Faculty Bibliography

Despite the wide acceptance of yeast two-hybrid (Y2H) system for protein-protein interaction analysis and discovery, conventional Y2H assays are not well suited for high-throughput screening of the protein interaction network ("interactome") on a genomic scale due to several limitations, including labor-intensive agar plating and colony selection methods associated with the use of nutrient selection markers, complicated reporter analysis methods associated with the use of LacZ enzyme reporters, and incompatibility of the liquid handling robots. We recently reported a robust liquid culture Y2H system based on quantitative analysis of yeast-enhanced green fluorescent protein (yEGFP) reporters that greatly increased the analysis throughput and compatibility with liquid handling robots. To further advance its utility in high-throughput complementary DNA (cDNA) library screening, we report the development of a novel surface display Y2H (sdY2H) library screening system with uniquely integrated surface display hemagglutination (sdHA) antigen and yEGFP reporters. By introduction of a surface reporter sdHA into the yEGFP-based Y2H system, positive Y2H targets are quickly isolated from library cells by a simple magnetic separation without a large plating effort. Moreover, the simultaneous scoring of multiple reporters, including sdHA, yEGFP, and conventional nutrient markers, greatly increased the specificity of the Y2H assay. The feasibility of the sdY2H assay on large cDNA library screening was demonstrated by the successful recovery of positive P53/T interaction pairs at a target-to-background ratio of 1:1,000,000. Together with the massive parallel DNA sequencing technology, it may provide a powerful proteomic tool for high-throughput interactome mapping on a genomic scale.

OBJECTIVE:To examine the diagnostic value (sensitivity and specificity of the ThinPrep Pap test in the detection of endometrial cancer and assess the morphologic features of endometrial cancer in ThinPrep tests. STUDY DESIGN/METHODS:In a retrospective, case-controlled study, we identified 60 Pap slides performed within 12 months of the tissue diagnosis of endometrial carcinoma and 56 comparison slides from patients without known carcinoma. The slides were blindly reviewed by the authors without knowledge of the surgical diagnosis. An independent diagnosis was given for the tests based on 14 diagnostic criteria from the 2001 Bethesda System and 6 additional criteria proposed by the authors. RESULTS:The sensitivity of detecting endometrial carcinoma was 88.3% (95% CI 77.8-94.2%) and specificity was 87.5% (95% CI 76.4-93.8%). The positive likelihood ratio was 7.067 (95% CI 3.513-14.217) and negative likelihood ratio was 0.133 (95% CI 0.066-0.269). Enlarged nuclei and the presence of nucleoli in endometrial cells were the most reliable indicators of endometrial cancer or atypical endometrial cells. CONCLUSION/CONCLUSIONS:The ThinPrep Pap test has high sensitivity and specificity in detecting or suggesting the presence of endometrial cancer. Certain cytomorphologic features are helpful in distinguishing benign and malignant endometrial lesions.

The aim of this retrospective study was to correlate cytological diagnoses of endometrial cancers in ThinPrep Pap tests with the histological diagnoses. ThinPrep specimens from 67 patients within 12 mo of the histological diagnosis of endometrial cancer were studied. Of this study sample, 89.6% had abnormal Pap tests. Abnormal Pap tests occurred in 96.8, 68.4, and 100% of patients with grades 1, 2, or 3 endometrial cancers, respectively. Of patients with endocervical involvement, 88.9% had positive or suspicious Pap tests, compared with 41.1% without endocervical involvement (LR = 7.85, P < 0.01). Of patients with > or =50% myometrial invasion, 78.9% had positive or suspicious Pap tests, compared with 34.8% with less than 50% invasion (LR = 10.97, P < 0.01). Positive or suspicious Pap tests were found in 59.5 and 32.1% of those with tumors > or =3 cm or <3cm, respectively (LR = 4.85, P < 0.05).

The survival, differentiation, and maintenance of responsive neurons are regulated by nerve growth factor (NGF), which is secreted by the target and interacts with receptors on the axon tip. It is uncertain how the NGF signal is communicated retrogradely from distal axons to neuron cell bodies. Retrograde transport of activated receptors in endocytic vesicles could convey the signal. However, little is known about endocytosis of NGF receptors, and there is no evidence that NGF receptors continue to signal after endocytosis. We have examined early events in the membrane traffic of NGF and its receptor, gp140(TrkA) (TrkA), in PC12 cells. NGF induced rapid and extensive endocytosis of TrkA in these cells, and the receptor subsequently moved into small organelles located near the plasma membrane. Some of these organelles contained clathrin and alpha-adaptin, which implies that TrkA is internalized by clathrin-mediated endocytosis. Using mechanical permeabilization and fractionation, intracellular organelles derived from endocytosis were separated from the plasma membrane. After NGF treatment, NGF was bound to TrkA in endocytic organelles, and TrkA was tyrosine-phosphorylated and bound to PLC-gamma1, suggesting that these receptors were competent to initiate signal transduction. These studies raise the possibility that NGF induces formation of signaling endosomes containing activated TrkA. They are an important first step in elucidating the molecular mechanism of NGF retrograde signaling.