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Research Area: The role of cell surface carbohydrates
in T cell development and function Research Interests: Cell surface glycosylation
is a complex and tightly regulated process, yet little is known
about the Research Area: Pathogenetic mechanisms in HIV infection
and inflammatory bowel disease Research Interests: Our group is devoted to identifying genetic traits and their functional cellular counterparts in immune function. This basic immunologic question is being addressed in the context of two diseases. First, our lab is engaged in the search for candidate microbial pathogens in inflammatory bowel disease. This search involves the use of disease specific marker antibodies for the detection of cognate microorganisms; and, representational display analysis, to identify candidate pathogens on the basis of marker genes expressed at lesional sites. Second, we are tackling the identity of host traits conferring susceptibility to HIV-1 pathogenesis. This work addresses role of viral gene products on function of infected host cells, and the mechanistic relationship of host immunodeficiency in B cell lymphomagenesis. Research Area: Melanocytic tumors and the factors which determine favorable and unfavorable outcome for individual patients. Research Interests: Dr. Cochran’s research and extensive publications focus on melanocytic tumors and the factors which determine favorable and unfavorable outcome for individual patients. Additionally, he focuses on the extent that therapy- or tumor-induced immune-suppression facilitates melanoma growth and spread. He is one of the original and leading proponents of the lymphatic mapping and sentinel node biopsy technique to stage and manage high-risk melanoma patients. His contributions to the field of melanoma prognosis and surgical management place him on the vanguard of this area of research. Research Area: Regulation of tight junction development Research Interests: The main interest of my laboratory is to understand molecular and cellular mechanisms underlying tight junction development in epithelial cells. The tight junction which is composed of several proteins forms a selective permeable barrier around epithelia. It regulates paracellular permeability of the tissue and separates the apical and basolateral plasma membrane domains of epithelial cells. How various tight junction proteins are assembled to form a functional tight junction in epithelial cells is poorly understood. Using Madin-Darby Canine Kidney (MDCK) cells as the model system and a variety of cell and molecular biological techniques we are trying to identify 1. Signal transduction mechanisms that initiate tight junction development. 2. Targeting of tight junction proteins to the tight junction and 3.Molecular mechanisms that regulate the levels of various tight junction proteins during tight junction development. In addition, we are also studying mechanisms leading to the loss of tight junction during carcinogenesis and the role of tight junction in cancer metastasis. Research Area: Molecular Pathology of Ataxia-Telangiectasia and Related Disorders Research Interests: Since 1985, we have been attempting to localize the gene(s) for ataxia-telangiectasia (AT) to a region small enough to clone and isolate so that we can develop a better understanding of this progressive and fatal disease of children. In July 1988, we localized the gene to chromosome 11q22-23 by linkage analyses. We formed an international consortium and analyzed over 200 families. In 1995, the ATM (A-T mutated) gene was cloned by the Israeli members of the consortium and found to have protein kinase homology. Our lab is presently focusing on: 1. Characterizing ATM mutations, most of which result in protein truncation, and developing "user-friendly" detection assays. 2. Using ATM cDNA and monoclonal antibodies to localize the ATM gene product in tissues from 11 autopsies of A-T patients as well as in cell extracts from >150 patients. 3. Cloning the >200 million-year-old Pufferfish ATM homology. 4. Cloning ATM cDNA into vacinnia vectors to allow study of ATM protein structure. Our long-term goals are gene-based therapy for A-T patients, and diagnostic testing for patients and carriers. Selected
