Current Topics

RESEARCH ADVANCES

Genes not GenomesCalled Key Driver ofBacterial Diversity

Marcia Stone

Mutation-encoding genes can sweepthough bacterial populations, drivingdifferentiation, according to B. JesseShapiro and his colleagues at the Mas-sachusetts Institute of Technology(MIT) in Cambridge. Analysis ofgenomic sequences from two recentlydiverged bacterial populations fromdifferent habitats suggests that genomicfragments rather than whole genomesdrive differentiation. “Despite differ-ences in how adaptive alleles are ac-quired, our results suggest that theyspread in a more uniform mannerwithin both bacterial and eukaryoticpopulations than previously imag-ined,” these researchers say. Details ap-peared in the April 6, 2012 Science(336:48–51).

Shapiro and his collaborators fo-cused on closely related, ecologicallydistinct strains of Vibrio cyclitrophi-cus living on particles of differentsize and composition in the AtlanticOcean. The bacterial cells had identical16S genes and more than 99% averageamino acid identity,making them idealfor studying early ecological differenti-ation, according to Shapiro. The maingenetic differences between these twopopulations were restricted to a fewsmall patches within the core genomes.Tight genotypic clusters emerge as aresult of preferential recombinationwithin, rather than between, cells ineach of the habitats without genome-wide selective sweeps. “We are veryexcited about this fınding because itexplains the organization of microbial

communities into ecologically differ-entiated genotypic clusters,” says Mar-tin F. Polz, one of two principal inves-tigators (PI) with whom Shapiroworks.

Quickly adaptable genomes appearto be shaped far more by horizontalgene transfer (HGT) than by clonal de-scent, says the other PI, Eric J. Alma.A few genome regions appear to haveswept through subpopulations in ahabitat-specifıc manner accompaniedby gradual separation of gene pools,he adds. This work confırms their ear-lier research showing that genes, notgenomes, serve as major units of bacte-rial evolution and that ecology governsHGT.

Because of the high rate of HGTin bacterial populations, a signifıcantpercentage of genes from closely re-lated bacterial strains can have differ-ent evolutionary histories. However,the Vibrio strains that they studiedshared a common ancestor until rela-tively recently and, since then, about99%, of the genome recombined, Sha-piro notes. “This is very much Darwin-ian because it’s driven by selection ofecologically important genes.”

“There is no question that genesmove around between all kinds oforganisms, even us,” says Norman R.Pace at the University of Colorado inBoulder. “But it’s also clear that manygenes, those most critical to the nucleicacids-based processing and informa-tion systems, for example, are phyloge-netically stable in the sense that theirphylogeny tracks with the rRNA tree.The bacterial cell has a core of genes . . .which are pretty stable. The rest aremore or less volatile because of sweepsas is so nicely described by Shapiro andcolleagues.”

“This is very exciting work becauseShapiro and colleagues show that bac-teria may be asexual but they’re notsimply clonal,” says R. Thane Papke oftheUniversity ofConnecticut in Storrs.“Therefore, theories of diversity based

MINITOPIC

FDA Approves Measuresinvolving HIV plusCMV Test

Officials of the Food and Drug Ad-ministration (FDA) in July approvedthe use of the antiviral drug Truvada(emtricitabine/tenofovir disoproxilfumarate) for reducing the risk ofbecoming infected by HIV—makingit the first drug approved for pre-exposure prophylaxis for this virus.The drug is produced by Gilead ofFoster City, Calif. In a related devel-opment earlier in July, agency offi-cials approved the first home-usediagnostic test for HIV, called theOraQuick In-Home HIV Test, whichdetects antibodies to the virus insaliva, and gives a readout in lessthan 1 hour. The test sensitivity is92% and its specificity is more than99%, according to OraSure Technolo-gies of Bethlehem, Penn. In anotherdevelopment involving a diagnostictest, FDA approved the first DNA-based test for detecting cytomega-lovirus (CMV). The COBAS AmpliPrep/COBAS TaqMan CMV Test, which ismanufactured by Roche MolecularSystems in Somerville, N.J., is meantfor use by health care professionals,particularly for monitoring organtransplant recipients undergoingCMV antiviral therapy.

