Prolonged α-amanitin treatment of cells for studying mutated polymerases causes degradation of DSIF160 and other proteins.

A useful method for studying the function of the mammalian RNA polymerase II takes advantage of the extreme sensitivity of its largest subunit, Rpb1, to α-amanitin. Mutations of interest are introduced into an α-amanitin-resistant version of Rpb1, which is then expressed ectopically in cells. The phenotypes of these cells are then examined after inhibiting the endogenous wild-type polymerase with α-amanitin. Here, we show that cells that are enabled to grow in α-amanitin by expression of an α-amanitin-resistant Rpb1 exhibit changes in cell physiology that can lead to misleading experimental outcomes. The changes we have characterized include the accelerated degradation of some proteins, such as DSIF160, and the reduced rate of synthesis of others. In one series of experiments, we examined an α-amanitin-resistant construct, with a mutant C-terminal domain (CTD), that was unable to direct poly(A)-dependent transcription termination in cells growing in α-amanitin. The potential interpretation that the termination defect in this construct is due to the mutation in the CTD was rejected when the construct was found to be termination-competent in cells grown in the absence of α-amanitin. Instead, it appears that certain termination factors become limiting when the cells are grown in α-amanitin, presumably due to the α-amanitin-induced degradation we have characterized and/or to the inadequate transcription of certain genes by the α-amanitin-resistant Rpb1-containing polymerase.

Improving accuracy of Tay Sachs carrier screening of the non-Jewish population: analysis of 34 carriers and six late-onset patients with HEXA enzyme and DNA sequence analysis.

The purpose of this study was to determine whether combining different testing modalities namely beta-hexosaminidase A (HEXA) enzyme analysis, HEXA DNA common mutation assay, and HEXA gene sequencing could improve the sensitivity for carrier detection in non-Ashkenazi (AJ) individuals. We performed a HEXA gene sequencing assay, a HEXA DNA common mutation assay, and a HEXA enzyme assay on 34 self-reported Tay-Sachs disease (TSD) carriers, six late-onset patients with TSD, and one pseudodeficiency allele carrier. Sensitivity of TSD carrier detection was 91% for gene sequencing compared with 91% for the enzyme assay and 52% for the DNA mutation assay. Gene sequencing combined with enzyme testing had the highest sensitivity (100%) for carrier detection. Gene sequencing detected four novel mutations, three of which are predicted to be disease causing [118.delT, 965A-->T (D322V), and 775A-->G (T259A)]. Gene sequencing is useful in identifying rare mutations in patients with TSD and their families, in evaluating spouses of known carriers for TSD who have indeterminate enzyme analysis and negative for common mutation analysis, and in resolving ambiguous enzyme testing results.

Impact of Implementing the Paris System for Reporting Urinary Cytology: A Single-institutional Experience With Emphasis on Diagnostic Yield of High-grade Urothelial Carcinoma and Low-grade Urothelial Neoplasm.

BACKGROUND/AIM: The Paris System (TPS) has recently been proposed as a method to standardize urinary cytology reporting. In this study, we evaluated the impact of implementing TPS compared to the traditional reporting system. PATIENTS AND METHODS: In total, 299 urine samples were reclassified according to TPS. We examined correlations between cytological and histological diagnoses, and calculated probabilities for detecting high-grade urothelial carcinoma (HGUC). RESULTS: TPS resulted in a decrease in the proportion of cases diagnosed as atypical urothelial cell (AUC) (43% to 31%). Among the AUC cases, the proportion of histologically confirmed HGUC cases rose (75% to 80%), as did the proportion of low-grade urothelial neoplasms (57% to 71%). All probabilities for detecting HGUC significantly increased using TPS. CONCLUSION: TPS improved the diagnostic yield of urinary cytology. The implementation of TPS is expected to be a major step towards standardizing urinary cytology reporting and providing clear information to clinicians.

Genomic targets in saliva.

Saliva, the most accessible and noninvasive biofluid of our body, harbors a wide spectrum of biological analytes informative for clinical diagnostic applications. While proteomic constituents are a logical first choice as salivary diagnostic analytes, genomic targets have emerged as highly informative and discriminatory. This awareness, coupled with the ability to harness genomic information by high-throughput technology platforms such as genome-wide microarrays, ideally positions salivary genomic targets for exploring the value of saliva for detection of specific disease states and augmenting the diagnostic and discriminatory value of the saliva proteome for clinical applications. Buccal cells and saliva have been used as sources of genomic DNA for a variety of clinical and forensic applications. For discovery of disease targets in saliva, the recent realization that there is a transcriptome in saliva presented an additional target for oral diagnostics. All healthy subjects evaluated have approximately 3,000 different mRNA molecules in their saliva. Almost 200 of these salivary mRNAs are present in all subjects. Exploration of the clinical utility of the salivary transcriptome in oral cancer subjects shows that four salivary mRNAs (OAZ, SAT, IL8, and IL1b) collectively have a discriminatory power of 91% sensitivity and specificity for oral cancer detection. Data are also now in place to validate the presence of unique diagnostic panels of salivary mRNAs in subjects with Sjögren's disease.

