Efficient fluorescence labeling of a large RNA through oligonucleotide hybridization

We present an efficient method of introducing fluorophore labels at selected locations in a large RNA. The method is based on specific and highly efficient hybridization between a fluorophore-containing DNA oligonucleotide and a modular hairpin loop replacing a functionally unimportant hairpin loop in the RNA. We demonstrate its feasibility using a 255-nucleotide RNA derived from the catalytic domain of RNase P from Bacillus subtilis. Hybridization of the DNA oligonucleotide to the modular hairpin loop minimally perturbs the structure and function of this RNA. This labeling scheme should be applicable in studies of RNA conformational dynamics by ensemble and single molecule fluorescence methods.     

Multiclass cancer classification and biomarker discovery using GA-based algorithms

MOTIVATION: The development of microarray-based high-throughput gene profiling has led to the hope that this technology could provide an efficient and accurate means of diagnosing and classifying tumors, as well as predicting prognoses and effective treatments. However, the large amount of data generated by microarrays requires effective reduction of discriminant gene features into reliable sets of tumor biomarkers for such multiclass tumor discrimination. The availability of reliable sets of biomarkers, especially serum biomarkers, should have a major impact on our understanding and treatment of cancer. RESULTS: We have combined genetic algorithm (GA) and all paired (AP) support vector machine (SVM) methods for multiclass cancer categorization. Predictive features can be automatically determined through iterative GA/SVM, leading to very compact sets of non-redundant cancer-relevant genes with the best classification performance reported to date. Interestingly, these different classifier sets harbor only modest overlapping gene features but have similar levels of accuracy in leave-one-out cross-validations (LOOCV). Further characterization of these optimal tumor discriminant features, including the use of nearest shrunken centroids (NSC), analysis of annotations and literature text mining, reveals previously unappreciated tumor subclasses and a series of genes that could be used as cancer biomarkers. With this approach, we believe that microarray-based multiclass molecular analysis can be an effective tool for cancer biomarker discovery and subsequent molecular cancer diagnosis.     

Fractionation of wheat straw by atmospheric acetic acid process

Fractionation of wheat straw was investigated using an atmospheric acetic acid process. Under the typical conditions of 90% (v/v) aqueous AcOH, 4% H(2)SO(4) (w/w, on straw), ratio of liquor to straw (L/S) 10 (v/w), pulping temperature 105 degrees C, and pulping time 3h, wheat straw was fractionated to pulp (cellulose), lignin and monosaccharides mainly from hemicellulose with yields of approximately 50%, 15% and 35%, respectively. Acetic acid pulp from the straw had an acceptable strength for paper and could be bleached to a high brightness over 85% with a short bleaching sequence. Acetic acid pulp was also a potential feedstock for fuels and chemicals. The acetic acid process separated pentose and hexose in wheat straw to a large extent. Most of the pentose (xylan) was dissolved, whereas the hexose (glucan) remained in the pulp. Approximately 30% of carbohydrates in wheat straw were hydrolyzed to monosaccharides during acetic acid pulping, of which xylose accounted for 70% and glucose for 12%. The acetic acid lignin from wheat straw showed relatively lower molecular weight and fusibility, which made the lignin a promising raw material for many products, such as adhesive and molded products.     

Estrogen receptor beta selective ligands: discovery and SAR of novel heterocyclic ligands

A series of ligands with varying heterocyclic cores and substituents that display a range of selectivity’s (up to >100x) for ER-β over ER- are reported.     

Progesterone inhibits the estrogen-induced phosphoinositide 3-kinase-->AKT-->GSK-3beta-->cyclin D1-->pRB pathway to block uterine epithelial cell proliferation

The mammalian cell cycle is regulated by the cyclin/cyclin-dependent kinase (CDK) phosphorylation of the retinoblastoma (pRB) family of proteins. Cyclin D1 with its CDK4/6 partners initiates the cell cycle and acts as the link between extracellular signals and the cell cycle machinery. Estradiol-17beta (E2) stimulates uterine epithelial cell proliferation, a process that is completely inhibited by pretreatment with progesterone (P4). Previously, we identified cyclin D1 localization as a key point of regulation in these cells with E2 causing its nuclear accumulation and P4 retaining it in the cytoplasm with the resultant inhibition of pRB phosphorylation. Here we show that E2 stimulates phosphoinositide 3-kinase to activate phosphokinase B/AKT to effect an inhibitory phosphorylation of glycogen synthase kinase (GSK-3beta). This pathway is suppressed by P4. Inhibition of the GSK-3beta activity in P4-treated uteri by the specific inhibitor, LiCl, reversed the nuclear accumulation of cyclin D1 and in doing so, caused pRB phosphorylation and the induction of downstream genes, proliferating cell nuclear antigen and Ki67. Conversely, inhibition of phosphoinositide 3 kinase by LY294002 or Wortmanin reversed the E2-induced GSK-3beta Ser9 inhibitory phosphorylation and blocked nuclear accumulation of cyclin D1. These data show the reciprocal actions of E2 and P4 on the phosphoinositide 3-kinase through to the GSK-3beta pathway that in turn regulates cyclin D1 localization and cell cycle progression. These data reveal a novel signaling pathway that links E2 and P4 action to growth factor-mediated signaling in the uterus.     

