Characterization of anti-NF-κB RNA aptamer-binding specificity in vitro and in the yeast three-hybrid system

RNA aptamers offer a potential therapeutic approach to the competitive inhibition of DNA-binding transcription factors. In previous reports we described in vitro selection and characterization of anti-NF-κB p50 and p65 RNA aptamers. We now describe the further characterization of these aptamers in vitro and in vivo. We show that sub-saturating concentrations of certain anti-p50 RNA aptamers promote complex formation with NF-κB p50 tetramers, whereas anti-p65 R1 RNA aptamers bind NF-κB dimers under all conditions tested. Yeast three-hybrid RNA aptamer specificity studies corroborate previous in vitro results, verifying that anti-p50 and anti-p65 R1 RNA aptamers are highly specific for NF-κB p502 and p652, respectively. These studies introduce a novel T-cassette RNA transcript that improves RNA display from a four-way RNA junction. Mutagenesis of the anti-p65 R1 aptamer reveals tolerated substitutions, suggesting a complex tertiary structure. We describe in vivo selections from a yeast three-hybrid RNA library containing sequences present early in the R1 SELEX process to identify novel anti-p65 RNA aptamers, termed Y1 and Y3. These aptamers appear to be compact bulged hairpins, reminiscent of anti-p50. Y1 competitively inhibits the DNA-binding domain of NF-κB p652 in vitro

Improved prediction of RNA tertiary structure with insights into native state dynamics

The importance of RNA tertiary structure is evident from the growing number of published high resolution NMR and X-ray crystallographic structures of RNA molecules. These structures provide insights into function and create a knowledge base that is leveraged by programs such as Assemble, ModeRNA, RNABuilder, NAST, FARNA, Mc-Sym, RNA2D3D, and iFoldRNA for tertiary structure prediction and design. While these methods sample native-like RNA structures during simulations, all struggle to capture the native RNA conformation after scoring. The authors propose RSIM, an improved RNA fragment assembly method that preserves RNA global secondary structure while sampling conformations. This approach enhances the quality of predicted RNA tertiary structure, provides insights into the native state dynamics, and generates a powerful visualization of the RNA conformational space

Characterization of anti-NF-iB RNA aptamerbinding

specificity in vitro and in the yeast three-hybrid syste

Distinct role of PLCβ3 in VEGF-mediated directional migration and vascular sprouting

Endothelial cell proliferation and migration is essential to angiogenesis. Typically, proliferation and chemotaxis of endothelial cells is driven by growth factors such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). VEGF activates phospholipases (PLCs) – specifically PLCγ1 – that are important for tubulogenesis, differentiation and DNA synthesis. However, we show here that VEGF, specifically through VEGFR2, induces phosphorylation of two serine residues on PLCβ3, and this was confirmed in an ex vivo embryoid body model. Knockdown of PLCβ3 in HUVEC cells affects IP3 production, actin reorganization, migration and proliferation; whereas migration is inhibited, proliferation is enhanced. Our data suggest that enhanced proliferation is precipitated by an accelerated cell cycle, and decreased migration by an inability to activate CDC42. Given that PLCβ3 is typically known as an effector of heterotrimeric G-proteins, our data demonstrate a unique crosstalk between the G-protein and receptor tyrosine kinase (RTK) axes and reveal a novel molecular mechanism of VEGF signaling and, thus, angiogenesis

High-Throughput Screening for Growth Inhibitors Using a Yeast Model of Familial Paraganglioma

