Carla Beckham

P Bodies, Stress Granules, and Viral Life Cycles

Eukaryotic mRNAs are in a dynamic equilibrium between different subcellular locations. Translating mRNAs can be found in polysomes, mRNAs stalled in translation initiation accumulate in stress granules and mRNAs targeted for degradation or translation repression can accumulate in P bodies. Partitioning of mRNAs between polysomes, stress granules, and P bodies affects rates of translation and mRNA degradation. Host proteins within P bodies and stress granules can enhance or limit viral infection, and some viral RNAs and proteins accumulate in P bodies and/or stress granules. Thus, an important interplay among P bodies, stress granules, and viral life cycles is beginning to emerge.

Interactions between Brome Mosaic Virus RNAs and Cytoplasmic Processing Bodies

ABSTRACT Cytoplasmic processing bodies are sites where nontranslating mRNAs accumulate for different fates, including decapping and degradation, storage, or returning to translation. Previous work has also shown that the Lsm1-7p complex, Dhh1p, and Pat1p, which are all components of P bodies, are required for translation and subsequent recruitment to replication of the plant virus brome mosaic virus (BMV) genomic RNAs when replication is reproduced in yeast cells. To better understand the role of P bodies in BMV replication, we examined the subcellular locations of BMV RNAs in yeast cells. We observed that BMV genomic RNA2 and RNA3 accumulated in P bodies in a manner dependent on cis-acting RNA replication signals, which also directed nonviral RNAs to P bodies. Furthermore, the viral RNA-dependent RNA polymerase coimmunoprecipitates and shows partial colocalization with the P-body component Lsm1p. These observations suggest that the accumulation of BMV RNAs in P bodies may be an important step in RNA replication complex assembly for BMV, and possibly for other positive-strand RNA viruses.

Expression of the Long Non-Coding RNA HOTAIR Correlates with Disease Progression in Bladder Cancer and Is Contained in Bladder Cancer Patient Urinary Exosomes.

Exosomes are 30-150nM membrane-bound secreted vesicles that are readily isolated from biological fluids such as urine (UEs). Exosomes contain proteins, micro RNA (miRNA), messenger RNA (mRNA), and long non-coding RNA (lncRNA) from their cells of origin. Although miRNA, protein and lncRNA have been isolated from serum as potential biomarkers for benign and malignant disease, it is unknown if lncRNAs in UEs from urothelial bladder cancer (UBC) patients can serve as biomarkers. lncRNAs are > 200 nucleotide long transcripts that do not encode protein and play critical roles in tumor biology. As the number of recognized tumor-associated lncRNAs continues to increase, there is a parallel need to include lncRNAs into biomarker discovery and therapeutic target algorithms. The lncRNA HOX transcript antisense RNA (HOTAIR) has been shown to facilitate tumor initiation and progression and is associated with poor prognosis in several cancers. The importance of HOTAIR in cancer biology has sparked interest in using HOTAIR as a biomarker and potential therapeutic target. Here we show HOTAIR and several tumor-associated lncRNAs are enriched in UEs from UBC patients with high-grade muscle-invasive disease (HGMI pT2-pT4). Knockdown of HOTAIR in UBC cell lines reduces in vitro migration and invasion. Importantly, loss of HOTAIR expression in UBC cell lines alters expression of epithelial-to-mesenchyme transition (EMT) genes including SNAI1, TWIST1, ZEB1, ZO1, MMP1 LAMB3, and LAMC2. Finally, we used RNA-sequencing to identify four additional lncRNAs enriched in UBC patient UEs. These data, suggest that UE-derived lncRNA may potentially serve as biomarkers and therapeutic targets.