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on clonality are inaccurate and bacte-rial speciation is far more like that ofeukaryotes than previously thought.It’s a breath of fresh air in a debate thatsorely needs these observations.”Marcia Stone is a science writerbased in New York City.

RESEARCH ADVANCES

Shifts in MicrobiotaInfluence Mating Choicesamong Fruit Flies

Shannon Weiman

Although commensal microbes areverymuch in the scientifıc andmedicalspotlight, their influences may far ex-ceed our wildest expectations, judgingfrom research presented at the 2012ASM General Meeting, held in SanFrancisco, Calif., last June. Commensalmicrobes can influence behavior, sex-ual selection, and other evolutionaryprocesses, says Eugene Rosenberg ofTel Aviv University in Tel Aviv, Israel,who spoke during the plenary session,“Who’s in Charge? How Microbes Af-fect Animal Behavior.”

Some flies choose their mates basedin part on diet-dependent, gut micro-bial signatures, Rosenberg says. Dro-sophila melanogaster fruit flies thatfeed on starch-containing growth me-dium exhibit positive sexual selection,choosing tomate with each other whilerejecting flies that are reared on a dif-ferent growth medium, called basicCMY, and vice versa. Rosenberg revis-ited these experiments, adding anti-biotics to the growth media to probethe underlying mechanism. Antibiotictreatment abolished mating prefer-ence—starch-fed flies mated at ran-domwith CMY-fed flies despite havinglived for generations on different diets.

To Rosenberg, these results pointedto gutmicrobiota as the likelymediatorof the changes in fly mating behavior.Indeed, Lactobacillus plantarum, astarch-metabolizing bacterium thattypically accounts for only 3% of gut

microbiota in CMY-fed flies, had ex-panded up to 26% of gut microbiotain starch-fed flies. When L. plantarumwas added to antibiotic-treated, ran-domly mating flies, this single bacterialspecies restored positive sexual selec-tion, Rosenberg says.

How can a fly possibly respond tothe microbial content of its mate’s in-testinal tract? “The odor of many ani-mals results from microbial modifıca-tions of compounds secreted by thehost, or released by microorganismsthemselves,” note Rosenberg and hiscollaborators. In the case of fruit flieson different diets, the researchers fındsignifıcant differences in fıve cuticularhydrocarbon pheromones from theflies that play a major role in their mat-ing behavior. Treating flies with anti-biotics reduces overall levels of phero-mones and reduces differences incuticular hydrocarbon profıles be-tween flies fed CMY and those fedstarch. These studies point to gut mi-crobiota as the likely source of thesecompounds that dictate sexual behav-ior, and the observed differences be-tween starch- and CMY-fed flies,Rosenberg says.

The scent glands of spotted hyenasin Kenya typically contain commensal

anaerobic bacteria that produce vola-tile odorants, such as alcohols, ketones,and short-chain fatty acids that arecommon components of scent mark-ings, according to another speaker inthe session, Kevin Theis of MichiganState University in East Lansing.Whileall spotted hyenas harbor the samecommensal species, the proportions ofthese bacteria vary according to traitsof the individual host, including age,sex, pregnancy, and clan association.These unique microbial profıles gener-ate signature mixtures of volatile com-pounds that can be “read” by a pass-erby.

Odor influences sexual behaviorfrom insects to large mammals. Thesestudies provide evidence that microbescan influence odor-related mate selec-tion behavior. Moreover, microbe-mediated sexual selection can lead torestricted genetic exchanges betweenpopulations, allowing for genetic di-vergence and, potentially, speciation,Rosenberg says. Thus, microbes maybe driving evolution by influencingsexual selective pressure in host popu-lations.

Shannon Weiman is a freelance writer in SanFrancisco, Calif.

Drosophila melanogaster fruit flies use gut microbial signatures as part of their criteria forchoosing mates. (Image © janeff/iStockphoto.)

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RESEARCH ADVANCES

. . . Changes to Micro-and Mycobiome AffectSome Cancers, Too

Marcia StoneEndogenous microbes may help indriving or suppressing cancer develop-ment and may also affect therapy out-comes, according toGiorgio Trinchierifrom the National Cancer Institute inFrederick, Md., part of the NationalInstitutes of Health. He and other ex-perts spoke last June during a Presi-dent’s Research Seminar at the Rocke-feller Research Laboratories in NewYork, N.Y.

