user’ regulary and predicubili, wwe measured Mand IT" fr diferent age and gender groups (ig. $10). T was suprising that wwe didnot observe gender or age-based die. ences in TT, but only e systematic, but Ststcaly insignificant, gender bused deren emerged in eaubriy. We also explored he impact of home, language groups, population wonusciencemag.org SCIENCE VOL 327 densi, and rural versus urban environment on predictability and found only insignificant varia- tions igs. S8, $12, and S13). Finally, we did no find siantcant changes in user regulary over the weekends compared with ther weekday mobility (ig. $8, which suggested that re laity st amposed by the work schedule, but petri is nrnsic to human atv. 1 surmary. the combination of the empit- cally determined user entropy and Fanos in- xuaiy indicates that thre vs & potential 93% average predictability in user maby, an excep tionally high value rooted inthe iret rg Inity oF human behavior Yeti is no the 93% preci that we find the mest suprising Feather its the ack of varity in pricy across the population, Inded, given the fattaied distribution of the distances over which users navel on a regular basis (Se fig. $2), most, individuals are well localized in fit neigh bochood, but afew eave widely Furthermore, suber of demographic nd extra parame, fiom age to population dent and the numberof towers visite, vary widely from user to user. is not unreasonable to expect, therefore, that, predictability shal als vary widely: For people ‘who travel li, it sould be easier foresee theirlecatin, whereas those who regulary cover hundreds of Kilometers should have a low prodicibiliy, Despite this inorent population hotrogenets, the maximal prodiclny varies very itle—indeed ATI) is narowly peaked 383% and we se no users whose predictably ‘would be under 80% “Alsough making explicit prdicions on wer ‘whereabouts is beyond ou goals her, apixop ate datseining algrits (19, 20, 27) could tum the prditabliy identified in ou stay ino actual mobility predictions. Most important, our ress indicat that when coms to process daiven by human mobility fem epidemic mode cling to urban planning ane tific engineering the development of accurate predictive models is a sientically grounded possibility, with potene Gal impact on ene welsing and pubic heats [At more fundamen level, they also inate that, despie our daepoted desire for change and sponta, our daily mobility is in it, characterized by deeper ruler References and notes 1 Calan, 5 Fan, ¥ Lot, er. Bo ys 3, 59 2009, 2. tan, Mt Be for Moti 8 20,38 908. REPORTS i 0. ocnan L ulapl el arse WC Gane © A Wg, tabs tne 83.779 2008 Stn, 0 Berar APs. 52 187 (2002, EN Wctgna HE Sly, Pp ee. 73, 206 BM, & , Oc. Gna, Ka, (hos Sltns Facts 34,129 200. Coz A.B Srbeny A), Yl, 2 Wepgea 15 kd. 819 207) Uta 0 race Geb, Pre Me Ae Sel OSA 14, 15124 (2008. Pang conte, CA His, AL, ab, Scie 324, 1072008 Aoi Notre 435,207 2009, 6 Gente, Lila Py Re Se Nox So Moter Pye 7, 012101 (2008 ‘cate, 6. Gl ys. Re Lr. 98, 208702 1.0. May. Me A. Pigalle iE pet. 42,36 2005, 0. bing. arn, eh Were 07, 487 2000. WS So H.5 KE Comma ug, 4862002 Ch ig, Rages Seer, Pace A 37,3017 WC Goal, PU, H. Mermann Py le 96, 8870 04. Aol Fein, Por Unites Compu 20, 255 (2008 1g A er oe el Sac 63,1057 (09 B Lanbiat eo Psa 307, 5517 206) Aerts and mthde ae late a sprog ‘atin Science Okie, [TR Aono, Aa, fai Nowe 399, 130099 Alot SoH. hen Rowet Comping n Cnputtiond Rance ping, Serie, 2008) I dager ey Pi Re Sat Mane, Sof ane Py. 93, 036127 2060. FM Fata Pars of hyormoian ie IT Pas 2d Wey, Me akan enn 96D USong. Rate Ran Xe, 23rd nat oo Crferereof teIEE Cnr ond Commies Scat WAPOCOM) 2,18 (2008. ‘We man Gana Wag) fog, {nd A ae casera commen on the nrg Ns wo ws sipped ye ames tedanel oneiton #38 Cerys Stug Comper pers, ASF win te Ioraton Thogy Rye OMA.O2737 06 SASTI6SO Cota eho lance spnires by he US. ‘ny Reseach atria ander aetna WSL:NF05-20053. 