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Epigenomics and sequencing: an intertwined and emerging big science of the next decade

Epigenomics and Sequencing 2007 Meeting on 'Chromatin Methylation to

Disease Biology and Theranostics, July 9–10 2007, Harvard Medical School,

Boston, MA, USA

Krishnarao Appasani

GeneExpression Systems, Inc., PO Box 540170, Waltham, MA 02454, USA

Tel.: +1 781 891 8181;

Fax: +1 781 891 8234;

E-mail: drappasani@expressgenes.com

The ‘Epigenomics and Sequencing2007’ meeting was the first inter-national event of a themed conferencethat was designed to focus on ‘Chro-matin Methylation to Disease Biology& Theranostics, as well as scientific andbusiness collaborations. The meetingwas organized by GeneExpression Sys-tems (MA, USA) at the prestigious Har-vard Medical School campus in Boston,MA, USA on July 9–10, 2007. The2-day intensive single-track meeting wasarranged in eight scientific sessions,attracted 100 scientists, and broughttogether industry leaders and entrepre-neurs, renowned international scientistsfrom academia, who contributed semi-nars, and product presentations that dis-played the latest tools in epigenetics andsequencing research. This report coversrepresentative presentations fromacademia, and from the biotechnologyand pharmaceutical industry.

The late Conrad Waddington, thelast Renaissance evolutionary biologist,coined the word ‘epigenetics’ in 1942.Epigentics is the phenomenon thatdescribes “the heritable changes in geneexpression that are not due to changesin DNA sequence”. Most of the non-heritable signals are resided and control-led by chromatin, which is the complexof DNA and protein that makes upchromosomes. Chromatin is easily visu-alized by cytological stains, hence itsname, which literally means coloredmaterial. Walther Flemming first usedthe term chromatin as follows:

“the nuclear-scaffold owes its capability of refraction, the way it behaves, and in

particular its colorability to a substance which, with regard to its latter attribute, I have termed chromatin. It is possible that this substance is really identical with the nuclein-bodies. … I’ll retain the name

chromatin as long as chemistry has decided about it, and I empirically refer to it as that substance in the cell’s nucleus that takes up

the dye upon staining the nucleus’ ‘Kerntinktionen’.”

Cytological stains distinguish the chro-matin compactness into euchromatinand heterochromatin. Chromatin thatpresents in a tight condensed form isalso known as heterochromatin (bio-logically inactive), and the non-condensed or extended loop form iscalled euchromatin, which is the tran-scriptionally active form. The nucleo-some is the fundamental unit ofchromatin structure, which comprises acore of eight histones around which146 bp of DNA are wrapped in 1.75spherical turns. Histones influenceevery aspect of DNA function. Thefunctions of chromatin are to packageDNA into a smaller volume to fit in thecell, to strengthen the DNA to allowmitosis and meiosis, and to serve as amechanism to control gene expression.Histones that are present in nucleo-somes undergo two biochemical modifi-cations: methylation and acetylation.The structure and function of chrom-atin varies considerably as the cell

10.2217/14622416.8.9.1109 © 2007 Future Medicine Ltd ISSN 1462-2416 Pharmaco

progresses through the cell cycle, by par-ticipating in DNA replication, repairand damage. In the past, three Nobelprizes were awarded (to Thomas Mor-gan, 1933, Aaron Klug, 1982, andRoger Kornberg, 2006) in the chroma-tin field for the elucidation of functionand structure. However, its involvementin development, differentiation, diseaseand genomic imprinting is still unclear,and a mystery to biologists.

In order to understand the recentdevelopments in chromatin biology andits emerging roles in various patho-logical states, this themed conferencewas organized. This meeting differenti-ates from other events owing to its care-ful integration of relevant emergingsequencing technologies, which is oneof the strong technology platforms thathas been continuously used today byvarious scientists to decipher the code ofepigenomes in normal and cancer cells.The potential of epigenomic research isseemingly unlimited. With this tool,scientists and clinicians are able to focusnot just on finding better treatments,but also on finding cures for many ofthe diseases ailing the world today,including cancer, neurodegenerative dis-eases, autoimmunity and inflammation.Modern biomedical science is finallybringing together the intellectual forcesof international academic researchers,industry scientists, and clinicians. Suchcollaborations are of high relevance foran emerging science such as epigenetics,and epigenomics holds great potentialfor therapeutics and understanding ofdevelopment and diseases.