Several examples stand out. For in-stance, commensal gut bacteria aredirectly linked with colitis-associatedcancers, while several pro- and anti-inflammatory bacteria modulate othergastrointestinal malignancies. In addi-tion, Helicobacter pylori is the well-known instigator for gastric cancer,

part of a relationship that Trinchiericalls the “canonical example” of a ma-lignancy associated with a single spe-cies. These bacteria are thought to up-set intestinal microbiota homeostasis,causing a state of “dysbiosis,” accord-ing to Gerardo Nardone from FedericoII University in Naples, Italy.

Gut commensals typically are re-stricted to the intestinal lumen, its epi-thelial surface, or underlying lymphoidtissues. Even though well contained,however, “the sheer number of mi-crobes in the intestinal tract makesan occasional breach inevitable,” saysLora V. Hooper from the Universityof Texas Southwestern Medical Centerin Dallas. Escaping cells are typicallyquickly eliminated via phagocytosis.However, some translocated micro-organisms are carried to mesentericlymph nodes by dendritic cells (DCs),where they stimulate immune re-sponses that damage or destroy mi-crobes roaming the body. Thus, the gutmicrobiome “affects all phases of can-cer, from initiation at the single-celllevel to early growth, progression, anddissemination,” says Trinchieri. De-tails appear in the 8 June 2012 Science(336:1268–1273) and the 2012 AnnualReview of Immunology (30:677–706).

In contrast, some commensal bacte-ria enhance the anti-inflammatoryactivity of adaptive immunity by di-recting T-regulatory (TREG) cells andinducing expression of IL-10, saysHooper. When colonized with 46strains of clostridia, for example, IL-10-secreting TREG cells expand ingerm-free mice. In another case,commensal flora protect transgenicmice against mammary carcinoma;moreover, treating them with anti-biotics speeds tumor growth. Similarly,antibiotics also sometimes interferewith anticancer therapies, suggestingthat commensals somehow regulatean individual’s response to treatment,according to Trinchieri, who is study-ing this phenomenon.

In addition to bacteria, themamma-lian gut contains fungi—the myco-

biome—that also interact with the hostimmune system, including throughan innate immune receptor called Dec-tin-1. It can signal cells to produceinflammatory cytokines and induceT helper 17, according to David M.Underhill at the Cedars-Sinai MedicalCenter in Los Angeles. Mice lackingthe Dectin-1 immune receptor geneshow increased susceptibility to chem-ically induced colitis, which is a riskfactor for colitis-associated cancers.Details appear in the June 8, 2012 Sci-ence (336:1314–1317).

RESEARCH ADVANCES

Genome for First RNAVirus of ArchaeaTentatively Identified

John OtrompkeArchaea from acidic hot springs in Yel-lowstone National Park apparentlyharbor RNA viruses, according toMark Young ofMontana State Univer-sity in Bozeman and his collaborators.“Scientists previously discovered DNAviruses of archaea, but before ourwork,no RNA viruses were discovered,” hesays. “This was a big blank spot on thescientifıc map, but there’s no biochem-ical reason for them not to exist.”Young spoke about this fırst RNA vi-rus, which was identifıed indirectly viametagenomic analysis, during the 2012annual meeting of the Canadian Soci-ety of Microbiologists, held last June inVancouver, British Columbia. Detailsappeared earlier this year in the Febru-ary 29, 2012 Journal of Virology (doi:10.1128/JVI.07196-11).

“There are about 5,000 viral para-sites on this planet that are known,but . . . only 30 to 40 of those are fromarchaea,” Young says. The genes of vi-ruses that infect archaea are unusual,suggesting that eukaryotes share anevolutionary relationship with archaeathat is closer than that with bacteria,he says. However, because archaea ortheir viruses cause no diseases in hu-mans, animals, or plants of agriculturalinterest, there is little interest in study-

MINITOPIC

Silk-Based MaterialStabilizes Vaccines,Antibiotics

A silk protein-based material stabi-lizes several types of antibiotics andvaccines, enabling some of them towithstand storage at 140°F (60°C),for more than 6 months, accordingto David Kaplan of Tufts Universityin Medford, Mass., and his collabora-tors. The silk films wrap around theantibiotics and vaccines, protectingthem while extending their shelf-lives. The chemistry and structure ofthe silk protein provide a stabiliz-ing environment for such molecules,the researchers say. Additionally, thesilk films protect one of the anti-biotics tested against the detrimen-tal effects of light. Details appear inthe July 9, 2012 Proceedings of theNational Academy of Sciences doi:10.1073/pnas.1206210109.