publications: Gatti RA, Boder E, Vinters HV, Sparkes RS, Norman A, Lange K: Ataxia-telangiectasia: an interdisciplinary approach to pathogenesis. Medicine 70: 99-117, 1991. Lange E, Borresen A-L, Chen X, Chessa L, Chiplunkar S, Concannon, Dandekar S, Gerken S, Lange K, Liang T, McConville C, Polakow J, Porras O, Rotman G, Sanal O, Sheikhavandi S, Shiloh Y, Sobel E, Taylor M, , Telatar M, Teraoka S, Tolun A, Udar N, Uhrhammer N, Vanagaite L, Wang Z, Wapelhorst B, Wright J, Yang H-M, Yang L, Ziv Y, Gatti RA. Localization of an ataxia-telangiectasia gene to a ~500 kb interval on chromosome 11q23.1: linkage analysis of 176 families in an international consortium. Amer J Hum Genet 57: 112-119, 1995. Savitsky K, Bar-Shira A, Gilad S, Rotman G, Ziv Y, Vanagaite L, Tagle DA, Smith S, Uziel T, Sfez S, Ashkenazi M, Pecker I, Frydman M, Harnik R, Patanjali SR, Simmons A, Clines GA, Sartiel A, Gatti RA, Chessa L, Sanal O, Lavin MF, Jaspers NGJ, Taylor AMR, Arlett CF, Miki T, Weissman SM, Lovett M, Collins FS, Shiloh Y: A single ataxia telangiectasia gene with a product similar to PI-3 kinase. Science 268: 1749-1753, 1995. Telatar M, Wang Z, Udar N, Liang T, Concannon P, Bernatovska-Matuszkiewicz E, Lavin M, Shiloh Y, Good RA, Gatti RA. Ataxia-telangiectasia: mutations in ATM cDNA detected by protein truncation screening. Amer J Hum Genet 59:40-44, 1996. Wright J, Teraoka S, Onengut S, Tolun A, Gatti RA, Ochs HD, Concannon P. A high frequency of distinct ATM gene mutations in ataxia-telangiectasia. Amer J Hum Genet 59:839-846, 1996.
Research Area: My laboratory's current research efforts are focused on 2 related themes in tumor biology: 1) identifying molecular protein profiles of tumor initiation and progression; and 2) understanding the function of epithelial membrane protein-2 (EMP2) in tumor progressive and escape from immune surveillance. Research Interest: UCLA EDRN. The first project is the UCLA Early Detection Research Network of which I am the co-Director. The Early Detection Research Network (EDRN) is a consortium launched by the NCI approximately 5 years ago. The EDRN is a main flagship of the NCI's war on cancer, and thus is a major focus of the NCI's strategic game plan. This program is focused on identifying viable strategies for detecting cancer. One of the missions of the UCLA EDRN is to evaluate / validate new tumor markers. Our vision is to discover molecular signatures of tumor development. In this regard, our research is almost directly analogous to tumor profiling using DNA arrays; our approach falls under the category of "proteomics". Our profiling effort is based on the hypotheses that: i) there are fundamental pathways common to most, if not all cancers (e.g., life-death decision, cell cycle control, DNA repair, etc.); ii) disruption of one or more of these pathways is caused by, and/or results in altered protein expression, modification, localization, and/or activity; iii) determination of these protein changes on a population basis would define statistical clinical relevance, and mechanistically, would help define the molecular / protein 'circuitry' of a tumor cell. One experimental approach we have used is to examine the expression of multiple protein within a given pathway using high density tissue microarrays. Tissue microarrays are "chips" with hundreds or thousands of individual tissue "spots" representing hundreds of different patients. Together, these samples can be tested for protein express, activation, and / or chromosomal abnormalities. There are many interesting implications to studying "molecular circuitry" in tumors. First, we will define protein profiles that are diagnostic for subsets of cancers and/or prognostic for outcomes. As such, our focused 'deliverable' is to define markers that will have clinically utility. Second, as we expand our protein profile database, multivariate and cluster-type analyses will suggest