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1.
Nat Genet ; 56(6): 1300-1309, 2024 Jun.
Article in English | MEDLINE | ID: mdl-38724748

ABSTRACT

Concurrent readout of sequence and base modifications from long unamplified DNA templates by Pacific Biosciences of California (PacBio) single-molecule sequencing requires large amounts of input material. Here we adapt Tn5 transposition to introduce hairpin oligonucleotides and fragment (tagment) limiting quantities of DNA for generating PacBio-compatible circular molecules. We developed two methods that implement tagmentation and use 90-99% less input than current protocols: (1) single-molecule real-time sequencing by tagmentation (SMRT-Tag), which allows detection of genetic variation and CpG methylation; and (2) single-molecule adenine-methylated oligonucleosome sequencing assay by tagmentation (SAMOSA-Tag), which uses exogenous adenine methylation to add a third channel for probing chromatin accessibility. SMRT-Tag of 40 ng or more human DNA (approximately 7,000 cell equivalents) yielded data comparable to gold standard whole-genome and bisulfite sequencing. SAMOSA-Tag of 30,000-50,000 nuclei resolved single-fiber chromatin structure, CTCF binding and DNA methylation in patient-derived prostate cancer xenografts and uncovered metastasis-associated global epigenome disorganization. Tagmentation thus promises to enable sensitive, scalable and multimodal single-molecule genomics for diverse basic and clinical applications.


Subject(s)
DNA Methylation , Prostatic Neoplasms , Humans , Animals , Male , Prostatic Neoplasms/genetics , Mice , Sequence Analysis, DNA/methods , Chromatin/genetics , DNA/genetics , DNA Transposable Elements/genetics , High-Throughput Nucleotide Sequencing/methods , CpG Islands/genetics , Cell Line, Tumor , CCCTC-Binding Factor/genetics , CCCTC-Binding Factor/metabolism , Transposases
2.
bioRxiv ; 2024 Mar 21.
Article in English | MEDLINE | ID: mdl-38562907

ABSTRACT

From extrachromosomal DNA to neo-peptides, the broad reprogramming of the cancer genome leads to the emergence of molecules that are specific to the cancer state. We recently described orphan non-coding RNAs (oncRNAs) as a class of cancer-specific small RNAs with the potential to play functional roles in breast cancer progression1. Here, we report a systematic and comprehensive search to identify, annotate, and characterize cancer-emergent oncRNAs across 32 tumor types. We also leverage large-scale in vivo genetic screens in xenografted mice to functionally identify driver oncRNAs in multiple tumor types. We have not only discovered a large repertoire of oncRNAs, but also found that their presence and absence represent a digital molecular barcode that faithfully captures the types and subtypes of cancer. Importantly, we discovered that this molecular barcode is partially accessible from the cell-free space as some oncRNAs are secreted by cancer cells. In a large retrospective study across 192 breast cancer patients, we showed that oncRNAs can be reliably detected in the blood and that changes in the cell-free oncRNA burden captures both short-term and long-term clinical outcomes upon completion of a neoadjuvant chemotherapy regimen. Together, our findings establish oncRNAs as an emergent class of cancer-specific non-coding RNAs with potential roles in tumor progression and clinical utility in liquid biopsies and disease monitoring.

3.
bioRxiv ; 2023 Jun 02.
Article in English | MEDLINE | ID: mdl-37398273

ABSTRACT

Large-scale sequencing efforts of thousands of tumor samples have been undertaken to understand the mutational landscape of the coding genome. However, the vast majority of germline and somatic variants occur within non-coding portions of the genome. These genomic regions do not directly encode for specific proteins, but can play key roles in cancer progression, for example by driving aberrant gene expression control. Here, we designed an integrative computational and experimental framework to identify recurrently mutated non-coding regulatory regions that drive tumor progression. Application of this approach to whole-genome sequencing (WGS) data from a large cohort of metastatic castration-resistant prostate cancer (mCRPC) revealed a large set of recurrently mutated regions. We used (i) in silico prioritization of functional non-coding mutations, (ii) massively parallel reporter assays, and (iii) in vivo CRISPR-interference (CRISPRi) screens in xenografted mice to systematically identify and validate driver regulatory regions that drive mCRPC. We discovered that one of these enhancer regions, GH22I030351, acts on a bidirectional promoter to simultaneously modulate expression of U2-associated splicing factor SF3A1 and chromosomal protein CCDC157. We found that both SF3A1 and CCDC157 are promoters of tumor growth in xenograft models of prostate cancer. We nominated a number of transcription factors, including SOX6, to be responsible for higher expression of SF3A1 and CCDC157. Collectively, we have established and confirmed an integrative computational and experimental approach that enables the systematic detection of non-coding regulatory regions that drive the progression of human cancers.