The iron chelator desferrioxamine inhibits atherosclerotic lesion development and decreases lesion iron concentrations in the cholesterol-fed rabbit

Several epidemiological studies have suggested that increased iron stores are associated with increased atherosclerotic events. In order to test the hypothesis that decreasing the vascular level of iron slows lesion growth, we examined the effects of the iron chelator Desferal (72 mg/kg/day, 5 days/week) on atherosclerosis and lesion iron content in cholesterol-fed New Zealand White rabbits. Rabbits were fed with a 1% w/w cholesterol diet for either 8 weeks (and for the last 5 weeks injected daily with Desferal) or 12 weeks (and for the last 9 weeks injected with Desferal). Controls were injected with saline. A significant reduction in average lesion area (p = 0.038) was observed in the 12-week treated animals compared with the 12-week controls. The average lesion iron level of the 12-week treated animals (58 ppm dry wt) was also significantly lower (p = 0.030) than in 12-week control animals (95 ppm dry wt), as measured using nuclear microscopy with the combination of scanning transmission ion microscopy, Rutherford back-scattering spectroscopy, and particle-induced X-ray emission. No reduction in lesion area or iron content was observed in the 8-week treated animals compared with controls, and no change in lesion zinc concentration was observed for either group. Our data strengthen the concept that iron contributes to the early stages of the development of atherosclerosis.     

Stabilization of cold-adapted protease MCP-01 promoted by trehalose: prevention of the autolysis

Protease MCP-01 is similar to other cold-adapted enzymes in that it is a cold-adapted serine protease having high specific activity and low thermostability at low and moderate temperature. Its thermolability and self-autolysis has resulted in difficulties in its purification, preservation and research on its structure and function. The disaccharide trehalose is known to effectively stabilize proteins. Its prevention effect on the autolysis of cold-adapted protease MCP-01 was monitored by capillary electrophoresis. In the absence of trehalose, protease MCP-01 autolyzed rapidly at 35 degrees C. However, when trehalose was added, autolysis was remarkably prevented and the loss of activity reduced. MCP-01 may be a useful model for basic research on the interaction of protein and trehalose.     

Qualitative and Quantitative PCR Methods for Event-specific Detection of Genetically Modified Cotton Mon1445 and Mon531

Based on the DNA sequences of the junctions between recombinant and cotton genomic DNA of the two genetically modified (GM) cotton varieties, herbicide-tolerance Mon1445 and insect-resistant Mon531, event-specific primers and probes for qualitative and quantitative PCR detection for both GM cotton varieties were designed, and corresponding detection methods were developed. In qualitative PCR detection, the simplex and multiplex PCR detection systems were established and employed to identify Mon1445 and Mon531 from other GM cottons and crops. The limits of detection (LODs) of the simplex PCR were 0.05% for both Mon1445 and Mon531 using 100 ng DNA templates in one reaction, and the LOD of multiplex PCR analysis was 0.1%. For further quantitative detection using TaqMan real-time PCR systems for Mon1445 and Mon531, one plasmid pMD-ECS, used as reference molecule was constructed, which contained the quantitative amplified fragments of Mon1445, Mon531, and cotton endogenous reference gene. The limits of quantification (LOQs) of Mon1445 and Mon531 event-specific PCR systems using plasmid pMD-ECS as reference molecule were 10 copies, and the quantification range was from 0.03 to 100% in 100 ng of the DNA template for one reaction. Thereafter, five mixed cotton samples containing 0, 0.5, 0.9, 3 and 5% Mon1445 or Mon531 were quantified using established real-time PCR systems to evaluate the accuracy and precision of the developed real-time PCR detection systems. The accuracy expressed as bias varied from 1.33 to 8.89% for tested Mon1445 cotton samples, and from 2.67 to 6.80% for Mon531. The precision expressed as relative standard deviations (RSD) were different from 1.13 to 30.00% for Mon1445 cotton, and from 1.27 to 24.68% for Mon531. The range of RSD was similar to other laboratory results (25%). Concluded from above results, we believed that the established event-specific qualitative and quantitative PCR systems for Mon1445 and Mon531 in this study are acceptable and suitable for GM cotton identification and quantification.     

An efficient preparation of polyanionic affinity agent and its evaluation for the measurement of glycated hemoglobin

An efficient method was developed for the preparation of polyanionic affinity agent (3), a key component in the measurement of glycated hemoglobin (GHb). Glycated hemoglobin is an important clinical marker for diagnosis of patients with diabetes and useful to monitor the management of disease. The affinity agent (3) was prepared based on coupling reaction between poly(acrylic acid) (1) and 3-aminophenylboronic acid (2) in water. The critical features of this polymeric affinity agent (3), such as size, boronic acid incorporation ratio and concentration, on the measurement of glycated hemoglobin were evaluated. It was found that the agent (3) prepared using poly(acrylic acid) (1) with 225 kDa molecular weight gave optimal GHb measurement. The performance test results demonstrated that the boronic acid incorporation ratio and concentration of affinity agent (3) play a critical role in the assay and determines the precision of glycated hemoglobin measurement.