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Single molecule sensors in which nanoscale pores within biological or artificial membranes act as mechanical gating elements are very promising devices for the rapid characterization and sequencing of nucleic acid molecules. The two terminal electrical measurements of translocation of polymers through single ion channels and that of ssDNA molecules through protein channels have been demonstrated, and have sparked tremendous interest in such single molecule sensors. The prevailing view regarding the nanopore sensors is that there exists no electrical interaction between the nanopore and the translocating molecule, and that all nanopore sensors reported to-date, whether biological or artificial, operate as a coulter-counter, i.e., the ionic current measured across the pore decreases (is mechanically blocked) when the DNA molecule transverses through the pore. We have fabricated nanopore "channel" sensors with a silicon oxide inner surface, and our results challenge the prevailing view of exclusive mechanical interaction during the translocation of dsDNA molecules through these channels. We demonstrate that the ionic current can actually increase due to electrical gating of surface current in the channel due to the charge on the DNA itself.As a first step toward the ultimate goal of single-molecule DNA sequencing using nanopore sensors, one must first identify the key mechanical and electrical variables that control the translocation of the molecules through the nanopores. First, a nanopore used for characterization and sequencing of a single molecule must have a diameter of less than the persistence length (∼50 nm for dsDNA) to avoid any signal averaging from thermally induced conformational changes. Second, the pores must be chemically stable and mechanically robust under a wide variety of conditions of use. Third, and finally, the mechanical and electrical interaction between the nanopores and the single molecules must be well characterized. Toward this end, the electrical detection of translocation of polymers through single ion channels has been demonstrated. 1-2 Subsequently, the pioneering studies of the use of R-hemolysin protein pore within a lipid bilayer for the translocation of single strands of DNA molecules using a voltage bias across the membranes sparked tremendous interest in such single molecule sensors. [3][4][5][6][7][8] The normal ionic current through the protein pore in a lipid bilayer would detectably reduce as a polyanionic chain of ssDNA molecules traversed through the pore, even allowing the distinction between polycytosine and polyadenine molecules, thus demonstrating the potential of single base discrimination in these sensors. 4,5 Despite these advantages, robust integration of these biological sensors within practical devices is quite problematic, and a mechanical pore 9,10 provides numerous advantages over biological pores. Besides the obvious advantages of being able to drastically change ambient conditions such as pH, electric field, and temperature without distorting the s...

• ALK-negative ALCLs have chromosomal rearrangements of DUSP22 or TP63 in 30% and 8% of cases, respectively. • DUSP22-rearranged cases have favorable outcomes similar to ALK-positive ALCLs, whereas other genetic subtypes have inferior outcomes.Anaplastic lymphoma kinase (ALK)-negative anaplastic large cell lymphoma (ALCL) is a CD30-positive T-cell non-Hodgkin lymphoma that morphologically resembles ALKpositive ALCL but lacks chromosomal rearrangements of the ALK gene. The genetic and clinical heterogeneity of ALK-negative ALCL has not been delineated. We performed immunohistochemistry and fluorescence in situ hybridization on 73 ALK-negative ALCLs and 32 ALK-positive ALCLs and evaluated the associations among pathology, genetics, and clinical outcome. Chromosomal rearrangements of DUSP22 and TP63 were identified in 30% and 8% of ALK-negative ALCLs, respectively. These rearrangements were mutually exclusive and were absent in ALK-positive ALCLs. Five-year overall survival rates were 85% for ALK-positive ALCLs, 90% for DUSP22-rearranged ALCLs, 17% for TP63-rearranged ALCLs, and 42% for cases lacking all 3 genetic markers (P < .0001).Hazard ratios for death in these 4 groups after adjusting for International Prognostic Index and age were 1.0 (reference group), 0.58, 8.63, and 4.16, respectively (P 5 7.10 3 10 25