20 vas supped by 2 eLnship ‘tom the hina Sholsip Cor Supporting Ontne Materat sceneag ngearer ul 59¢820180¢2 Herat ond ethase SOM Tet fences 2 jure 2009; scepled 28 Deeb 2008 19 FEBRUARY 2010 1021Nowisthe time to draw upon every tool of US. power to promote Curinterestsin the world, We should make maximum use ofa core ‘trenath of our country — leadership in science and technology. ‘We,the undersigned Demnacrats and Republicans, beliave the ‘Administration and Congress should olavate the role of Science Diplomacy n US. national security and foreign policy and work to: | > strengthen inks between US. and foreign scientific Communities as akey part of US. diplomacy, Offer scenic cooperation and technological assistance 5. bridge to opening broader dialogue with former adversaries and as an incentive to prevent conflict > Bring the world’s top scientists and engineers together to tackle pressing lobal challenges like energy security, climate change, poverty, disease, and WMO proliferation; and » Provide funding for exchange programs, collaborative research technical assistance and capacity building to fully qualified US. governmental and non-governmental ‘organizations Science Diplomacy Zhongguancun Life Science Park, Beijing, China novo Hoxdisk Novo Nordisk China RAD center i an integrated part o Novo Nordit’s R&D organization, Currently, the R&D center has four research departments: Molecular Biolog), Protein Chemisty, Cell Biology. snd the newly frnod Diaces Ressrch. The cen i expanding ite Protein competentes fo suppont engsng posts an ant bat tt fof new molecular gets for dhe treatment of dabetes,autoimanune and inammtry dseases. We are seckng innovative and energetic seni who plan to advance thei ever in China 0 i in the ‘deere helo 1, SolentistSenlor Sclntist, Antibody Phage Display (Job Code: My effort on antibody diseovery generation via ph rons atc am eit aie at ted Din Molecular Biology, Blachemisty or Inmunclogy and>3 years A proven tack record with phage display or other display platforms + Solid knowledge and hands-on s-depthenperenee in various moleculat ‘engineering. expresion, punficstion and charateiaton 2, Selenist in Molesular bi Protein Expeession (2 positions) Responsibility Play a ciect role in developing and optimiring recombinant protein provdction, protein engineeing ad assay development Reguivemen + "PhD. im Moleular Biology, Biochemistry o relevant biological cence, line development o throughput eel culture + Expertknomledgs of aplirabe state-of-art cell cloup or through technologies and methods in molecular biology In addon spect equ or the oui Job Code M2: Experscce sn manology ov aflammatory tesa 3. 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Pie electronical send cuvelam vi to Kemet ‘Lyons Jones, M.D. Chai ofthe Search Commit Is iens@ueideds, oe by sce mal ts ‘9500 Gilman Deve, La Jos, CA 920930828, Appian sil e sonsidered tne Apt 80,2010, ‘uni afind apple hs ben sete. eS 0 AB Ain’ Ea Opp Epa wh oe ‘nian nent eld ty POSTDOCTORAL FELLOWSHIP ‘Molecular Biology ‘Research and Teaching The Department of Biology a Viana Univer sisyinnitesapcations for Bosdoxtoral Fes (Gb pore teshing, 80 percent research) in mo” lecular bcs, sing Agent 2010, One-year po sion, renewable for up to to yeas Puig ae ik for eaablahingcesen in rscrch a each ings ental io apliations to sun wndcrgradte tenure track pots Teaching asapument ince Sneha cra in melee tcnaques/escch Sind amedeclr ology course inate of pecan {ora ceaneaughe asgment in mecha ole ‘pete rescore aren, and ever opportuni ‘ar alaboration with Bulan the Depron est (sre website: ep: /wr: biology rlanovac) APHD. 6 reoued Appicans mas ao oie 3 ‘website: ups obs llnova ada. 