The two steps of poly(A)-dependent termination, pausing and release, can be uncoupled by truncation of the RNA polymerase II carboxyl-terminal repeat domain.

The carboxyl-terminal repeat domain (CTD) of RNA polymerase II is thought to help coordinate events during RNA metabolism. The mammalian CTD consists of 52 imperfectly repeated heptads followed by 10 additional residues at the C terminus. The CTD is required for cleavage and polyadenylation in vitro. We studied poly(A)-dependent termination in vivo using CTD truncation mutants. Poly(A)-dependent termination occurs in two steps, pause and release. We found that the CTD is required for release, the first 25 heptads being sufficient. Neither the final 10 amino acids nor the variant heptads of the second half of the CTD were required. No part of the CTD was required for poly(A)-dependent pausing--the poly(A) signal could communicate directly with the body of the polymerase. By removing the CTD, pausing could be observed without being obscured by release. Poly(A)-dependent pausing appeared to operate by slowing down the polymerase, such as by down-regulation of a positive elongation factor. Although the first 25 heptads supported undiminished poly(A)-dependent termination, they did not efficiently support events near the promoter involved in abortive elongation. However, the second half of the CTD, including the final 10 amino acids, was sufficient for these functions.

Characterization of salivary RNA by cDNA library analysis.

Oral fluid (saliva) meets the demands for a noninvasive and accessible diagnostic medium. Recent reports by our group and others described the presence and use of human RNA in saliva as a diagnostic or forensic tool, including the use for oral cancer detection. To gain insights into the integrity of salivary RNA, we examined in detail the integrity of salivary RNA by generating a cDNA library from pooled supernatant saliva of 10 healthy donors. From a library with a primary library titer of 1.3 x 10(6) cfu/mL of which 95% of the clones had inserts, we successfully sequenced 117 random colonies containing recombinant clones. BLAST search results indicated that all of these clones contained sequences of human origin. Most of the salivary RNAs appeared to be endonucleolytically cleaved at random positions as indicated by comparisons to respective full length parental RNAs from the Genbank. Twelve of the insert sequences matched to the normal salivary core transcriptome sequences, which are highly abundant mRNAs present in healthy individuals. This study provides an in-depth molecular analysis of the saliva transcriptome and should be a useful resource for future basic and translational studies of RNA in human saliva. In addition, this paper presents unequivocal evidence for the presence of RNA in saliva as determined by the use of diverse techniques such as reverse transcriptase quantitative polymerase chain reaction (RT-qPCR), in vitro translation, and the construction of a salivary cDNA library.

Fibrinogen Seoul (FGG Ala341Asp): a novel mutation associated with hypodysfibrinogenemia.

Dysfibrinogenemia is a coagulation disorder caused by a variety of structural abnormalities in the fibrinogen molecule that result in fibrinogen function. The molecular basis of hypodysfibrinogenemia was investigated in a 66-year-old woman with peripheral artery obstructive disease and in her family members. Plasma level of functional fibrinogen determined using the Clauss method was lower (75 mg/dL; normal, 140-460 mg/dL) than that measured with immunologic nephelometric assay (137 mg/dL; normal, 180-400 mg/dL). Similar results were also observed in two family members through two generations. DNA was extracted from whole blood, and the coding regions and intron/exon boundaries of gamma chain gene (FGG) were amplified. A novel (Fibrinogen Seoul) heterozygous FGG mutation (GCT->GAT, Ala341Asp) was identified in all three affected family members. Thrombin-catalyzed polymerization was found to be defective on the analysis of purified fibinogen from the propositus. Molecular modeling also showed a conformational change of fibrinogen structure.

Measurement of cetuximab and panitumumab-unbound serum EGFR extracellular domain using an assay based on slow off-rate modified aptamer (SOMAmer) reagents.

BACKGROUND: Response to cetuximab (Erbitux®) and panitumumab (Vectibix®) varies among individuals, and even those who show response ultimately gain drug resistance. One possible etiologic factor is differential interaction between the drug and target. We describe the development of an assay based on Slow Off-rate Modified Aptamer (SOMAmer(™)) reagents that can distinguish drug-bound from unbound epidermal growth factor receptor (EGFR). METHODS: This quantitative assay uses a SOMAmer reagent specific for EGFR extracellular domain (ECD) as a capturing reagent. Captured SOMAmer is quantitated using PCR. Linearity and accuracy (recovery) of the assay were assessed using normal sera and purified EGFR ECD. RESULTS: This EGFR ECD assay showed linearity between 2.5 and 600 ng/mL. Average recovery was 101%. The assay detected EGFR but showed little cross-reactivity to other ErbB proteins: 0.4% for ErbB2, 6.9% for ErbB3, and 1.3% for ErbB4. Preincubation of normal serum with either cetuximab or panitumumab resulted in a dose-dependent decrease in EGFR ECD levels measured using the SOMAmer assay; preincubation did not affect measurement with an ELISA. CONCLUSIONS: This SOMAmer-based serum EGFR ECD assay accurately and specifically measures EGFR in serum. Detection of significant amounts of drug-unbound EGFR in patients undergoing cetuximab or panitumumab treatment could be an indicator of poor drug response. Further studies are needed to evaluate the utility of the assay as an indicator of drug efficacy or as a guide to dosing.