novel, previously unappreciated, protein interactions. The mechanism of interaction will be testable experimentally. The Molecular Mechanism of Tumor Progression: Evasion of host immune surveillance and resistance to therapeutic treatment. My second research focus is on the mechanism of tumor progression, specifically the mechanism by which tumor cells become resistance to immune surveillance and/or therapeutic treatment. Through a productive collaboration with Dr. Jonathan Braun, we have identified a GAS3 family member, Epithelial Membrane Protein-2 (EMP2), in a screen for functional tumor suppressor genes. Reduced expression of EMP2 ehanced tumorigenicity in vivo, and profoundly decreased cell susceptibility to cytotoxic lymphocyte (CTL)-mediated killing in vitro. Biochemical studies indicated that EMP2 may play an important and previously unappreciated role in trafficking of surface proteins to specific lipid raft domains. Accordingly, the focus of our research is to define the biochemical role of EMP2 on subcellular trafficking of surface proteins; its mechanism in cytotoxic cell targeting and immunogenicity; and, its use in disease stratification of a native human cancer. Research Area: Carcinogenesis, Hypoxia, Development Research Interests: Dr. Hankinson’s research focuses on the mechanism of carcinogenesis by polycyclic aromatic hydrocarbons (found in cigarette smoke and smog) and dioxin (a widespread pollutant), and related compounds. Carcinogenesis by these compounds depends upon their binding to the aryl hydrocarbon receptor (AHR) and the subsequent dimerization of AHR with the ARNT protein. He is studying the molecular mechanism of activation of gene transcription by the liganded AHR/ARNT dimer (including the role of coactivator proteins in this process) and is analyzing a number of novel dioxin-inducible genes his group has discovered. In addition, he also studies the roles of AHR and ARNT in animal models of carcinogenesis. ARNT also dimerizes with HIF-1a to form HIF-1 (Hypoxia Inducible Factor), which is the master regulator of the hypoxic response. Dr. Hankinson is studying the molecular mechanism of gene activation by HIF-1 and the role of HIF-1 in tumor angiogenesis and growth. Current research focuses on the following :
Selected publications: Roth, M.D., Marquez-Magallanes, J.A., Yuan, M., Sun, W., Tashkin, D.P., Hankinson, O. (2001). Induction and Regulation of the Carcinogen-Metabolizing Enzyme, CYP1A1, by Marijuana Smoke and Delta(9)-Tetrahydrocannabinol. Am. J. Respir. Cell. Mol. Biol. 24: 339-344. Heo, Y., Saxon, A., Hankinson, O. (2001). Effect of Diesel Exhaust Particles and their Components on Allergen-Specific IgE and IgG1 response in mice. Toxicol. 159: 143-158. Lei, X.-D., Chapman, B., Hankinson, O. (2001). Loss of CYP1A1 Messenger RNA Expression Due to Nonsense-Mediated Decay. Mol. Pharmacol. 60: 388-393. Anttila, S.L., Tuominen, P., Hirvonen, A., Karjalainen, A., Hankinson, O., Elovaara, E. (2001). CYP1A1 levels in lung tissue of tobacco smokers and polymorphisms of CYP1A1 and aromatic hydrocarbon receptor. Pharmacogenetics. 11: 501-509. Yoon, D.Y., Buchler, P., Saarikoski, S.T., Rivera, S.P., Hines, O.J., Reber, H.A., Hankinson, O. (2001). Identification of genes differentially induced by hypoxia in pancreatic cancer cells. Biochem. & Biophys. Res. Comm. 288: 882-886. Rivera, S.P., Saarikoski, S.T., Hankinson, O. (2002). Identification of a novel dioxin-inducible cytochrome P450. Mol. Pharm. 61: 255-259. Wang, S., and Hankinson, O. (2002). Functional involvement of the BRM/SWI2-related gene 1 protein (BRG-1) in cytochrome P4501A1 transcription mediated by the aryl hydrocarbon receptor complex. J. Biol. Chem. 277: 11821-11827. Beischlag, T.V., Wang, S., Torchia, J., Reisz-Porszasz, S., Muhammad, K., Nelson, W.E., Probst, M.R., Rosenfeld, M.G., Hankinson, O. (2002). Recruitment of the NCoA/SRC-1/p160 Family of Transcriptional Co-activators by the Aryl Hydrocarbon Receptor/Aryl Hydrocarbon Receptor Nuclear Translocator Complex. Mol. Cell. Biol. 22: 4319-4333. Saarikoski, S.T., Rivera, S.P., Hankinson, O.