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ing these viruses except among a hand-ful of scientists doing basic research.

Young and his collaborators reliedon metagenomic analysis to assembleRNA segments from viral popula-tions isolated from hot spring samples.One of the two putative RNA virusesin question has a genome containingapproximately 5,600 nucleotides. It aswell as another less fully analyzed par-tial genome in their samples includesa gene that appears to encode an RNA-dependent RNA polymerase (RdRp),which is considered a hallmark of pos-itive-strand RNA viruses. The RdRpgenes of archaeal viruses “form aunique group distinct from the RdRpsofRNAviruses of Eukarya andBacteria. . . and even more distant from knownbacterial RNA viruses,” he and his col-laborators note. “These positive-strandRNA viruses might be direct ancestorsof RNA viruses of eukaryotes.”

Viruses of archaea were fırst identi-fıed in Japan, then later in Iceland,Kamchatka in Russia, and also in Las-sen Volcanic National Park in north-ern California as well as in Yellow-stone, according to virologist KennethStedman of Portland State Universityin Oregon. “The results in Young’swork are very exciting,” he says, while

also cautioning that the evidence forthis RNA being the genome of an ar-chaeal virus is indirect. “Right now allthey have is sequences,” he says. “Theyfound RNA in an ecosystem wherethere are quite a lot of archaea, butthere are certainly not only archaeapresent there.”Whether their genomesconsist of DNA or RNA, he adds, anyviruses that infect archaea should notbe called archaea “phage,” arguing thatthis term should remain reserved forthe viruses that infect bacteria.John Otrompke is a writer based in Chicago.

RESEARCH ADVANCES

Seeking Insights intoResistance to Two Antibiotics

Jeffrey L. FoxA longstanding riddle underlying theresistance of particular bacterial patho-gens to polymyxin B—itself a vin-tage antimicrobial peptide—yielded toanalysis revealing a newly recognizedself-modifıcation process in gram-neg-ative bacteria, according to StephenTrent of the University of Texas (UT)at Austin and his collaborators. Mean-while, a clinical trial evaluating a newboron-containing antibacterial agent,

designated GSK2251052 (also calledGSK052 or AN3365), was suspendedearly this year when patients with uri-nary tract infections being treated de-veloped resistance to this experimentaldrug, according to ClinicalTrials.gov,

Hot springs in Yellowstone National Park have been important sites for research into Archaea;now researchers have found RNA viruses for the first time in archaea living there. (U.S. ParkService photo.)

MINITOPIC

Influenza Virus CarriesModulator Gene;Flu Dose Matters

The influenza virus encodes a gene,designated PA-X, that modulates aninfected host’s immune responses,according to Paul Digard of theRoslin Institute at the University ofEdinburgh, Scotland, and his collab-orators at several institutions in theUnited Kingdom and the UnitedStates. ”The flu virus has a very, verysmall genome—just 12 genes,” saysone of those collaborators, AndrewFirth of the University of Cambridge.“Finding a new gene makes a prettysignificant change to our under-standing of this virus.” That gene,within segment 3 of the viral ge-nome, is part of a second openreading frame that is accessed viaribosomal frameshifting. When ac-tive, it decreases pathogenicity inmice but, if unexpressed, leads toincreases in host inflammatory re-sponses as well as in apoptotic andT cell-signaling pathways. Detailsappear in the July 13, 2012 Sciencedoi: 10.1126/science.1222213. Sepa-rately, the number of flu particlesinvolved in causing an infection af-fects its course, according to MartinRichter of the Université de Sher-brooke and Centre de RechercheClinique Étienne-Le Bel in Québec,Canada, and his collaborators. Forinstance, high viral concentrationsstimulate a better host immune re-sponse and broader protectionagainst other flu strains. Details ap-pear in the July 2012 Journal ofLeukocyte Biology (doi:10.1189/jlb.1011490).

