The Actinobacillus actinomycetemcomitans cytolethal distending toxin (Cdt) is a potent immunotoxin that induces G2 arrest in human lymphocytes. We now show that the CdtB subunit exhibits phosphatidylinositol (PI)-3,4,5-triphosphate phosphatase activity. Breakdown product analysis indicates that CdtB hydrolyzes PI-3,4,5-P3 to PI-3,4-P2 and therefore functions in a manner similar to phosphatidylinositol 5-phosphatases. Conserved amino acids critical to catalysis in this family of enzymes were mutated in the cdtB gene. The mutant proteins exhibit reduced phosphatase activity along with decreased ability to induce G2 arrest. Consistent with this activity, Cdt induces time-dependent reduction of PI-3,4,5-P3 in Jurkat cells. Lymphoid cells with defects in SHIP1 and/or ptase and tensin homolog deleted on chromosome 10 (PTEN) (such as Jurkat, CEM, Molt) and, concomitantly, elevated PI-3,4,5-P3 levels were more sensitive to the toxin than HUT78 cells which contain functional levels of both enzymes and low levels of PI-3,4,5-P3. Finally, reduction of Jurkat cell PI-3,4,5-P3 synthesis using the PI3K inhibitors, wortmannin and LY290004, protects cells from toxin-induced cell cycle arrest. Collectively, these studies show that the CdtB not only exhibits PI-3,4,5-P3 phosphatase activity, but also that toxicity in lymphocytes is related to this activity.

Convergent evidence implicates the TERE1 protein in human bladder tumor progression and lipid metabolism. Previously, reduced TERE1 expression was found in invasive urologic cancers and inhibited cell growth upon re-expression. A role in lipid metabolism was suggested by TERE1 binding to APOE, a cholesterol carrier, and to TBL2, a candidate protein in triglyceride disorders. Natural TERE1 mutations associate with Schnyder's corneal dystrophy, characterized by lipid accumulation. TERE1 catalyzes menaquinone synthesis, known to affect cholesterol homeostasis. To explore this relationship, we altered TERE1 and TBL2 dosage via ectopic expression and interfering RNA and measured cholesterol by Amplex red. Protein interactions of wild-type and mutant TERE1 with GST-APOE were evaluated by binding assays and molecular modeling. We conducted a bladder tumor microarray TERE1 expression analysis and assayed tumorigenicity of J82 cells ectopically expressing TERE1. TERE1 expression was reduced in a third of invasive specimens. Ectopic TERE1 expression in J82 bladder cancer cells dramatically inhibited nude mouse tumorigenesis. TERE1 and TBL2 proteins inversely modulated cellular cholesterol in HEK293 and bladder cancer cells from 20% to 50%. TERE1 point mutations affected APOE interactions, and resulted in cholesterol levels that differed from wild type. Elevated tumor cell cholesterol is known to affect apoptosis and growth signaling; thus, loss of TERE1 in invasive bladder cancer may represent a defect in menaquinone-mediated cholesterol homeostasis that contributes to progression.

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Step and Flash Imprint Lithography (S-FIL ® ) in conjunction with Sacrificial Imprint Materials (SIM) shows promise as a cost effective solution to patterning sub 45 nm features and is capable of simultaneously patterning two levels of interconnect structures, which provides a high throughput and low cost BEOL process. This paper describes the integration of S-FIL into an industry standard Cu/low-k dual damascene process that is being practiced in the ATDF at Sematech in Austin. The pattern transferring reactive ion etching (RIE) process is the most critical step and was extensively explored in this study. In addition to successful process development, the results provide useful insight into the optimal design of multilevel templates which must take into account the characteristics of both the imaging material and the dielectric layer.The template used in this study incorporates both the via and trench levels of an M2 (Metal 2) test vehicle that incorporates via chains with varying via dimensions, Kelvin test structures, serpentines, etc. The smallest vias on the template are 120 nm vias with an aspect ratio of 2.0 and the smallest dense lines are 125 nm/175 nm with an aspect ratio of 2.9. Two inter-level dielectrics (ILD), Coral ® and Black Diamond ® were studied. No trench etch stop was incorporated in the ILD film stack. A multi-step, in-situ etching scheme was developed that achieves faithful pattern transfer from the sacrificial imprint material (SIM) into the underlying low k ILD with surprisingly wide process latitude. This multi-step scheme includes the following etch steps: a residual layer open, a via etch, a trench descum, a trench etch, and an SIM removal ash. Among these steps, the trench etch was found to be the most challenging to develop and it holds the key to producing high aspect ratio dual damascene features. An etching chemistry based on two fluorocarbon gases, CF 4 and C 4 F 8 , was found to be very effective in delivering the desired etch profiles with optimal sidewall angle, minimal facet formation. The optimized etch process can be exploited to provide substantial size reduction and/or increased aspect ratio relative to the template. In this way structures with final critical dimensions of 95 nm in vias with aspect ratio of 3.0 and 67 nm/233 nm in dense lines with aspect ratio of 3.6 were demonstrated with wide process latitude. This enables manufacturing of the template at larger dimensions, which simplifies both fabrication and inspection.The successful development of the dual damascene RIE process at the second metal (M2) level was demonstrated in a mixed and matched build with an ATDF standard first layer metal (M1) process. The M1 dielectric was TEOS and was patterned by 248 nm lithography. The M2 and Via levels used Coral as ILD and both levels were patterned simultaneously by S-FIL using Molecular Imprint Imprio 55 and Imprio 100 imprint tools. This electrical test vehicle provided solid evidence that S-FIL is fully compatible with industry standard dual damascene process.