Epigenetics is one of the fundamentalmechanisms that is involved in embryodevelopment and differentiation of celltypes. Applications of genomicsapproaches in the study of epigenetics to‘study the totality of epigenetic marks ina given cell type’ is now popularly termed‘epigenomics’. Epigenetic modifications

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provide a cellular memory for transcrip-tional control in all cell types. Theseoperate in four mechanisms such as:RNAi, histone code, nucleosomalremodeling, and CpG methylation.There is a growing body of evidence thatthe impact of epigenetic imbalances goesfar beyond cancer. Altered methylationpatterns have been reported in severalhuman diseases such as cardiovascular,diabetes, neurodegenerative and inflam-matory diseases. This field has an impactin the modern biomedicine and com-mercial enterprise for the development ofnew theranostics (therapeutics anddiagnostics) for several human diseases.

Chromatin biologyThe conference was opened by ShujiOgino (Brigham & Women’s Hospital,Boston, MA, USA), a physician-scien-tist, who gave an overview on the epi-genomic profiling of colorectal cancer(CRC). He summarized the ‘NursesHealth Study’, an epidemiology study inwhich approximately 121,000 patientsparticipated, those who were exposed toseveral epidemiological factors (such asdiet, lifestyle, family history and so on)among those several molecular altera-tions were observed. His group’s workalso focused on the study of CpG islandmethylator phenotype (CIMP) in CRC.To discriminate biologically significantmethylation from insignificant (low-level) methylation, methylation-specificreal-time TaqMan® PCR (MethyLight)quantitative assays were carried out inhuman tumor tissues. Quantitative PCRon carefully selected CpG islands ena-bled them to precisely diagnose CIMPstatus in a cost-effective way, and helpedto assess effects of CIMP on clinical out-comes – patient survival and treatmentefficacy. It was also observed that therewas a high incidence of microsatelliteinstability in 15% of CRCs. Folate B6and B12 were shown to prevent CRCpossibly through the prevention of DNAmethylation. Ogino, in collaborationwith others, developed 16 marker panels(some including RUNX3, CACNA1G,IGF2, MLH1, NEU-ROG 1 and so on)for determining the status of survival inCRC patients.

siRNAs targeted to gene promoterscan direct epigenetic modifications thatresult in transcriptional gene silencingin human cells. It is not clear whetherthe antisense strand of the siRNAsbinds DNA or an RNA transcript cor-responding to the promoter region.Kevin Morris (Scripps Research Insti-tute, La Jolla, CA, USA) presenteddirect evidence that promoter-specificRNA, a new species of previouslyuncharacterized low-copy RNApolymeraseII expressed RNAs, isrequired for siRNA-directed epigeneticmodifications and transcriptionalsilencing of siRNA-targeted promoters.His results clearly demonstrated that anRNA component is involved in writingthe histone code. Additionally, distinctmechanistic differences between tran-scriptional gene silencing and post-transcriptional gene silencing werereported in human cells by ChristopherAdams (Invitrogen Corporation,Carlsbad, CA, USA). Adams observedan epigenetic off-target effect at thelocal chromatin of the correspondinggene. Heterochromatin is an alteredchromatin structure that is epi-genetically inherited and plays impor-tant roles in gene regulation andgenome stability. The long-standingparadigm that heterochromatin is tran-scriptionally inert has been challengedby recent studies suggesting that RNAi-dependent gene silencing in hetero-chromatin can be mediated primarilyby degradation of nascent transcriptswithin these domains. Marc Bühler(Harvard Medical School) presentedhighly compelling data and convincedall the audience that transgenes insertedin heterochromatic repeats are directtargets of RNAi. Additionally, a role foranother RNA-degradation pathwaythrough TRAMP (involving its subunitCid14) and exosome complexes inrobust silencing of heterochromaticgenes was also presented.