4.
Nat Cancer ; 4(5): 682-698, 2023 05.
Article in English | MEDLINE | ID: mdl-37169843

ABSTRACT

Antisense RNAs are ubiquitous in human cells, yet their role is largely unexplored. Here we profiled antisense RNAs in the MDA-MB-231 breast cancer cell line and its highly lung metastatic derivative. We identified one antisense RNA that drives cancer progression by upregulating the redox enzyme NADPH quinone dehydrogenase 1 (NQO1), and named it NQO1-AS. Knockdown of either NQO1 or NQO1-AS reduced lung colonization in a mouse model, and investigation into the role of NQO1 indicated that it is broadly protective against oxidative damage and ferroptosis. Breast cancer cells in the lung are dependent on this pathway, and this dependence can be exploited therapeutically by inducing ferroptosis while inhibiting NQO1. Together, our findings establish a role for NQO1-AS in the progression of breast cancer by regulating its sense mRNA post-transcriptionally. Because breast cancer predominantly affects females, the disease models used in this study are of female origin and the results are primarily applicable to females.


Subject(s)
Breast Neoplasms , Neoplasms, Second Primary , Skin Neoplasms , Animals , Mice , Female , Humans , Breast Neoplasms/genetics , RNA, Antisense , Quinones/metabolism , NAD(P)H Dehydrogenase (Quinone)/genetics , Melanoma, Cutaneous Malignant
5.
J Oral Maxillofac Surg ; 81(4): 511-514, 2023 04.
Article in English | MEDLINE | ID: mdl-36587930

ABSTRACT

Recent innovations in camera and display technologies have created a new potential modus for the microvascular surgeon. An exoscope consists of cameras mounted to a flexible arm that broadcasts the image to a nearby high-definition monitor. This article details the experience using this emerging technology to perform microvascular anastomosis for 46 cases of head and neck free flaps reconstruction. The exoscope is compared against the operating microscope and we conclude that the exoscope is a viable substitute when performing microvascular anastomosis with some advantages in the areas of surgeon comfort and viewing angulation.


Subject(s)
Free Tissue Flaps , Plastic Surgery Procedures , Humans , Free Tissue Flaps/blood supply , Microsurgery/methods
6.
Oral Maxillofac Surg Clin North Am ; 33(3): 305-315, 2021 Aug.
Article in English | MEDLINE | ID: mdl-34116906

ABSTRACT

Facial trauma remains a common reason for visits to the emergency department or urgent care facility. The ear remains susceptible to trauma given its delicate anatomy and position in the maxillofacial region. Understanding the anatomy and recognizing the circumstances regarding the mechanism of injury help dictate treatment. The goals of treatment should remain to restore the physiologic form and function of the ear. Middle ear injuries should also be addressed during the process. Although primary repair remains feasible in most cases, there are instances when delayed and staged reconstruction is necessary to achieve successful results.


Subject(s)
Plastic Surgery Procedures , Ear, Middle , Humans
7.
Nature ; 575(7782): 371-374, 2019 11.
Article in English | MEDLINE | ID: mdl-31546247

ABSTRACT

Nucleic acid-sensing Toll-like receptors (TLRs) are subject to complex regulation to facilitate the recognition of microbial DNA and RNA while limiting the recognition of an organism's own nucleic acids1. Failure to properly regulate these TLRs can lead to autoimmune and autoinflammatory diseases2-6. Intracellular localization of these receptors is thought to be crucial for the discrimination between self and non-self7, but the molecular mechanisms that reinforce compartmentalized activation of intracellular TLRs remain poorly understood. Here we describe a mechanism that prevents the activation of TLR9 from locations other than endosomes. This control is achieved through the regulated release of the receptor from its trafficking chaperone UNC93B1, which occurs only within endosomes and is required for ligand binding and signal transduction. Preventing release of TLR9 from UNC93B1, either by mutations in UNC93B1 that increase affinity for TLR9 or through an artificial tether that impairs release, results in defective signalling. Whereas TLR9 and TLR3 are released from UNC93B1, TLR7 does not dissociate from UNC93B1 in endosomes and is regulated by distinct mechanisms. This work defines a checkpoint that reinforces the compartmentalized activation of TLR9, and provides a mechanism by which activation of individual endosomal TLRs may be distinctly regulated.