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PROFESSOR fheuity postion in the Tochemisye and Cal ogy Deparment of Re rivers: We ack an outstanding sent With ‘xpi fh roa arcs of expcinemial and the reel bengal cach, sch partion cope ineel, developmental, and/or cancer biology. Can tides nenginteniiplinaryapproschet and/or ‘node erganting are epee crconraged to ap Candidates must have 8 PhDs postdoctoral a: Dg and conunding reach porewial, Seccahl ‘ales ae expected to devlop and main a ‘isos retcnch program support by extraml nding and to purtcpare a pradate sed weer ‘sont sation. Review of tppicattons Wil com fence Meh 12010, and consinee uni the poston i ke. Peue comps a PDE containing a one lee, culo tse sumiary of pst ecard sate Ineo rr serch pon ete ena cron {ermal bebsenrehereeed Also plex srange For four letzers of veterence to be scat the she emai adres, couse 0 iy Aine A ASSISTANT/ASSOCTATE PROFESSOR of ‘ToxIcoLocy, Universgy of Connections Store, CT The Department of Pharmaccticl Science atthe ‘Universi of Connecti, School of Pharmacy ‘pres spiisiton for tenor tack fly poston Inncofewalology atthe Austant or Asochte ro Fesor evel, storing Aug 23,2010, We ae sock. 2 uviolognt with 3 stomg background in research Inn aching with foc ot the tone elects of ne hina on the sent and/or peripheral nwo Sem and/or devlupmetal neoroxsology. Ts Wl complement the interes he ul in the Dgormene of Pharmacsoasl Sciences wih i ‘hk negative approaches to dso mechani Sfdreg or tonicnt action, dev dscuvey and des, tn pharmaceutical echdlogy. The Deparment ironed in new 200,000 aguire foot stro he ane lina i he aconce quad of the Univray of Gannett an encores fay iter plinary interactions with her programa of the Unters? Sich as Phslogy’/Neumibyologh and the Molecular find Call Biology Urogran, The svesfl carte iPepected to desiop sarong, exramuraly inde ‘acarch pera a oct partipte in each tng athe graduate apd presauna levee A compe. ine slay along with strap funds wl be prided (Gralcrtone Appice mos poses a PhD degree ‘oe egunafen, rung ord and writen camimunicton Sassi a soong tackgnd in researc and ech tng wi focus othe tena ts of tents the conn an perineal neous stem ane {scopes netromnigy ‘Apicings should submit ciicuam vt, a bi seems of recrch and teching ntest at mates tad fl sdremes of tece references, We wend pe fer rss sr applic cecal at emai Fel lebel@aconnedu ur by surfice mil sent Leslie Lael, University of Consectieut, School of army, Department of Pharmsceati Senn, 69 Norsh Eaperlc Road, Unit 3092, Stor, CT 106269:80925 Review of appisions all aren oat 16, 2010, and conn unl the ponion eo moe Bl sara ‘ml th shir, ad 19 FEBRUARY 2010 VOL327 SCIENCE www.sciencecareers.orgulm university uu til ulm The Medical Faculty of Ulm University invites applications for the position of a Cee muha C me) (Head of the Institute) We are seeking a candidate with an outstanding international research record in the field of mo. lecular immunology. Teaching experience as well as excellent third party funding are also required. The successful candidate should cooperate wit research centres of the Medical Faculty and Univer: sity (among others DFG-funded Collaborative Research Centres, Clinical Research Units, Max- Planck-Research Group) and should interact with institutions of the University Hospital. In addition, ‘we expect the candidate to actively participate in the development of navel interdisciplinary research centres/units. Candidates must have a MD and/or PhD, or equiv alent doctoral degree. Teaching obligations require the candidate to have a postdoctoral lecture qual- ification (Habilitation in German or comparable qualification like Junior Professor). The major teaching language is German. Lim University and its University Hospital are certified in a family program and offer a dual ‘career service program. Ulm University aims to increase the number of ‘women among the faculty staff and therefore explicitly encourages the application of female scientists. Applications including the application form, full CV, a complete list of grants and public and a summary of current research activities and objectives should be sent to (and received not later than March 45, 2040): Ulim University, Professor Dr. K.