(2002). Mitogen-inducible gene-6 (mig-6), adipophilin and tuftelin
are inducible by hypoxia. FEBS Lett. 530: 186-90. Additional Web Page Links: Research Area: Targeted molecular therapy of glioblastoma Research Interest: The research in my laboratory focuses on Glioblastoma (GBM), the most common malignant brain tumor of adults. GBMs are among the most lethal of all cancers. They invade the surrounding brain as infiltrating single cells, which renders them surgically unresectable and accounts, in part, for their poor prognosis. In addition, these tumors are among the most radio- and chemotherapy resistant tumors. Clearly, the treatment of this tumor requires new approaches. My laboratory focuses on: 1) identifying molecular subsets of these tumors with distinct signal transduction abnormalities, 2) developing targeted molecular therapies that will inhibit the growth/survival/invasion of these distinct molecular subsets and 3) developing tools to identify these molecular subsets in patient biopsies. Because genetically-based constitutive activation of signaling pathways in tumor cells tends to promote enhanced reliance of those tumor cells upon such pathways, targeted inhibition may be a highly effective, and selective, therapy for some types of cancer. The tumor suppressor gene PTEN is disrupted in nearly 40% of GBMs leading to constitutive activation of the Akt pathway. In collaboration with our colleague Charles Sawyers, we have developed a pre-clinical GBM model derived from primary patient tumor cells and have demonstrated that PTEN deficient/Akt active GBMs are exquisitely sensitive to the growth inhibiting properties of the rapamycin analogue CCI-779. We can show that targeted inhibition of the Akt pathway at the level of mTOR selectively blocks the growth of these PTEN deficient GBM cells (Smith et al., in preparation.) and we have developed immunohistochemical reagents to evaluate activation of this pathway in patient biopsy samples (Choe et al, in preparation). This work forms the basis for two ongoing clinical trials.Our laboratory is also in the process of identifying other signaling pathways that are genetically disrupted in GBMs. We have used an expression profiling strategy on patient biopsy samples to demonstrate that EGFR over-expressing GBMs have a distinctive gene expression signature and to identify novel molecular subsets of EGFR-negative GBMs. This work, which has been accepted at Oncogene (pending revision) is supported by our finding that EGFR expressing GBMs have a distinctive MMP activation profile (Choe et al., Clinical Cancer Research, 8(9): 2894-2901). This approach also helps to identify additional signal transduction pathways that can be targeted in each of these distinct molecular GBM subtypes. Research Area: Signal transduction, Cell Cycle, Leukemia, Targeting Proteins for Ubiquitination and Proteolysis Research Interests: Hematopoiesis is defined as the development of blood cells. My laboratory is interested in the mechanisms by which normal blood cells become cancerous. White blood cell production is regulated by hematopoietic growth factors, such as Granulocyte-macrophage colony-stimulating factor (GM-CSF) and Granulocyte colony-stimulating factor (G-CSF). We previously showed that GM-CSF activation results in phosphorylation of several kinases and transcription factors, including the cAMP responsive element binding protein known as CREB. Our laboratory discovered that CREB is overexpressed in bone marrow cells from patients with acute leukemia, but not in normal bone marrow. Our goal is to understand the mechanism of CREB overexpression and its contribution to the leukemogenic process. We are also studying the role of the cell cycle protein, p55Cdc, during chemotherapy-induced apoptosis. p55Cdc regulates the spindle assembly (mitotic) checkpoint that is responsible for insuring that all cells receive the same amount of genetic material during cell division. Work in Drosophila and yeast demonstrate that p55Cdc is critial for normal cell cycle progression. In mammalian cells, overexpression of p55Cdc results in increased apoptosis. We are currently investigating the role of p55Cdc in apoptotic pathways regulating cell death in leukemias. Finally, in collaboration with Ray Deshaies (Caltech) and Craig Crews (Yale) we are developing a novel approach to cancer therapy by targeting proteins to the heterotetrameric complex, SCF (Skp1-cullin-Fbox-hrt1). We plan to use a chimeric molecule, termed Protac (Proteolysis Targeting Chimeric Pharmaceutical) that contains a moiety that is recognized by a component of the SCF at one end and the target-binding protein at the other end. We have successfully used this approach with the anti-angiogenic factor, MetAP-2 and its binding protein ovalicin, as "proof of principle" and demonstrated that MetAP-2 can be recruited to the SCF for ubiquitination. We are now testing various Protac derivatives to target cancer promoting proteins for proteolysis. Research Area: Epigenetics and signal transduction mechanisms in B-cell development and neoplasia. Research Interests: Our group studies the basic biological mechanisms involved in B lymphocyte development and neoplasia utilizing AIDS-related lymphoma (AL) as a model system. AL occurs in 5-7 million HIV-infected individuals worldwide. These tumors are