Chromatin biochemistryIt is generally recognized that disruptionor remodeling of chromatin structuremay be a prerequisite step for most of theDNA transactions. Original models for

the mechanistic role of post-translationalhistone modifications proposed thatmarks on histone tails might directlyinfluence chromatin structure. CraigPeterson (University of MassachusettsMedical School, Worcester, MA, USA)adopted new chemical strategies using‘chromatin remodelers’ and concludedthat the ‘code’ has to be read in the con-text of each gene’s regulatory circuitry.He also developed a ‘native chemicalligation’ method for ‘designer histones’in order to generate full-length proteinsfrom unprotected peptides. In addition,his group had showed ‘a nail of twonails’ approach using H3510-phos(coats mitotic chromosomes) andH4K16Ac (a hallmark protein for thedetermination of dosage compensationin flies) – H4 tail is indeed required forchromatin folding in vitro. In conclu-sion, the results indicate that only asmall number of histone marks (e.g.,H4K16Ac) directly regulate chromatinstructure. Peterson also developed ‘anarray of 12 nucleosomes’ to study theassembly of nucleosomes in solutionstate using electron microscopy.

Cytosine methylation could beinheritable, but inheritance of histonemodifications is questionable. Histoneproteins not only regulate the chroma-tin structure, but also recruit remode-ling enzymes. These studies can beexplored in depth by using the chroma-tin immunoprecipitation (ChIP) tech-nique in which purified DNA can befragmented and immunoprecipitatedwith antibodies specific for histone pro-teins. Bradley Bernstein (MassachusettsGeneral Hospital, Boston and BroadInstitute of Harvard and MassachusettsInstitute of Technology, Cambridge,MA, USA) used genome-wide mappingapproaches to dissect chromatin andgenerate chromatin state maps by a sin-gle-molecule-based DNA deep-sequencing method (Solexa, now partof Illumina, Inc., San Diego, CA,USA), and accurately reproduced thearray data. According to him, ChIP-seqtechnology is inexpensive (US$1000per sample) and powerful in identifyinggenome-wide repeats and alleles, andrequires only nanogram quantities of

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DNA. The Bernstein approach helpedto reach the following six conclusions inembryonic pluripotent stem cells: • Helped distinguish CpG-poor

(K4me3 = active) and CpG-rich(K4me3 + K27me3 = repressed)promoters;

• In embryonic stem cells, CpG-richpromoters (bivalent in nature) becamerepressed and specified lineage-specificgenes; however, they became active inmultipotent stem cells;

• Genome-wide K4me3 and K36me3identified genes and promoters thatare in use;

• K9me3 localized at telomeres,satellites and long-term repeats;

• It determined allele-specific read-outsfor chromatin modifications;

• It helped to identify sequencedeterminants of the epigeneticreprogramming.DNA methylation is one of the

important mechanisms that play signifi-cant roles in mammalian development,maintaining a stable genome, providingsignal(s) for selective gene silencing andserving as an epigenetic marker forgenomic imprinting in mammals. Thebiology of genomic imprinting wasreported for the first time during theearly 1980s as an important fundamen-tal process limited to development –later it was reported in many forms ofcancers. Recently, it was observed in sev-eral pediatric diseases such as Beckwith-Wiedemann syndrome (overgrowth andmetabolic defects), Algelman syndrome(speech impairment, mental retarda-tion), Prader Willi syndrome (obesityand muscle hypotonia) and transientneonatal diabetes. DNA methyltrans-ferases (such as Dnmt1, Dnmt3a andDnmt3b) are important genes those areinvolved in DNA hypomethylation andcause global gene activation. LaurieJackson-Grusby (Children’s Hospital,Boston, MA, USA) presented a system-atic strategy to test the loss of imprint-ing model using knockout mice,chimera analysis and observed that tran-sient demethylation caused paternaloverexpression. Using Ptch-p35 double-mutant mice studies, she also presented

that epigenetic changes owing to bothDNA hyper- and hypo-methylationcould promote tumorigenesis, possiblyby the silencing of tumor suppressorgenes. She concluded her presentationwith some unanswered questions, suchas: what are the endogenous mecha-nisms of ‘epigenetic instability’? Doesthis equate with plasticity? Will study-ing epigenetic regulators of plasticityhelp to develop targets for therapeutics?