-M. Debatin, Dean of the Medical Faculty, D-89069 Ulm. ns The Univesity is an equal opportunity employer and handicapped applicants with ar equivalent qualifications will be given preference PRESIDENT AND CHIEF EXECUTIVE OFFICER THE METHODIST HOSPITAL RESEARCH INSTITUTE ‘The Methodist Hospital Research institute (IMEI) is 4 comerstone in The Methodist Hospital's strategy for Continued development as an academic medical center, Since its inception in 200, the Research Insitute has receives! more than $21 million in fining from the Notional Institutes of Health, and its peer-reviewed! funding is increasing exponentially ench year, TIMER] ‘oversees al clinkal tials at The Methodlst Hospital, {with 700 clinical rials currently under way and 1,700, ‘credentialed esearch team members, including more {han 40 principal invetatore The Research tate {saffiliates with Weill Cornell Medical College and the University of Hosston, With the planned retirement ofthe founding director of the Inte Methodist iin newt ofa collaborative leader to guide TRIE tote next level of accomplishment ‘The Metis Hospital Research atte Clinical and Translational Reseatch Buding, anew 44040 square foot {acl will open this year and will add substantially tothe esearch insttute's space portfolio, The bili inches Spenlaboeatny pact caged toheure90 papel Stventigtors, large and somal animal vivarium, coe faites tocenhance interisipinary research Good Nanfcturing Practice acy sate neat maging equipment neon dott for Both prelinicl an clinica studies, and 8 BSL laboratory for infectious disease research Candidates must have an earned doctoral degree and a recone! of personal research achievement commensttate swith a Weill Comell Medical College appointment at the ‘ak of fll Professor. Additional relevant esucation oF experience will be advantageous. Previous experience in research administration ix preferred The Methexlist Hospital isthe lagship of The Methodist “Hospital System antl located in the hestt of The Texas Meslical Center The Methodist Hospital is the only hospital in Texas named! to ULS.Nea & World Reports 2008 “America’s Best Hospitals” Honor Roll Join us as we celebrate our 30th year asa lending healthcare provider i our community and the wld! The saccessha Eandidate will receive a generous salary fringe benefits, and relocation package. To apply or nominate ‘candidate in conidences email CV to NI Michelle Bach, Executive Recruitment for ‘The Methodist Hospital Research Institat, = mabsehldtmbs.ong. WORK FES Meth@list’ The Methodist Hospital =SSS= Research Institute Houston TexasWhitacre Chair in Energy Science and Engin ring The Department of Chemical Poginering at Texas Tech University is actively seeking wo Ml the newly endowed Whitacre Chair in Sustainable Energy. Candidates fr the position will be exeeptional ingividls whose research areas are algned withthe university goals ‘of erating a strong mult departmental reseateh base in Sustainable nergy at Texas Tech University. The selctd individual wil be an intematonally recognized leader in hs other eld as deinonststed bysuch metres asthe peer reviewed publication and citation records, atonal recognitions such s major awards or fellowships in technical Socetos and a strong tack record of competitive esearch funding The Department of Chemical Engineering at Texas Teen University ‘as embarked on buildings nationally recognized rescarch-intensive department wth srenghs in Polymers and Materials, Bioengineering and Biotechnology, Computational Chemical Engineering, and Pro ‘ess Systems and Enginoering. The succesful cadiate ws enpected 1 expand the revearch portfolio of the deparmieat int Sustainable nor. The postion isan Endowed Chit the ove of Ful Professor and incumbents willbe expected to participate in esearch land teaching at this fv ‘ill have very tong records of scholarship supported by ext Funding and that externally sponsored research willbe brought to Texas Toch University Interested candidates should apply online at_ hetps:ti jobs texastech.eda’. Please use equistion mumber 80764 Incr ‘ue candidates should include detailed CV, a research statement nd list of five referees, For frther information, please ease the Scurch committee chair, DeRaghu Rengasamy a raghrengasamy 818 Equal Opparsunty Employer Professor and Head, Division of Cell Biology and Biophysics Applications ae invited for Profesor and Head