Preparative separation of pyrrolizidine alkaloids by high-speed counter-current chromatography

We have applied a high-speed counter-current chromatography (CCC) technique to the separation and purification of pyrrolizidine alkaloids from Amsinckia tessellata, Symphytum spp., Trichodesma incanum (Boraginaceae), and Senecio douglasii var. longilobus (Asteraceae). Alkaloidal fractions were separated in a solvent system composed of a chloroform mobile phase and 0.2 M potassium phosphate buffer, of an optimum pH, as the stationary phase. Up to 800 mg of sample could be successfully separated in a single run, with excellent resolution of alkaloids. Lycopsamine and several of its acetylated derivatives were resolved from alkaloidal fractions of Amsinckia and Symphytum. However, diastereomeric pairs such as 7-acetyl-lycopsamine and 7-acetyl-intermedine, could not be separated. The presence of diastereoisomers was determined by gas chromatography-mass spectrometry. Trichodesma contained predominantly trichodesmine, which was resolved from a small quantity of incanine. we report the electron impact mass spectrum of incanine for the first time. Resolving power of CCC was sufficient to separate the closely related alkaloids senecionine and seneciphylline from Senecio, in addition to florosenine and retrorsine, Pyrrolizidine alkaloid compositions of the four species, determined by mass spectral techniques, were consistent with literature, except for the lack of riddelliine and the presence of the otonecine-based florosenine in Senecio douglasii var. longilobus.

Gender and Renal Cancer: Do Variations in Clinical Presentation and Imaging Patterns Explain Observed Differences Between Males and Females?

OBJECTIVES To determine whether gender variations in imaging and healthcare access are contributing to observed differences in renal cancer, we examine the initial events in the diagnosis of renal masses in a cohort of patients and correlate it with detailed data on imaging patterns over the same period. METHODS A total of 308 patients diagnosed with a renal mass over 11 years were reviewed. Information on symptoms, imaging, diagnosing physician, demographics, and pathology was gathered. Data on imaging for 1 862 485 patients at our institution over the same period were also collected. The data were analyzed for temporal trends, gender variations, and differences between incidental and nonincidental masses. RESULTS Females presented with smaller masses (4.8 vs 6.0 cm, P = .0064), and were less likely to have clear cell tumors (58.7% vs 63.4%, P = .049). A total of 66.9% of female and 61.1% of male cases were incidental (not significant). In both males and females, primary care physicians were the most common diagnosing physicians (47.4% and 49.6%, respectively). Gynecologic complaints were an uncommon cause of diagnosis for women (5.3%). Computerized tomography was the most common diagnosing modality for both males and females (69.1% and 63.2%, respectively). Ultrasound as the diagnosing modality did not reach statistical significance between males and females (23.4% and 28.6%, respectively). During the 11- year period, women underwent more imaging studies overall than men (19.7% difference), but the difference was lower when only considering studies that can diagnose renal masses (6.4% difference). CONCLUSIONS Gender variations in imaging rates and presentation for obstetrics/gynecology concerns by females did not lead to a significant difference in incidental diagnosis and do not appear adequate to explain gender differences in renal cancer presentation.

Membrane-association of mRNA decapping factors is independent of stress in budding yeast

Recent evidence has suggested that the degradation of mRNA occurs on translating ribosomes or alternatively within RNA granules called P bodies, which are aggregates whose core constituents are mRNA decay proteins and RNA. In this study, we examined the mRNA decapping proteins, Dcp1, Dcp2, and Dhh1, using subcellular fractionation. We found that decapping factors co-sediment in the polysome fraction of a sucrose gradient and do not alter their behaviour with stress, inhibition of translation or inhibition of the P body formation. Importantly, their localisation to the polysome fraction is independent of the RNA, suggesting that these factors may be constitutively localised to the polysome. Conversely, polysomal and post-polysomal sedimentation of the decapping proteins was abolished with the addition of a detergent, which shifts the factors to the non-translating RNP fraction and is consistent with membrane association. Using a membrane flotation assay, we observed the mRNA decapping factors in the lower density fractions at the buoyant density of membrane-associated proteins. These observations provide further evidence that mRNA decapping factors interact with subcellular membranes, and we suggest a model in which the mRNA decapping factors interact with membranes to facilitate regulation of mRNA degradation.