Tumor suppressor genes such as p53may be inappropriately modified in can-cer cells. Therefore, understanding theirpathways could lead to the identifica-tion of drug targets. To determine therole of histone demethyl transferases,lysine methylation was explored byFrancois Gaudet (Novartis Institutes forBiomedical Research, Cambridge, MA,USA) using a knockout mice approach.He created Set7/9 mice and demon-strated that reduced Set7/9 caused loss ofp53 function. The mice study demon-strated that loss of a single Set7/9 alleleleads to transformation susceptibility,which can be extended to human cancers.This opens a great opportunity todevelop histone-modifying enzyme-baseddrug targets.

PharmacoEpigenomics:To aid the development of high-throughput assays for drug-discoveryprojects, Jeffrey Falk (Aviva SystemsBiology, San Diego, CA, USA)presented a novel promoter array tech-nology, the chromatin immuno-precipitation–DNA selection andligation method. This method facili-tated genome-wide profiling of epi-genetic modifications, DNAmethylation sites and transcription fac-tor/promoter interactions involved invarious pathways. His presentation alsocovered global promoter mapping andlocus tiling arrays to provide newinsight into transcriptional and epige-netic mechanisms involved in cancerand stem cell differentiation. Develop-ment of histone deacetylase inhibitors(HDACi) based on small molecules isthe main interest of several biotechnol-ogy and pharmaceutical companies. Jef-frey Besterman’s (MethylGene Inc.,

Montreal, Canada) presentation coveredthe development of one such drug can-didate, MGCD0103, which is underclinical trials.

Marina Bibikova (Illumina, Inc., SanDiego, CA, USA) and her colleaguesdeveloped a DNA-methylation detec-tion method based on genotyping ofbisulfite-converted genomic DNA. Inthis assay, non-methylated cytosines areconverted to uracil when treated withbisulfite, while methylated cytosinesremain unchanged. The detection of themethylation status of a particular cyto-sine in the genome can be carried outusing a genotyping assay for a C/Upolymorphism. The GoldenGate Assayfor methylation was developed by Illu-mina and combines a high level of assaymultiplexing and scalable automationfor sample handling and data processingwith a miniaturized bead-based arrayplatform. Bibikova also discussed therecently launched Methylation CancerPanel I to interrogate 1505 CpG sitesselected from 807 genes. The methyla-tion data generated by array was vali-dated by methylation-specific PCRs andbisulfite sequencing. She concluded thatthe GoldenGate® Assay for methylationwill provide powerful insight intoepigenetic mechanisms of gene regula-tion that can be applied to diagnosis,prognosis and treatment of diseases.

Technology developmentDNA-methylation analysis based onbisulfite conversion can provide detailedquantitative information about methyla-tion patterns. Benjamin Schroeder(Applied Biosystems, Inc., Foster City,CA, USA) presented a capillary electro-phoresis-based sequencing method.Using Pyrosequencing™ methylationanalysis, Matthew Poulin (EpigenDx,Inc., Worcester, MA, USA) quantita-tively analyzed both global and gene-specific methylation (MGMT, p16 andRASSF1) of DNA from different tissuepreparations, as well as a variety of celllines. Rene Cortese’s (Epigenomics AG,Berlin, Germany) presentation coveredthe DNA-methylation profiles ofhuman chromosomes 6, 20 and 22 in 12different healthy tissues and primary

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cells by direct bisulfite sequencing. Theanalysis of changes in DNA methylationis challenging owing to the lack of stand-ardized methods for providing reproduc-ible data from limited sample material(e.g., patient biopsies). Gerald Schock(Qiagen, GmbH, Germany) presentedan improved methylation analysisthrough prevention of DNA fragmenta-tion during cytosine conversion. SteveRapko and his colleagues (GenzymeBiosurgery, Cambridge, MA, USA) ana-lyzed DNA from 47 strains of chondro-cytes and 18 strains of synoviocytes atvarious passages in monolayer culture, aswell as DNA derived from cartilage tis-sue. Using bisulfite sequencing in con-junction with a statistically derivedclassifier system, he analyzed DNAmethylation over seven genomic regions.