high-grade B-cell malignancies that arise in the unique setting of severe immunodeficiency, chronic antigenic stimulation and altered patterns of cytokine production. These unusual features strongly suggest that novel transforming genes and mechanisms function in AL formation. This hypothesis is supported by the extensive surveys of known oncogenes, tumor suppressor genes and candidate tumorigenic herpesviruses, such as EBV and HHV-8, which have not revealed a consistent link between individual AL lesions. A subtraction-based gene discovery program, combined with high-throughput candidate gene screening, has resulted in over 200 AL-specific genes being identified, 65 of which are unique and thus far uncharacterized. Early studies of one gene, TCL1, suggests that it functions to promote cell survival and proliferation by interacting with the AKTcell signaling pathway. TCL1 is normally down-regulated during B-cell differentiation; however, in both AL and non-AL, TCL1 levels remain aberrantly high suggesting a role in promoting mature B-cell lymphomas . TCL1 transgenic mice have been generated that develop lymphoma, establishing a model system to study the normal and abnormal biochemical and signaling functions of TCL1. Further characterization of this model indicates that it is one of the first to accurately model the vast majority of B- cell cancers that arise in humans, which has been a major goal in hematology/oncology research for many years. Deciphering the molecular mechanisms that contribute to the development of AIDS-related and non-AIDS B-cell cancer in the context of normal B lymphocyte differentiation is complex and requires a multifaceted approach. Major current themes in the lab include understanding how TCL1 dysregulation contributes to lymphomagenesis, characterization of additional differentially-expressed genes in B-cell lymphomas and new investigations of novel epigenetic mechanisms controlling gene expression in health and disease. Recently, in studies linked to TCL1 gene regulation, we have discovered a novel type of DNA methylation that appears to have a role in chromatin structure and gene expression. Methylation of the internal cytosine in CC(A/T)GG motifs causes gene silencing. Major questions we are currently addressing include identification of the methyltransferase and associated proteins involved in this new epigenetic regulation and its significance in B lymphocyte development and neoplasia. Research Area: Molecular Mechanism of Carcinogenesis Research Interests: Our work centers mostly on basic mechanisms and genetic control of homologous and illegitimate recombination, events involved in carcinogenesis. Effects of cancer predisposing mutations on the frequency of spontaneous and carcinogen induced mitotic recombination in vitro and in vivo, investigation of the biological effects of "nonmutagenic" carcinogens. Genetic instability and deletions are involved in carcinogenesis. We have previously constructed and/or used assays (DEL assays) that select for DNA deletion events in yeast (Schiestl et al. 1988, Schiestl 1989, Schiestl et al. 1989b,c, Schiestl and Reddy 1990, Carls and Schiestl 1994, Brennan et al. 1994), in human cells (Aubrecht et al. 1995) and in vivo in the mouse (Schiestl et al. 1994, Schiestl et al. 1997, Schiestl et al. 1997, Schiestl et al. 1998). DEL events in all three formats are inducible by a wide variety of proven carcinogens, including carcinogens that are negative in many other short-term tests. We have shown that many Salmonella assay negative carcinogens induce oxidative stress in yeast. (Brennan and Schiestl 1997, 1998). We are interested in determining the differential effects of "mutagenic versus nonmutagenic" carcinogens in the mouse using a variety of assays including gene expression profiling. The first model to study recombination in vivo is the pun mouse which has a 70 kb internal gene duplication disruption in the p coat color gene. Reversion to the wildtype sequence occurs by recombination between the two copies and is inducible by carcinogens (Schiestl et al. 1994, Schiestl et al. 1997). We have crossed the pun mutation into different DNA repair deficient backgrounds and found exciting results that p53 is involved in ionizing radiation induced recombination (Submitted) implying that it may be involved in the processing of double-strand breaks. As second model we have constructed a transgenic mouse that carries a duplication of exons 2 and 3 of the Hprt gene and we have already developed a sensitive histochemical staining method to identify deletion events in different tissues of the mouse. We will study the genetic control of such deletion events. p53, ERCC1, ATM, and SCID mutations cause the human diseases Li-Fraumeni syndrome, Xeroderma Pigmentosum, Ataxia Telangiectasia and severe combined immuno deficiency respectively. Furthermore we received mRAD52 and Ku80 mice. We are currently investigating effects of these mutations on spontaneous, carcinogen induced recombination events. Mechanism of Persistent Genetic Instability Multiple genetic changes are required for the development of a malignant tumor cell and many environmentally-induced cancers show a delayed onset of more than 20 years following exposure. The frequency of such changes found in cancer cells is higher than can be explained through