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which is maintained by the NationalInstitutes of Health.

Soon after Anacor Pharmaceuticalsof PaloAlto, Calif., developedAN3365,the company entered a partnershipwith GlaxoSmithKline (GSK), head-quartered near London, United King-dom, to bring the promising com-pound into clinical trials. The boron-containing molecule blocks proteinsynthesis by inhibiting aminoacyltRNA synthesis in gram-negative bac-teria—specifıcally, by binding to theediting domain of particular tRNAmolecules and interfering with addi-tions of the amino acid leucine (Mi-crobe, November 2010, p. 466–468).Currently, GSK researchers are con-tinuing to study GSK052 and intendto publish their results, according toJohn Tomayko, who is GSK SeniorDirector of Infectious Diseases Medi-cine Discovery and Development inCollegeville, Pa.

More than 50 years ago, researchersrealized thatVibrio choleraeO1 El Tor,unlike close relatives of this gram-neg-ative bacterium, is resistant to poly-myxin, but the mechanism underlyingthis resistance remained a mystery,according to Trent and his collabora-tors. Recently they learned that the ElTor strains of V. choleraemodify com-ponents of their cell wall—specifıcally,adding the amino acid glycine orthe dipeptide diglycine to the lipid Aanchor of lipopolysaccharide. Suchchanges confer a neutral instead ofnegative charge to the cell surface,thereby rendering these bacteria resis-tant to the cationic polymyxin B pep-tide, the UT researchers report. Detailsappear in theMay 29, 2012 Proceedingsof the National Academy of Sciences(doi:10.1073/pnas.1201313109).

Resistance to polymyxin sets El Torapart from classical O1V. cholerae bio-types, and acquiring this resistance“could be a key for fıtness,” helping toexplain the worldwide emergence of ElTor, which is the pathogen responsiblefor the current—and seventh—cholerapandemic, Trent says. In 2010, 48

countries reported, in total, more than300,000 cases of cholera, mainly inthe Americas, particularly Haiti, and inAfrica, according to the World HealthOrganization, which points out thatthese fıgures vastly underestimatecases, particularly from Asia.

A key difference between those twobiotypes is that classical strains carry amutation that apparently blocks themfrom transferring glycyl residues to thelipid A anchor of LPS, according toTrent and his collaborators. Another“cool” feature of this resistancemecha-nism is that it closely resembles whathappens within many gram-positivebacteria, in which the amino acid D-

alanine, when added to outer-surfaceteichoic acids, can confer resistance topeptide antibiotics, he says. Thus, bothgram-positive and –negative bacteriaappear to share a “charge-based [sur-face] remodeling strategy.”

The El Tor fındings are “quite inter-esting, and I like the connection to thegram-positive modifıcation of teichoicacid,” says Lynn Silver, a New Jersey-based consultant who focuses on anti-biotics. “Resistance to polymyxin by avariety of gram negatives is becoming ahot topic.”

Jeffrey L. Fox is the Microbe Current Topics andFeatures Editor.

RESEARCH ADVANCES

Marine CyanobacteriumProduces Novel, Dual-ActingDrug Prospects

Carol Potera

A cyanobacterium that resides in coralreefs produces chemical compoundswith both anti-inflammatory and anti-infectious properties, according toWilliam Gerwick of the Scripps Insti-tution of Oceanography in San Diego,

Darkly colored cyanobacteria overtake a Hawaiian coral reef. While the cyanobacteria aredamaging to the reefs, researchers have found that the cyanobacterium Leptolyngbya crossbyanaproduce compounds with anti-inflammatory and anti-infective properties. (Photo © Jennifer E.Smith, Center for Marine Biodiversity and Conservation, Scripps Institution of Oceanography,University of California, San Diego.)

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Calif. Potential therapeutics could bedeveloped for either indication, but aproduct with both capabilities “wouldbe a very powerful combination,” hesays, citing as an example an acne treat-ment that would block microbial colo-nization while reducing inflammationof the skin.