of the Division of Cel Bialogy and Biophysica he School of Biological Sciences, University ‘of Missouri-Kansas Ci. The success candidate shoul Fave record of txcelene in rsserch with sustainod extramural fading. The cand date ‘rll be expected o plays leer role in rehate ad undergrads ‘uation, faculty mentorship, determining future research directions, and in the overall development and growth othe Schoo. The Schoo of Broigical Sciences positioning elo bocome s eegioal leader in ih aewe of srutural, moovula and cellar bolog, std microbic- ‘gy-andweleemes applications from aalifod cand dats in those aes, Hoover, outstanding scientists for al res of bse ie scien are encouraged to apply. The sbceesl candidate wil reve an endovted professorship accompanied by « generous annul research stipend, 3 ompetive 12-monh slay, renovated research space, a sit-up pack fee commensurate with rink, andthe avait of exelent research ‘ppor facies within the School of Biological Sciences, Candas should have a PhD. and curenly hold a tenured scademic position st ‘Toapply, pleas submitlectroniclly (MS Word or pl) & CV. statement present and future research interes, andthe nares and sddresaes of 5 reorences 0: Ms. Micacla Eseareno (escarenoma uimke-edu). ‘materials wil be handled with sit confidentiality. The poston wil sin open ut led UNKC isa Eau decest Equal Opportait Afrmative Action Eimployer thas fly commie 0 achieving adverse fact and staf Women, minorities, veterans, and individ wth dabiities are encour app ASSISTANT, ASSOCIATE, FULL PROFESSOR Cur) Hunter College of The City University of New York invites applications fora tenure-track facully positon in bioinformatics and high performance computing with expertise in the development, adaptation and implementation of HPC algorithms applied to su dataintensive biological areas as (bt not imited to} next-generation sequencing biomesial ontologies, and systems biology, The successful candidate wil be appointed tothe dactralfaculy at the CUNY Graduate Center and wil participate actively in Hunters ‘NlH-unded Quantitative Biology (QuBi) Project, The anticipated start dates Fall2010. The appointment will be made In the Department of Computer Science or Biological Sclencas ‘depending on qualifications. Preference willbe given to candidates with welrestabished strong records of peer-reviewed publications and external funding, but exceptional junior candidates wil also be Conse Ealvaton ongoing an il conn ute poston isle. Hunter's computing and biomedical research community, is supported by extensive funding fom NIM and NSF. Hunter College is strategically located in Manhattan near ather major biomedical research centers and universities and atiacts an engaging and tliverse student bod For information on howto apply, vist: hips. hunter.cuny.ecu/qubi/ Questions may be emailed to: bioinformaties@hunter.cuny edu The iy Universi of New York opment OpporniyAtienative HUNTER ist ‘merce with Diabites Act Employer CLINICAL ASSISTANT PROFESSOR OF BIOINFORMATICS 1p TA CMe ello exe NE ENSe PEt a ‘The Department of Biology at New York University invites applicotions for 8 Cleiea! Assistont Professor snpamtmant £0 Start September I, 2010, pending Busgetary ana samiisratve approval. Responsbilties inchide developing and teaching © Departments Aesdemic Atfws portolin Teaching ites vl include four course oferings armually (2 per semester in adation to the above scasemia sdminisvativeresponbilties. Pe Feseorch, teaching and administrative experience is preterred Successtul candidates wll have 2 PRD in Biclogy and/or Informatics at wellas prior teaching experience. The Department of Boloay (ht //bioloayasnyi.edu) offers an Outstanding and Coles esearch environment. Candidates should submit 2 single BOF fe containing 2 Cover txperlencs to Bology informatiessearchanyusedu. The following Sress can ba vane for the cover letter Or. Gloris Coruzs Department af Biology, New York University, 1009 silver Center, 100 Washington Square East, New York, MY 10003. "hres POF letters of reterence should aiso be eubmtted separately £0 biology informatissearchanyu.edu Closing date Tora mavens /sMarch 31, 2010. 