The genetics of peripheral T-cell lymphomas are poorly understood. The most well-characterized abnormalities are translocations involving ALK, occurring in approximately half of anaplastic large cell lymphomas (ALCLs). To gain insight into the genetics of ALCLs lacking ALK translocations, we combined mate-pair DNA library construction, massively parallel ("Next Generation") sequencing, and a novel bioinformatic algorithm. We identified a balanced translocation disrupting the DUSP22 phosphatase gene on 6p25.3 and adjoining the FRA7H fragile site on 7q32.3 in a systemic ALK-negative ALCL. Using fluorescence in situ hybridization, we demonstrated that the t(6;7)(p25.3;q32.3) was recurrent in ALKnegative ALCLs. Furthermore, t(6;7)(p25.3; q32.3) was associated with down-regulation of DUSP22 and up-regulation of MIR29 microRNAs on 7q32.3. These findings represent the first recurrent translocation reported in ALK-negative ALCL and highlight the utility of massively parallel genomic sequencing to discover novel translocations in lymphoma and other cancers. IntroductionRecurrent chromosomal translocations are common pathogenetic events in hematologic malignancies. 1 Among peripheral (postthymic) T-cell lymphomas, however, the only well-characterized translocations are those involving the anaplastic lymphoma kinase gene ALK. 2 ALK is an important prognostic marker and therapeutic target in T-cell anaplastic large cell lymphomas (ALCLs) 3,4 ; however, approximately half of ALCLs lack ALK expression, despite nearly identical morphology and phenotype. 5 ALKnegative ALCLs can occur either cutaneously or systemically. We previously identified recurrent IRF4 translocations in cutaneous ALCLs, 6 but recurrent translocations in the more lethal, systemic form of ALK-negative ALCL have not been reported.Massively parallel ("Next Generation") DNA sequencing technology represents a quantum advance in the ability to understand cancer genomes. To identify translocations in ALK-negative ALCL, we performed massively parallel sequencing of a mate-pair DNA library constructed from a systemic ALK-negative ALCL. Using a unique bioinformatic algorithm for translocation discovery, we identified a translocation, t(6;7)(p25.3;q32.3), and demonstrated this translocation in additional ALK-negative ALCLs. This represents the first recurrent translocation reported in systemic ALKnegative ALCL, and demonstrates the utility of mate-pair library sequencing as a tool for translocation discovery. MethodsBriefly, mate-pair library construction followed the manufacturer's protocol (Illumina) using approximately 5-kb genomic DNA fragments. Sequencing was performed on an Illumina GAIIx, and results were mapped to the genome using a binary indexing algorithm. 7 Candidate translocations were validated by polymerase chain reaction (PCR), Sanger sequencing, and fluorescence in situ hybridization (FISH). Gene and microRNA expression levels were assessed using quantitative real-time PCR. The study was approved by the Mayo Clinic Institutional Review Board. Detail...

Peripheral T-cell lymphomas (PTCLs) are aggressive malignancies of mature T lymphocytes with 5-year overall survival rates of only ϳ 35%. Improvement in outcomes has been stymied by poor understanding of the genetics and molecular pathogenesis of PTCL, with a resulting paucity of molecular targets for therapy. We developed bioinformatic tools to identify chromosomal rearrangements using genomewide,

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Most human tumors have abnormal numbers of chromosomes, a condition known as aneuploidy. The mitotic checkpoint is an important mechanism that prevents aneuploidy by restraining the activity of the anaphasepromoting complex (APC). The deubiquitinase USP44 was identified as a key regulator of APC activation; however, the physiological importance of USP44 and its impact on cancer biology are unknown. To clarify the role of USP44 in mitosis, we engineered a mouse lacking Usp44. We found that USP44 regulated the mitotic checkpoint and prevented chromosome lagging. Mice lacking Usp44 were prone to the development of spontaneous tumors, particularly in the lungs. Additionally, USP44 was frequently downregulated in human lung cancer, and low expression correlated with a poor prognosis. USP44 inhibited chromosome segregation errors independent of its role in the mitotic checkpoint by regulating centrosome separation, positioning, and mitotic spindle geometry. These functions required direct binding to the centriole protein centrin. Our data reveal a new role for the ubiquitin system in mitotic spindle regulation and underscore the importance of USP44 in the pathogenesis of human cancer.

Epigenetic modifications in eukaryotic genomes occur primarily in the form of 5-methylcytosine (5 mC). These modifications are heavily involved in transcriptional repression, gene regulation, development and the progression of diseases including cancer. We report a new single-molecule assay for the detection of DNA methylation using solid-state nanopores. Methylation is detected by selectively labeling methylation sites with MBD1 (MBD-1x) proteins, the complex inducing a 3 fold increase in ionic blockage current relative to unmethylated DNA. Furthermore, the discrimination of methylated and unmethylated DNA is demonstrated in the presence of only a single bound protein, thereby giving a resolution of a single methylated CpG dinucleotide. The extent of methylation of a target molecule could also be coarsely quantified using this novel approach. This nanopore-based methylation sensitive assay circumvents the need for bisulfite conversion, fluorescent labeling, and PCR and could therefore prove very useful in studying the role of epigenetics in human disease.