Julia Schliwka (Febit biotech GmbH,Heidelberg, Germany) presented amicrofluidic Geniom one® technologyplatform that is based on an on-chipformat. Geniom primer arrays presentthe key solution for large-scale sequenc-ing upstream handling, targeting anydesired fragment within large genomesor complex sample compositions. Epi-genetic states guide the interpretation ofthe genome. The heritable modifica-tions of DNA and DNA-associated pro-teins involved are end points ofintracellular signaling, and are thus thelink between the genome and the sur-rounding environment; intra- as well asextra-cellular. DNA-methylation is animportant part of the epigenetic patternand can be used to monitor physio-logical and pathological states. TomasEkström (Karolinska Institutet, Stock-holm, Sweden) developed a new tech-nique, the luminometric methylationassay (LUMA), to show that globalDNA methylation can be used formortality prognosis in chronic kidneydisease. Investigations in vitro alsodemonstrated that inhibition of histonedeacetylation caused rapid global andspecific demethylation. MenzoHavenga (Crucell Holland BV, Leiden,The Netherlands) presented an innova-tive STAR technology platform forexpressing recombinant proteins ofepigenetic interest.

Sequencing approaches in epigenomicsAlex Meissner (Whitehead Institute forBiomedical Research of MIT,Cambridge, MA, USA) presented ashotgun bisulfite sequencing approachusing 454 or Solexa (now part of Illu-mina). These sequences were thenaligned and mapped back to the refer-ence genome. For genome-wide meth-ylation analysis of embryonic cells, hisgroup constructed shotgun bisulfitesequencing libraries from randomlysheared fragments. He concluded thatshotgun bisulfite sequencing datadetected biologically relevant epigeneticDNA methylation markers. Industrykeynote speaker Maithreyan Srinivasan(454 Life Sciences–Roche Diagnostics,Branford, CT, USA) presented the 454Sequencing™ (pyrophosphate-basedsequencing optimized for beads in pico-liter wells) platforms that involvedsubjecting template DNA to solid-phase amplification onto beads inemulsions. Beads that contain ampli-fied template DNA are deposited intothe wells of a picotiter plate, and anaverage of 100 or 250 bases weredetected per read. In addition, he pre-sented data obtained from investiga-tions that detect rare drug-resistantHIV mutants in patient samples.

Amit Meller (Boston University, Bos-ton, MA, USA) developed a novelmethod for high-throughput single-mol-ecule DNA sequencing using ‘nanoporearrays’. This method involves two steps.First, the DNA is enzymatically con-verted to an alternative form, in whicheach nucleotide is systematically substi-tuted with two short, specific segments(∼10 nt long), named ‘base-units’. Thistransformation magnifies the footprint ofeach of the four different nucleotides inDNA, facilitating the hybridization oftwo-color fluorescent tags to each of thebase units in the converted DNA. Sec-ond, the converted DNA is electro-phoretically threaded through a2 nm-nanopore fabricated in thin solid-state membranes, mounted on top of amicroscope capable of resolving individ-ual fluorophores. When the DNA isthreaded through the pore, the tags are

stripped off, one at a time, in an‘unzipping’ process that his team haswell characterized. In the near future, hisgroup aim to develop high-density arrays(typically 100 × 100 nanopores on a100 µm × 100 µm chip), yielding aDNA-sequencing throughput of approx-imately 2.5 Mbase/s. Cytosine methyla-tion changes are stable and thought to beamong the earliest events in tumor-igenesis. Theoretically, DNAs carryingtumor-specifying methylation patternsescape the tumors and may be found cir-culating in the sera from cancer patients,thus providing the basis for developmentof noninvasive clinical tests for early can-cer detection. Indeed, using PCR-basedtechniques, several groups reported thedetection of tumor-specifying methyl-ated DNA in the sera from cancerpatients with varying clinical success.However, by design, such analyticalapproaches allow assessment of the pres-ence of molecules with only one methyla-tion pattern, leaving the bigger pictureunexplored. Therefore, Jeffrey Jeddeloh(Orion Genomics, LLC, St Louis, MO,USA) presented a ‘massively parallelbisulphite pyrosequencing’ method thatrevealed the molecular complexity ofbreast cancer-associated cytosine-methyl-ation patterns obtained from tissue andserum DNA.