Subject(s)
Membrane Transport Proteins/metabolism , Toll-Like Receptor 9/metabolism , Animals , Cell Line , Humans , Membrane Glycoproteins/genetics , Membrane Glycoproteins/metabolism , Membrane Transport Proteins/genetics , Mice , Mice, Inbred C57BL , Protein Binding , Protein Transport , Signal Transduction , Toll-Like Receptor 3/genetics , Toll-Like Receptor 3/metabolism , Toll-Like Receptor 7/genetics , Toll-Like Receptor 7/metabolism , Toll-Like Receptor 9/genetics
8.
J Oral Maxillofac Surg ; 77(11): 2269-2278, 2019 Nov.
Article in English | MEDLINE | ID: mdl-31348875

ABSTRACT

Glioblastoma multiforme is the most common primary brain tumor. It is locally aggressive but rarely spreads outside the central nervous system. We present an unusual case of a 57-year-old woman who had presented 1 year after surgical resection and adjuvant therapy, with evidence of recurrent tumor invading through the skull base into the orbital apex, masticator, and pterygoid space. We have also reviewed all available case reports on local invasion of glioblastoma that occurred in absence of treatment and recurrence that developed away from the initial surgical location.


Subject(s)
Brain Neoplasms , Glioblastoma , Brain Neoplasms/diagnosis , Brain Neoplasms/surgery , Female , Glioblastoma/diagnosis , Glioblastoma/surgery , Humans , Middle Aged , Neoplasm Recurrence, Local , Skull Base
9.
Circ Res ; 120(2): 354-365, 2017 Jan 20.
Article in English | MEDLINE | ID: mdl-27923814

ABSTRACT

RATIONALE: Endothelial-mesenchymal transition (EndoMT) is implicated in myofibroblast-like cell-mediated damage to the coronary arterial wall in acute Kawasaki disease (KD) patients, as evidenced by positive staining for connective tissue growth factor (CTGF) and EndoMT markers in KD autopsy tissues. However, little is known about the molecular basis of EndoMT involved in KD. OBJECTIVE: We investigated the microRNA (miRNA) regulation of CTGF and the consequent EndoMT in KD pathogenesis. As well, the modulation of this process by statin therapy was studied. METHODS AND RESULTS: Sera from healthy children and KD subjects were incubated with human umbilical vein endothelial cells. Cardiovascular disease-related miRNAs, CTGF, and EndoMT markers were quantified using reverse transcriptase quantitative polymerase chain reaction, ELISA, and Western blotting. Compared with healthy controls, human umbilical vein endothelial cell incubated with sera from acute KD patients had decreased miR-483, increased CTGF, and increased EndoMT markers. Bioinformatics analysis followed by functional validation demonstrated that Krüppel-like factor 4 (KLF4) transactivates miR-483, which in turn targets the 3' untranslated region of CTGF mRNA. Overexpression of KLF4 or pre-miR-483 suppressed, whereas knockdown of KLF4 or anti-miR-483 enhanced, CTGF expression in endothelial cells in vitro and in vivo. Furthermore, atorvastatin, currently being tested in a phase I/IIa clinical trial in KD children, induced KLF4-miR-483, which suppressed CTGF and EndoMT in endothelial cells. CONCLUSIONS: KD sera suppress the KLF4-miR-483 axis in endothelial cells, leading to increased expression of CTGF and induction of EndoMT. This detrimental process in the endothelium may contribute to coronary artery abnormalities in KD patients. Statin therapy may benefit acute KD patients, in part, through the restoration of KLF4-miR-483 expression. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT01431105.