4% NEW YORK UNIVERSITYwww.uva.ni/vacancies ‘The Faculty of Science of the Univesity of Amsterdam attaches great importance tothe contribution af female scentists In research and education. For this reason we are offering: 5 MAcGILLAVRY FELLOWSHIPS FOR WOMEN (TENURE TRACK) os. sor in a concerted recruftment programme named after the ground breaking ‘rystallographer Carolina MacGllary, an UvA alumnus. We are looking {for women who excel in one ofthe faculty's disciplines and who aspire to a career as ful professor at the University of Amsterdam, ‘The scientific disciplines of the Faculty are + Blolagial science and Biomedical Science + Earth Sciences (Physical Geography) ‘Informatics and Logic ‘The fellomship comprises a tenure-track, whereby the fellow starts as Physics assistant professor (UD) in a temporary postion, with the prospect of + Chemisty ‘2 permanent appointment as associate professor (UHO) 2s well 3s 3 + Astronomy subsequent career path leading to @ fll professorship. Depending on the fellows qualifications, entrance ata higher levels also possible, ‘+ Mathematics and Statistics More information: At the website wwwwsclence uva.nimgt [At the website wwawiwva nlvacancies (subject: Maccillavey fellowship) We offer among other things a suitable startup package, and assistance in finding a job for your partner, child care and accommodation. Fellows ‘can ako count on excellent guidance fey University OF AMSTERDAM SE John Innes Centre FACULTY POSITION IN CANCER RESEARCH INDEPENDENT To] FELLOWSHIPS Inechonsmee! The eh nes Oznite (1), Norwich, UKs a wi leading centre tr epelene in pat and microbial sciences bao onthe Norah Research Park, We ate irwiting aplicatns from outstanding researcher wo ele ol, or wish to apy for Independent Research tub seercn Fellonshis, to afend a Corference at he JC on 7th June 2010. [Athe meeting yx wil ave an opty o present your pcposed orn mrs eaonme ie on usonangenstnervonmen deo shige) racwamortpios, Tu creer cand b ered bariepatomqrodatevomingstonrams. oautodqualeacns elles PMD tent ath adh rt espn Sr Brey eyo uc pr wre sos Aare Ces we il adel rote Canin wit top pests an rf Siootiy ore wt Flora te Ft nd paca edt ‘nde sncnoueppotariacnseMovadn Pe ns 29a stay fy sae 2 Sry adage re tre oc in ‘etiedwavsaetgin eib ay 2p 2010 en ord etre ip immo toa Gp mr tocraase fa. Gynt Enalger Teregeien oddone sth reser expense, nay andres rg Te open ea My 2010 ormaton ‘hestScecePancnbeloeedat pucenrprt dade) THELVEISTYOF TEAS MDANDERSON: CANCERCENTER Making Cancer History”kz. Shseeer “The Meal Facto the Unversity of Heidelberg ony with the German Cancer Reseach ener (KZ) ineter spelaton forthe fovowig postion Full Professorship (W3) for Molecular Oncology of Gastrointestinal Tumours (K.-H. Bauer Endowed Professorship) ‘he successful candidate wil be appointed ae Drector ofa research division atthe Geman Cancer sare cote (OR), wel 5 the Head of he secon of Paces Carnoma Resear st the Chi for General, scr and Farspanation Samer We encourage candidates with an exelent nemationa siti repvatin inthe fi of gastitestinal rsloge Eipedence Inthe Hasan of excelent Bese research to i Snngnee to paripae my ate enti ahr an resachprojee Deter the German neat Feseath Centr DKF, the Raion Center uma Oseases NT at the Surpcal Unies Hosa feteber Cancites fortis poston are requted to hos habitation (ostdacror lecture qualfeston) or Saubaent scenic qualification several Yeats of experience I te mening of Yseach BoUups and isbot procurement sna implomentason at ett hes {ee ondtns of eon ae et dou nS 7 ane scion, reo te den Weenie rata lw on high education (Landeshocs San Diego ° Presenting Sponsor OSA ‘The Optical Society By “ge @ weimnorrz Canada SUBARU | aSSOCIATION ak & = POT => te ponnecione UCSD yam THE ROYAL A SOCIETY ug PATHWAY GENOMICS THE 388 KAVLI FOUNDATION In addition generous funding for AAAS Awards is provided by the Kavli Foundation and Affymetrix. 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