Mathias Ehrich (Sequenom, Inc.,San Diego, CA, USA) presented resultsfrom a study using a mass spectrome-try-based method that allowed large-scale quantitative methylation profil-ing with resolution at the individualCpG level. He applied this method in ahypothesis-driven approach andselected more than 500 candidate pro-moter regions for methylation analysisand demonstrated the effectiveness ofcandidate gene approaches in thediscovery of novel epigenetic biomark-ers. Alon Goren (Hebrew UniversityMedical School, Jerusalem, Israel) sum-marized his group’s results on DNA rep-lication timing of the human β-globindomain, which was controlled by his-tone modification at the origin, suggest-ing that histone modification at theorigin serves as a switch for controllingreplication timing.

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Epigenomics in disease biologySkin is the most commonly exposedorgan to cancer. David Fisher (Dana-Far-ber Cancer Institute, Boston, MA, USA)summarized his objectives in understand-ing the melanoma, or skin cancer.Melanocyte-inhibiting transcription fac-tor (MITF) plays an important role inthis particular carcinogenesis. Using ‘lossof function’ and ‘gain of function’ ofMITF experiments allowed his team toidentify several proliferation/survivalgenes (such as Bcl2, CDK2 and c-met ).To understand the localization of thesegenes on chromatin, his team hadadopted a mechanistic ‘nucleosomal posi-tioning’ approach. In addition, his group,in collaboration with NimbleGen Sys-tems (Madison, WI, USA) screened anarray and examined approximately3600 human promoters; these were fur-ther validated by the H3 ChIP method.In conclusion, MITF regulates theexpression of the silver gene in melano-cytes; however, it does not operate innon-melanocytes. Manel Esteller (Span-ish National Cancer Center, Madrid,Spain) highlighted ‘hyper methylome’ ofcancer cells. He demonstrated that micro-RNAs such as miR-124a are silenced bymethylation. Using bisulfite sequencing,he presented most compelling evidencethat the DNA methylome of humanviruses (such as EBV, HBV and HPV16)are dynamically changing as the diseasesprogress. Identification of genetic andenvironmental causes of complex

diseases, such as schizophrenia, multiplesclerosis, diabetes, cancer and numerousothers, seems to be a much more compli-cated task when compared with cloningthe genes in simple Mendelian condi-tions. The slow progress in research ofcomplex diseases could be due to limita-tions of the basic strategy. It can be arguedthat, in comparison with DNA sequence-based factors, epigenetic changes are moreconsistent with the non-Mendelianaspects of complex diseases. Artturas Pet-ronis (University of Toronto, Toronto,Canada) used a 12K CpG island micro-array-based DNA methylation profilingmethod and observed a number of epige-netic differences in the brains of individu-als affected with major psychiatric diseaseversus controls, which were then verifiedusing bisulfite sequencing.

Epigenetic markers exhibit highsensitivity and specificity for differenttumor types and can be assayed inbiofluids and other specimens collectedby noninvasive technologies. MukeshVerma (National Institutes of Health,Bethesda, MD, USA) highlighted themission goals of the Cancer Institute insupporting research and developmentof appropriate diagnostic assays and‘overall roadmap’ approaches of theNIH in funding projects in theepigenetics area.

Outstanding accomplishmentsOn behalf of the scientific committee,Krishnarao Appasani (GeneExpression

Systems) honored Manel Esteller, thekeynote speaker, with an ‘EpigenomicsInnovator Award’ for his contributions onthe study of epigenomes of cancer cells.

Final thoughtsThe message from the meeting is thereare a lot of issues that have to beaddressed before we take histonedeacetylase-based therapeutics into theclinic to treat human diseases. Most ofthe attendees felt that this was a“unique, coherent, well-organized, tar-get meeting for learning cutting-edgetechnology and meeting authorities inthe epigenomics and sequencing field”.Additionally, attendees felt that themeeting had a great mix of industryand academia and enabled a goodexchange of research results and poten-tial collaborative opportunities. I hopethat the potential applications of epige-nomics research in basic biology, agri-culture and biomedicine will become areality in the coming years; however,development of drugs and therapeuticswill take more time.

Financial disclosureThese opinions are exclusively of the authorsand do not reflect those of GeneExpressionSystems, Inc.

The authors have no relevant financial inter-ests, including employment, consultancies, hono-raria, stock ownership or options, experttestimony, grants or patents received or pending,or royalties related to this manuscript.

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