Subject(s)
Atorvastatin/administration & dosage , Connective Tissue Growth Factor/biosynthesis , Epithelial-Mesenchymal Transition/physiology , Gene Targeting/methods , MicroRNAs/biosynthesis , Mucocutaneous Lymph Node Syndrome/blood , Mucocutaneous Lymph Node Syndrome/therapy , Animals , Cattle , Child, Preschool , Connective Tissue Growth Factor/genetics , Dose-Response Relationship, Drug , Endothelial Cells/drug effects , Endothelial Cells/metabolism , Epithelial-Mesenchymal Transition/drug effects , Female , Human Umbilical Vein Endothelial Cells/drug effects , Human Umbilical Vein Endothelial Cells/physiology , Humans , Hydroxymethylglutaryl-CoA Reductase Inhibitors/administration & dosage , Infant , Kruppel-Like Factor 4 , Male , Mice , Mice, Transgenic , MicroRNAs/administration & dosage , MicroRNAs/genetics , Mucocutaneous Lymph Node Syndrome/genetics
10.
J Oral Maxillofac Surg ; 74(11): 2123-2127, 2016 Nov.
Article in English | MEDLINE | ID: mdl-27566985

ABSTRACT

Oral and Maxillofacial Surgeons have increasing opportunities to train in head and neck oncologic and reconstructive surgery. The development of a fellowship training programs within our speciality has had a transformative effect on the speciality. This survey by the commitee on Maxillofacial Oncology and Reconstructive is aimed to evaluate the impact of fellowship training on the careers of the fellows and speciality from 2005-2015.


Subject(s)
Fellowships and Scholarships , Head and Neck Neoplasms/surgery , Plastic Surgery Procedures/education , Practice Patterns, Physicians'/statistics & numerical data , Surgery, Oral/education , Humans , Surveys and Questionnaires , United States
11.
Sci Rep ; 6: 30064, 2016 07 25.
Article in English | MEDLINE | ID: mdl-27452728

ABSTRACT

Tumor infiltrating lymphocytes (TILs) have been associated with favorable prognosis in multiple tumor types. The Cancer Genome Atlas (TCGA) represents the largest collection of cancer molecular data, but lacks detailed information about the immune environment. Here, we show that exome reads mapping to the complementarity-determining-region 3 (CDR3) of mature T-cell receptor beta (TCRB) can be used as an immune DNA (iDNA) signature. Specifically, we propose a method to identify CDR3 reads in a breast tumor exome and validate it using deep TCRB sequencing. In 1,078 TCGA breast cancer exomes, the fraction of CDR3 reads was associated with TILs fraction, tumor purity, adaptive immunity gene expression signatures and improved survival in Her2+ patients. Only 2/839 TCRB clonotypes were shared between patients and none associated with a specific HLA allele or somatic driver mutations. The iDNA biomarker enriches the comprehensive dataset collected through TCGA, revealing associations with other molecular features and clinical outcomes.


Subject(s)
Breast Neoplasms/genetics , Exome/genetics , Genes, T-Cell Receptor beta/genetics , Lymphocytes, Tumor-Infiltrating/immunology , Receptors, Antigen, T-Cell, alpha-beta/genetics , T-Lymphocytes/immunology , Adaptive Immunity/genetics , Complementarity Determining Regions/genetics , Female , Gene Expression Profiling , Humans , Lymphocytes, Tumor-Infiltrating/cytology , T-Lymphocytes/cytology
12.
J Transl Med ; 13: 122, 2015 Apr 16.
Article in English | MEDLINE | ID: mdl-25889843