random mutation and it was proposed that a sub-population of cells develop a mutator phenotype. Such a persistent elevated level of genetic instability is also a major contributor to the progressive, multistage development of malignant disease. This phenotype, sometimes called delayed reproductive death, has indeed been observed in mammalian cells after treatment with ionizing radiation but the mechanism has not been defined. We have observed a similar genomic instability more than 50 cell divisions after exposure to ionizing radiation in yeast. These effects cannot be due to the initial damage because of their persistence over many generations. Mutations in a single gene leading to an elevated level of genetic instability also cannot account for these effects because they occur in up to 70% of the exposed cells. It is more likely that a difference in gene expression accounts for the high frequency of deletions (HFD) phenotype. We will investigate the mechanism of these delayed inheritable changes with conventional genetic tools as well as determining the gene expression profile (6200 genes) for yeast HFD cultures and control cultures to identify genes which may be involved in the maintenance or destabilization of genetic integrity. Finally, we will alter the expression of genes that are up or down regulated in HFD clones, and determine the effect of the altered gene expression on the initiation and/or inheritance of the HFD phenotype. This project should characterize the phenomenon of persistently elevated genetic instability, give insights into its mechanism and might also provide molecular targets for intervention to reverse the phenotype. Interindividual Differences in DNA double-strand break repair efficiency We have also constructed plasmid model systems to determine the efficiency of cells for DNA double strand break repair by homologous versus illegitimate recombination. We are currently determining interindividual differences in DNA double strand break repair between cells from different control people and cells from 17 cancer patients that showed unexpected hypersensitivity to radiation treatment. In this project we are collaborating with Drs. Jack Little from our department, Tom Lindahl from the ICRF in London and Mark Meuth from Sheffield Univ. England. This project is also carried out in collaboration with Gene Therapeutic Inc. to determine polymorphisms in 18 genes involved in double strand break repair by high throughput DNA sequencing. We are also collaborating with Carl Barrett from the NIEHS to determine gene expression profiles of the different human cell lines in response to radiation exposure. This approach will hopefully link phenotypes in double-strand break repair with genotypes in DSB repair genes and may uncover new genetic risk factors for cancer. Investigation of the mechanism, genetic control and inducibility of illegitimate recombination and restriction enzyme-mediated recombination in yeast and in mammalian cells. A system to study illegitimate integration of transformed DNA fragments in yeast has been developed (Schiestl and Petes 1991). In the presence of a restriction enzyme in the transformation mixture, the DNA fragments integrated into the respective genomic restriction sites by Restriction Enzyme-Mediated Integration (REMI) (Schiestl and Petes 1991). These original findings have, in the meantime, found widespread use for insertional mutagenesis and RFLP mapping in different organisms. In the absence of the restriction enzymes, the DNA fragments integrated by illegitimate integration falling into different classes. About 40% of integration events happen by microhomology mediated integration (Schiestl et al. 1993). Another 40% are mediated by topoisomerase I (Zhu and Schiestl 1996). Mutations of the yeast RAD50 gene reduce the frequency of illegitimate recombination 100 fold, but actually increase the frequency of homologous integration (Schiestl et al. 1993). We also found that restriction enzymes increase the frequency of integration of DNA fragments into human cells and that XRCC5 (Ku70) is involved in REMI (Submitted). We have achieved regulated expression of the human topoisomerase I in a yeast strain deleted for its own TOP1 gene. Most surprisingly, after overexpression of the human gene the frequency of illegitimate integration increased 20 fold and all additional events had target sites in the rDNA. Addition of a topoisomerase I inhibitor abolished this increase. We are currently developing a selective assay for illegitimate recombination (without the need for yeast transformation). With this assay we want to isolate mutants that increase (hyperrecombination) or decrease (hyporecombination) the frequency of IR and we also want to determine environmental factors that may alter the frequency of IR. Current results also show that ionizing radiation and other mutagens induce illegitimate integration. Thus we are able to map DSBs (e.g. meiotic, X-ray induced etc.) in yeast just by transforming a fragment, cloning and sequencing of the integration junctions and comparing those with the sequences in the database. This will yield the genomic distribution and sequence specificity of such illegitimate integration events that were attracted by genomic double-strand breaks. |
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