<div><p>Classical tumor suppressor genes block neoplasia by regulating cell growth and death. A remarkable puzzle is therefore presented by familial paraganglioma (PGL), a neuroendocrine cancer where the tumor suppressor genes encode subunits of succinate dehydrogenase (SDH), an enzyme of the tricarboxylic acid (TCA) cycle of central metabolism. Loss of SDH initiates PGL through mechanisms that remain unclear. Could this metabolic defect provide a novel opportunity for chemotherapy of PGL? We report the results of high throughput screening to identify compounds differentially toxic to SDH mutant cells using a powerful <i>S. cerevisiae</i> (yeast) model of PGL. Screening more than 200,000 compounds identifies 12 compounds that are differentially toxic to SDH-mutant yeast. Interestingly, two of the agents, dequalinium and tetraethylthiuram disulfide (disulfiram), are anti-malarials with the latter reported to be a glycolysis inhibitor. We show that four of the additional hits are potent inhibitors of yeast alcohol dehydrogenase. Because alcohol dehydrogenase regenerates NAD⁺ in glycolytic cells that lack TCA cycle function, this result raises the possibility that lactate dehydrogenase, which plays the equivalent role in human cells, might be a target of interest for PGL therapy. We confirm that human cells deficient in SDH are differentially sensitive to a lactate dehydrogenase inhibitor.</p> </div

The NAFLD Fibrosis Score: A Noninvasive System That Identifies Liver Fibrosis in Patients with NAFLD

Patients with nonalcoholic fatty liver disease (NAFLD) and advanced liver fibrosis are at the highest risk for progressing to end-stage liver disease. We constructed and validated a scoring system consisting of routinely measured and readily available clinical and laboratory data to separate NAFLD patients with and without advanced fibrosis. A total of 733 patients with NAFLD confirmed by liver biopsy were divided into 2 groups to construct (n = 480) and validate (n = 253) a scoring system. Routine demographic, clinical, and laboratory variables were analyzed by multivariate modeling to predict presence or absence of advanced fibrosis. Age, hyperglycemia, body mass index, platelet count, albumin, and AST/ALT ratio were independent indicators of advanced liver fibrosis. A scoring system with these 6 variables had an area under the receiver operating characteristic curve of 0.88 and 0.82 in the estimation and validation groups, respectively. By applying the low cutoff score (-1.455), advanced fibrosis could be excluded with high accuracy (negative predictive value of 93% and 88% in the estimation and validation groups, respectively). By applying the high cutoff score (0.676), the presence of advanced fibrosis could be diagnosed with high accuracy (positive predictive value of 90% and 82% in the estimation and validation groups, respectively). By applying this model, a liver biopsy would have been avoided in 549 (75%) of the 733 patients, with correct prediction in 496 (90%). Conclusion: a simple scoring system accurately separates patients with NAFLD with and without advanced fibrosis, rendering liver biopsy for identification of advanced fibrosis unnecessary in a substantial proportion of patients

Experimental yeast growth curves on (A) galactose medium; (B) glycerol medium; (C) 1∶1 galactose:glycerol medium.

<p>WT (solid black), <i>sdh2Δ</i> (dashed red). <i>nhp6AΔ</i>, (solid grey).</p

Structures and yeast growth curves in galactose:glycerol (1∶1) medium showing differential growth inhibition of <i>sdh2Δ</i> mutant yeast for six nitro-alkene compounds (potential Michael acceptors) from main library HTS.

<p>A. 6035147 (12 µM). B. 6988322 (25 µM). C. 7241889 (6 µM). D. 7279172 (50 µM). E. 7289669 (3 µM). F. 7312219 (25 µM). Solid lines: growth in the absence of drug for WT (black) and <i>sdh2Δ</i> mutant (red). Dashed lines: growth in the presence of the indicated drug concentration.</p

Structures and yeast growth curves in galactose:glycerol (1∶1) medium showing differential growth inhibition of <i>sdh2Δ</i> mutant yeast for four non-reactive compounds from main library HTS.

<p>A. 7619814 (50 µM). B. 7172827 (50 µM). C. 7783421 (25 µM). D. 4032-1245 (6 µM). Solid lines: growth in the absence of drug for WT (black) and <i>sdh2Δ</i> mutant (red). Dashed lines: growth in the presence of the indicated drug concentration.</p