Human mRNA DeXD/H-box helicases are ubiquitous molecular motors that are required for the majority of cellular processes that involve RNA metabolism. One of the most abundant is eIF4A, which is required during the initiation phase of protein synthesis to unwind regions of highly structured mRNA that would otherwise impede the scanning ribosome. Dysregulation of protein synthesis is associated with tumorigenesis, but little is known about the detailed relationships between RNA helicase function and the malignant phenotype in solid malignancies. Therefore, immunohistochemical analysis was performed on over 3000 breast tumors to investigate the relationship among expression of eIF4A1, the helicase-modulating proteins eIF4B, eIF4E and PDCD4, and clinical outcome. We found eIF4A1, eIF4B and eIF4E to be independent predictors of poor outcome in ER-negative disease, while in contrast, the eIF4A1 inhibitor PDCD4 was related to improved outcome in ER-positive breast cancer. Consistent with these data, modulation of eIF4A1, eIF4B and PCDC4 expression in cultured MCF7 cells all restricted breast cancer cell growth and cycling. The eIF4A1-dependent translatome of MCF7 cells was defined by polysome profiling, and was shown to be highly enriched for several classes of oncogenic genes, including G-protein constituents, cyclins and protein kinases, and for mRNAs with G/C-rich 5′UTRs with potential to form G-quadruplexes and with 3′UTRs containing microRNA target sites. Overall, our data show that dysregulation of mRNA unwinding contributes to the malignant phenotype in breast cancer via preferential translation of a class of genes involved in pro-oncogenic signaling at numerous levels. Furthermore, immunohistochemical tests are promising biomarkers for tumors sensitive to anti-helicase therapies.

Four-stranded G-quadruplex DNA secondary structures have recently been visualized in the nuclei of human cultured cells. Here, we show that BG4, a G-quadruplex-specific antibody, can be used to stain DNA G-quadruplex structures in patient-derived tissues using immunohistochemistry. We observe a significantly elevated number of G-quadruplex-positive nuclei in human cancers of the liver and stomach as compared to background non-neoplastic tissue. Our results suggest that G-quadruplex formation can be detected and measured in patient-derived material and that elevated G-quadruplex formation may be a characteristic of some cancers.

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The Patnaik histologic grading system is commonly used to predict the behavior of cutaneous mast cell tumors (MCTs) in dogs, but it is less useful for grade 2 MCTs because they exhibit considerable variation in biological behavior. In this retrospective study, immunohistochemical staining for Ki-67, proliferating cell nuclear antigen (PCNA), and survivin and a standardized argyrophilic staining of nucleolar organizer regions (AgNOR) protocol were performed on 121 archived paraffin-embedded specimens of canine cutaneous MCTs, for which clinical follow-up data were available. Cox regression models indicated that the Ki-67 score (hazard ratio, 1.92; P , .001) and mean AgNOR score (hazard ratio, 2.57; P , .001) were significantly associated with Patnaik grade and survival time. A binary Ki-67 variable (cutoff point Ki-67 score 5 1.8) was a significant predictor of survival for dogs with grade 2 MCTs. The estimated 1-, 2-, and 3-year survival probabilities for dogs with grade 2 MCTs and Ki-67 scores less than 1.8 were 0.92, 0.86, and 0.77, respectively (SEs, 0.08, 0.14, and 0.23, respectively; median not estimable). The corresponding survival probabilities for dogs with grade 2 MCTs and Ki-67 scores higher than 1.8 were 0.43, 0.21, and 0.21, respectively (SEs, 0.19, 0.18, and 0.18, respectively; median survival time, 395 days). No significant association was identified between survival and survivin score or PCNA score. This study shows that both mean AgNOR score and Ki-67 score are prognostic markers for canine MCTs. The Ki-67 score can be used to divide Patnaik grade 2 MCTs into 2 groups with markedly different expected survival times.

We retrospectively demonstrated that an outbreak of severe respiratory disease in a pack of English foxhounds in the United Kingdom in September 2002 was caused by an equine influenza A virus (H3N8). We also demonstrated that canine respiratory tissue possesses the relevant receptors for infection with equine influenza virus.

Estrogen Receptor-β (ERβ) has been implicated in many cancers. In prostate and breast cancer its function is controversial, but genetic studies implicate a role in cancer progression. Much of the confusion around ERβ stems from antibodies that are inadequately validated, yet have become standard tools for deciphering its role. Using an ERβ-inducible cell system we assessed commonly utilized ERβ antibodies and show that one of the most commonly used antibodies, NCL-ER-BETA, is non-specific for ERβ. Other antibodies have limited ERβ specificity or are only specific in one experimental modality. ERβ is commonly studied in MCF-7 (breast) and LNCaP (prostate) cancer cell lines, but we found no ERβ expression in either, using validated antibodies and independent mass spectrometry-based approaches. Our findings question conclusions made about ERβ using the NCL-ER-BETA antibody, or LNCaP and MCF-7 cell lines. We describe robust reagents, which detect ERβ across multiple experimental approaches and in clinical samples.

Blocking Notch signaling in pancreatic cancer promotes hypoxia and cell death.

The purpose of this study was to evaluate the pharmacokinetic profile of intranasal lorazepam in comparison to currently established administration routes. Eleven healthy volunteers completed this randomized crossover study. On three occasions, each separated by a 1-week washout, subjects received a 2 mg dose of lorazepam via the intranasal, intravenous, or intramuscular route. Blood samples were collected serially from 0 to 36 hours. Noncompartmental methods were used to determine pharmacokinetic parameters. Lorazepam was well absorbed following intranasal administration with a mean (%CV) bioavailability of 77.7(11.1). Intranasal administration resulted in a faster absorption rate than intramuscular administration. Elimination profiles were comparable between all three routes. The concentration-time profile for intranasal delivery demonstrated evidence of a double peak in several subjects, suggesting partial oral absorption. Females were found to have significantly higher AUC values than males for all three delivery routes. Overall, this study demonstrated favorable pharmacokinetics of intranasal lorazepam in relation to standard administration methods. Intranasal delivery could provide an alternative, noninvasive delivery route for lorazepam.