ABSTRACT

BACKGROUND: Malignant mesothelioma (MM) arises from mesothelial cells that line the pleural, peritoneal and pericardial surfaces. The majority of MMs are pleural and have been associated with asbestos exposure. Previously, pleural MMs have been genetically characterized by the loss of BAP1 (40-60%) as well as loss of NF2 (75%) and CDKN2A (60%). The rare peritoneal form of MM occurs in ~10% cases. With only ~300 cases diagnosed in the US per year, its link to asbestos exposure is not clear and its mutational landscape unknown. METHODS: We analyzed the somatic mutational landscape of 12 peritoneal MM of epitheloid subtype using copy number analysis (N = 9), whole exome sequencing (N = 7) and targeted sequencing (N = 12). RESULTS: Peritoneal MM display few copy number alterations, with most samples having less than 10 Mbp total changes, mostly through deletions and no high copy number amplification. Chromosome band 3p21 encoding BAP1 is the most recurrently deleted region (5/9), while, in contrast to pleural MM, NF2 and CDKN2A are not affected. We further identified 87 non-silent mutations across 7 sequenced tumors, with a median of 8 mutated genes per tumor, resulting in a very low mutation rate (median 1.3 10(-6)). BAP1 was the only recurrently mutated gene (N = 3/7). In one additional case, loss of the entire chromosome 3 leaves a non-functional copy of BAP1 carrying a rare nonsense germline variant, thus suggesting a potential genetic predisposition in this patient. Finally, with targeted sequencing of BAP1 in 3 additional cases, we conclude that BAP1 is frequently altered through copy number losses (N = 3/12), mutations (N = 3/12) or both (N = 2/12) sometimes at a sub-clonal level. CONCLUSION: Our findings suggest a major role for BAP1 in peritoneal MM susceptibility and oncogenesis and indicate important molecular differences to pleural MM as well as potential strategies for therapy and prevention.


Subject(s)
Genetic Predisposition to Disease , Lung Neoplasms/genetics , Mesothelioma/genetics , Mutation/genetics , Peritoneal Neoplasms/genetics , Tumor Suppressor Proteins/genetics , Ubiquitin Thiolesterase/genetics , DNA Copy Number Variations/genetics , Humans , Mesothelioma, Malignant
13.
Free Radic Biol Med ; 64: 61-8, 2013 Sep.
Article in English | MEDLINE | ID: mdl-23727269

ABSTRACT

Endothelial functions are highly regulated by imposed shear stress in vivo. The characteristics of shear stress determine mechanotransduction events that regulate phenotypic outcomes including redox and inflammatory states. Recent data indicate that microRNAs (miRs) in vascular endothelial cells play an essential role in shear stress-regulated endothelial responses. More specifically, atheroprotective pulsatile flow (PS) induces miRs that inhibit mediators of oxidative stress and inflammation while promoting those involved in maintaining vascular homeostasis. Conversely, oscillatory flow (OS) elicits the opposing networks. This is exemplified by the PS-responsive transcription factor Krüppel-like factor 2 (KLF2), which regulates miR expression but is also regulated by OS-sensitive miRs to ultimately regulate the oxidative and inflammatory state of the endothelium. In this review, we outline important findings demonstrating the multifaceted roles of shear stress-regulated miRs in endothelial redox and inflammatory balance. Furthermore, we discuss the use of algorithms in deciphering signaling networks differentially regulated by PS and OS.


Subject(s)
Endothelial Cells/metabolism , Endothelium, Vascular/metabolism , Kruppel-Like Transcription Factors/metabolism , Mechanotransduction, Cellular , MicroRNAs/metabolism , Endothelial Cells/cytology , Endothelium, Vascular/cytology , Gene Expression Regulation , Humans , Inflammation/genetics , Inflammation/metabolism , Inflammation/pathology , Kruppel-Like Transcription Factors/genetics , MicroRNAs/genetics , Oxidation-Reduction , Oxidative Stress , Pulsatile Flow , Stress, Mechanical
14.
Proc Natl Acad Sci U S A ; 110(8): 3161-6, 2013 Feb 19.
Article in English | MEDLINE | ID: mdl-23382195

ABSTRACT

B-cell lymphoma-6 protein (Bcl-6) is a corepressor for inflammatory mediators such as vascular cell adhesion molecule-1 and monocyte chemotactic protein-1 and -3, which function to recruit monocytes to vascular endothelial cells upon inflammation. Poly [ADP ribose] polymerase 1 (PARP-1) is proinflammatory, in part through its binding at the Bcl-6 intron 1 to suppress Bcl-6 expression. We investigated the mechanisms by which PARP-1 dissociates from the Bcl-6 intron 1, ultimately leading to attenuation of endothelial inflammation. Analysis of the PARP-1 primary sequence suggested that phosphorylation of PARP-1 Serine 177 (Ser-177) by AMP-activated protein kinase (AMPK) is responsible for the induction of Bcl-6. Our results show that AMPK activation with treatment of 5-aminoimidazole-4-carboxamide ribonucleotide, metformin, or pulsatile shear stress induces PARP-1 dissociation from the Bcl-6 intron 1, increases Bcl-6 expression, and inhibits expression of inflammatory mediators. Conversely, AMPKα suppression or knockdown produces the opposite effects. The results demonstrate an anti-infamatory pathway linking AMPK, PARP-1, and Bcl-6 in endothelial cells.