Mats of the cyanobacterium Lep-tolyngbya crossbyana were growingonto and overwhelming coral reefs inHonaunauBay inKona,Hawaii, a pop-ular dive site, at least as early as 2008,says his Scripps colleague JenniferSmith. Those bacterial blooms weresmothering and bleaching corals onwhich they grew, while damagingnearby corals with which they had no

direct contact. This damage at a dis-tance suggested to Smith that L. cross-byanamay be leaching toxic chemicalsthat can diffuse through sea water,prompting her to send samples to Ger-wick for chemical analysis.

Postdoctoral researcher HyukjaeChoi isolated three natural productsfrom L. crossbyana, which were namedhonaucin A, B, and C in honor of thebay where the cyanobacteria that pro-duce them were collected. The hon-aucins consist of hydroxy-butyrolac-tone joined via ester linkage tochlorocrotonic acid.

All three of these honaucins blockquorum sensing, whichwas a surrogatemeasure of their anti-infectiveness in a

dose-dependent manner, according toGerwick. The compounds were testedin Vibrio harveyi BB120 cultures thatuse bioluminescence as a marker forquorum sensing. When tested inmouse macrophage cells for anti-in-flammatory activity, the honaucins re-duce nitric oxide levels and the subse-quent stimulation of proinflammatorycytokines, especially interleukin-1�, hesays. Promising though these fındingsmay be for humanmedicine, they offerno clues about how to protect coralreefs against damaging cyanobacteria,he adds, noting that environmentalpollutants are the likely stimulus forgrowth of those cyanobacteria.

Several brominated and iodinatedhonaucins are being evaluated forchanges in their anti-infective andanti-inflammatory activities, Gerwicksays. For example, one such analog,4�-bromohonaucin, has enhanced ac-tivity and is being further developed.Details appear in the May 25, 2012Chemistry & Biology [doi:10.1016/j.chembiol.2012.03.014].

“Marine natural products offer theunique opportunity to discover novelmechanisms of action that can betranslated into treating human dis-ease,” says Keith Glaser, an adjunctprofessor of pharmacology at Mid-western University in Downers Grove,Ill. The honaucins offer a novel scaffoldfor blocking inflammatory mecha-nisms and pathogens that drive inflam-matory responses, he says. Moreover,Gerwick and his collaborators took thetrouble early on to address what hasproved a major shortcoming for manyothers seeking to develop natural prod-ucts—ensuring an adequate supply ofraw materials—by isolating honaucinsand developing derivatives for furtherdevelopment, he adds. “This maintainsour natural marine resources for thefuture discovery of more novel com-pounds.”

Carol Potera is a freelance writerin Great Falls, Mont.

MINITOPIC

Human Microbiota Data Bonanza

During June and July, researchers from a variety of universities and otherinstitutions who are analyzing the human microbiome released their latest data ina giant wave. Highlights from reports delivered during the 2012 ASM GeneralMeeting, held in San Francisco, Calif., last June or published in the June 8, 2012Science, June 14, 2012 Nature, and several journals within the Public Library ofScience include:

• More than 10,000 microbial species were found among 242 healthy U.S.volunteers—129 males and 113 females—in samples collected from 15anatomic sites in men and 18 in women (including three vaginal sites),according to officials from the National Institutes of Health (NIH), who areoverseeing the Human Microbiome Project.

• The human microbiome contributes some 8 million protein-coding genes—about 360 times more bacterial than human genes per individual host, whoseown genome is endowed with about 22,000 protein-coding genes.

• The gut bacterium Bifidobacterium dentium proves capable of secreting largeamounts of �-aminobutyric acid (GABA), a neurotransmitter of both the hostenteric and central nervous systems, according to Karina Pokusaeva andJames Versalovic of Baylor College of Medicine in Houston, Tex., and theircollaborators. GABA modulates pain and may also inhibit inflammation, theysay.

• The nasal microbial populations of patients with chronic sinus conditions aredepleted of diversity and frequently overgrown with Corynebacterium spp.,according to Nabeetha Nagalingam of the University of California, SanFrancisco, and collaborators.

• Mice colonized with bacteria ordinarily found in the human gut developimmune responses much like those of germ-free mice, suggesting that gutmicrobiomes are very much attuned to and may function properly only whenmatched with their appropriate host species, according to Dennis Kasper ofHarvard Medical School in Boston and his collaborators. Their findings appearin the June 22, 2012 Cell doi:10.1016/j.cell.2012.04.037.

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