A universal pre-analytic solution for concurrent stabilization of salivary proteins, RNA and DNA at ambient temperature.

OBJECTIVE: Saliva is a biofluid that can be obtained from individuals without supervision by health care providers. To maximize this clinical advantage, it is highly desirable to have a global salivary analyte stabilizer for proteins, RNA and DNA at ambient temperature. DESIGN: Whole saliva, saliva supernatant and saliva filtrate (5.0 microm) were treated with RPS at room temperature (RT) for up to 6 days and then subjected to SDS-PAGE. Immunoblotting of beta-actin and cystatin C were used to evaluate protein stability. For salivary DNA/RNA, whole saliva was incubated with RPS at RT for up to 10 weeks. After extracting total DNA/RNA in samples at week 0, 2, 6 and 10, DNA stability was assayed by chromosome 18 DNA qPCR and RNA stability by beta-actin mRNA RT-qPCR. RESULTS: beta-actin completely degraded in all types of saliva samples after 6-day incubation at RT. However, 24.0%, 91.4% and 89.3% of beta-actin remained intact with RPS for whole saliva, saliva supernatant and filtrate, respectively. Similarly, 70.3% of cystatin C in supernatant remained intact in the presence of RPS. For salivary DNA/RNA, the cycle threshold (Ct) values showed no significant change for chromosome 18 DNA and beta-actin mRNA in RPS-incubated saliva during the 10-week time course while significant increase in Ct values were observed in controls without RPS for both beta-actin mRNA and DNA. CONCLUSIONS: RPS provided effective concurrent stabilization to salivary DNA/RNA in whole saliva for up to 10 weeks and proteins in saliva filtrate for 6 days at RT. We also achieved separation of saliva supernatant from cellular elements by a simple filtration step (bypassing the need for centrifugation).

Salivary microRNA: discovery, characterization, and clinical utility for oral cancer detection.

PURPOSE: We have previously shown that a transcriptome is found in saliva and subpanels of these mRNAs can be used as oral cancer biomarkers. In this study, we measured the presence of microRNAs (miRNA) in saliva and determined their potential as an additional set of oral cancer biomarkers. EXPERIMENTAL DESIGN: A total of 314 miRNAs were measured using reverse transcriptase-preamplification-quantitative PCR in 12 healthy controls. Degradation pattern of endogenous and exogenous saliva miRNAs were measured at room temperature over time. Selected miRNAs were validated in saliva of 50 oral squamous cell carcinoma patients and 50 healthy matched control subjects. RESULTS: We detected approximately 50 miRNAs in both the whole and supernatant saliva. Endogenous saliva miRNA degraded much slower compared with exogenous miRNA. Two miRNAs, miR-125a and miR-200a, were present in significantly lower levels (P < 0.05) in the saliva of oral squamous cell carcinoma patients than in control subjects. CONCLUSIONS: Both whole and supernatant saliva of healthy controls contained dozens of miRNAs, and similar to saliva mRNAs, these miRNAs are stable. Saliva miRNAs can be used for oral cancer detection.

Characterization of RNA in saliva.

BACKGROUND: We have previously shown that human mRNAs are present in saliva and can be used as biomarkers of oral cancer. In this study, we analyzed the integrity, sources, and stability of salivary RNA. METHODS: We measured the integrity of salivary RNA with reverse transcription followed by PCR (RT-PCR) or RT-quantitative PCR (RT-qPCR). To study RNA entry sites into the oral cavity, we used RT-PCR analysis of salivary RNA from the 3 major salivary glands, gingival crevice fluid, and desquamated oral epithelial cells. We measured stability of the salivary beta-actin mRNA by RT-qPCR of salivary RNA incubated at room temperature for different periods of time. We measured RNA association with other macromolecules by filtering saliva through pores of different sizes before performing RT-qPCR. To assess RNA-macromolecule interaction, we incubated saliva with Triton X-100 for different periods of time before performing RT-qPCR. RESULTS: In most cases, we detected partial- to full-length salivary mRNAs and smaller amounts of middle and 3' gene amplicons compared with the 5'. RNA was present in all oral fluids examined. Endogenous salivary beta-actin mRNA degraded more slowly than exogenous beta-actin mRNA, with half-lives of 12.2 and 0.4 min, respectively (P <0.001). Salivary RNA could not pass through 0.22 or 0.45 mum pores. Incubation of saliva with Triton X-100 accelerated degradation of salivary RNA. CONCLUSIONS: Saliva harbors both full-length and partially degraded forms of mRNA. RNA enters the oral cavity from different sources, and association with macromolecules may protect salivary RNA from degradation.