Overall Survival after Partial Versus Radical Nephrectomy for a Small Renal Mass: Systematic Review of Observational Studies

Introduction: In EORTC trial 30904 of partial versus radical nephrectomy overall survival was significantly better in the radical nephrectomy arm. However, many observational studies reported better survival after partial than radical nephrectomy. We present an updated systematic review of observational studies of overall survival after partial versus radical nephrectomy with assessment of quality of evidence. Methods: The literature search was performed until December 31, 2013, and all studies reporting overall survival after partial vs radical nephrectomy were included in the initial review. Further inclusion criteria for complete review were malignant tumors 7 cm or smaller, or benign tumors of any size, and survival analysis performed with adjustment for confounding variables. Studies not meeting these criteria were excluded from full review because of selection bias in favor of patients treated with partial nephrectomy who were younger and with less advanced tumors. Results: A total of 34 studies were included in the initial review and 13 were included in the full review. The 13 studies were based on the SEER database (6) or on institutional cohorts (7). In 8 of the 13 studies the estimated hazard ratios were significantly below 1, indicating better overall survival after partial nephrectomy, while in the remaining 5 studies estimated HR was not significantly different from 1. Median HR was 0.80 (interquartile range 0.57 to 0.96, absolute range 0.40 to 1.10). Conclusions: In most observational studies overall survival was better after partial than after radical nephrectomy. However, because residual confounding could be present despite adjustment for measured covariates, another randomized trial of partial vs radical nephrectomy may be needed to confirm or refute the findings of EORTC 30904.

MP98-13 HOTAIR AFFECTS BLADDER CANCER EPITHELIAL-TO-MESENCHYME TRANSITION THROUGH BOTH THE CANONICAL WNT-PATHWAY AND EXTRACELLULAR VESICLES

INTRODUCTION AND OBJECTIVES: Reactive oxygen species (ROS) have been identified as important chemical mediators in cell growth and differentiation. The glutathione redox system is the main mechanism protecting against damage caused by ROS in the human body. In this study, we investigated the role and therapeutic potential of the glutathione redox system in bladder cancer. METHODS: The expression levels of glutathione peroxidase 2 (GPX2) and Ki-67 proteins were analyzed in human transurethral resection (TUR) specimens by immunohistochemistry; correlations between the GPX2 expression and prognosis were also analyzed. In addition, male F344 rats were given 0.05% BBN in drinking water and 0.1% Phenyl isothiocyanate in their diet for 36 weeks. Bladder tissue samples were collected from each animal for analyses. Furthermore, the rat cell line, BC31, and human cell lines, T24, RT4, TCC-SUP, and 5637, were transfected with GPX2 siRNA and negative control siRNA (NC). Subsequently, cell proliferation rates and ROS levels were investigated by cell counting, DCFH assay, western blotting, and flow cytometry. siRNAor NCtransfected BC31 cells were subcutaneously implanted into nude mice. RESULTS: GPX2 was strongly expressed in low grade and low MIB-1 index cancers. PFS and CSS rates were significantly better in patients with higher GPX2 than in those with lower GPX2. Furthermore, GPX2 expression was significantly lower in the normal epithelium of the control group of animals with bladder cancer when compared with those in the treated group, and GPX2 expression was significantly higher in urothelial cancer than in the normal epithelium. BC31 and RT4 cells strongly expressed GPX2 when compared with the other cell lines. Silencing of GPX2 caused significant growth inhibition, and the DCFH assay revealed significant reductions in ROS levels in the siRNAtreated cells. Caspase-dependent apoptosis was fund to be the cause for the decrease in proliferation rates in the siRNA group. Interestingly, tumor growth was significantly inhibited in the BC31-implanted nude mice using the siRNA strategy for Gpx2. CONCLUSIONS: Our findings demonstrated that GPX2 plays several important roles in carcinogenesis through the regulation of apoptosis against intracellular ROS, and may be considered as a novel marker or therapeutic target in bladder cancer.