Subject(s)
AMP-Activated Protein Kinases/metabolism , Poly(ADP-ribose) Polymerases/physiology , Proto-Oncogene Proteins c-bcl-6/physiology , Animals , Cells, Cultured , Introns , Mice , Phosphorylation , Poly (ADP-Ribose) Polymerase-1 , Proto-Oncogene Proteins c-bcl-6/genetics , Transcription, Genetic , Transcriptional Activation
15.
Head Neck ; 30(11): 1531-4, 2008 Nov.
Article in English | MEDLINE | ID: mdl-18327774

ABSTRACT

BACKGROUND: Fibular free flaps are an alternative method in the management of chronic osteomyelitis of the mandible without osteoradionecrosis. METHODS: A prospective review of 2 cases of chronic osteomyelitis of the mandible managed with a fibular free flap was conducted. Patient satisfaction and aesthetic results were examined. Follow-up to 9 months was achieved and radiographic studies conducted. The current literature on the treatment of chronic osteomyelitis of the mandible and the use of free flaps in its management was reviewed. RESULTS: The 2 patients with chronic osteomyelitis of the mandible whose conservative management failed were effectively treated with the use of a fibular free flap reconstruction. CONCLUSION: Microvascular reconstruction with a fibular free flap should be considered as a treatment option in the management of chronic osteomyelitis of the mandible.


Subject(s)
Fibula/transplantation , Mandible/surgery , Oral Surgical Procedures/methods , Osteomyelitis/surgery , Surgical Flaps , Adolescent , Adult , Chronic Disease , Female , Humans , Patient Satisfaction , Plastic Surgery Procedures/methods , Treatment Outcome
17.
J Clin Ultrasound ; 30(2): 59-67, 2002 Feb.
Article in English | MEDLINE | ID: mdl-11857510

ABSTRACT

PURPOSE: Emergency abdominal sonography has become a common modality worldwide in the evaluation of injuries caused by blunt trauma. The sensitivity of sonography in the detection of hemoperitoneum varies, and little is known about the accuracy of sonography in the detection of injuries to specific organs. The purpose of this study was to determine the overall accuracy of sonography in the detection of hemoperitoneum and solid-organ injury caused by blunt trauma. METHODS: From January 1995 to October 1998, 3,264 patients underwent emergency sonography at our institution to evaluate for free fluid and parenchymal abnormalities of specific organs caused by blunt trauma. All patients with intra-abdominal injuries (IAIs) were identified, and their sonographic findings were compared with their CT and operative findings, as well as their clinical outcomes. RESULTS: Three hundred ninety-six (12%) of the 3,264 patients had IAIs. Sonography detected free fluid presumed to represent hemoperitoneum in 288 patients (9%). The sonographic detection of free fluid alone had a 60% sensitivity, 98% specificity, 82% positive predictive value, and 95% negative predictive value for diagnosing IAI. The accuracy was 94%. Seventy patients (2%) had parenchymal abnormalities identified with sonography that corresponded to actual organ injuries. The sensitivity of the sonographic detection of free fluid and/or parenchymal abnormalities in diagnosing IAI was 67%. CONCLUSIONS: Emergency sonography to evaluate patients for injury caused by blunt trauma is highly accurate and specific. The sonographic detection of free fluid is only moderately sensitive for diagnosing IAI, but the combination of free fluid and/or a parenchymal abnormality is more sensitive.


Subject(s)
Abdominal Injuries/diagnostic imaging , Wounds, Nonpenetrating/diagnostic imaging , Abdominal Injuries/diagnosis , Abdominal Injuries/surgery , Adolescent , Adult , Aged , Aged, 80 and over , Child , Child, Preschool , Emergencies , Hemoperitoneum/diagnostic imaging , Humans , Infant , Infant, Newborn , Middle Aged , Prospective Studies , Sensitivity and Specificity , Tomography, X-Ray Computed , Ultrasonography , Wounds, Nonpenetrating/diagnosis , Wounds, Nonpenetrating/surgery
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