An economical and efficient protocol for total RNA isolation from Jatropha curcas

Jatropha curcas is an important non feed crop, increasingly important as a biofuel crop. It is hardy and resistant to different stress conditions in the field. In the wastelands of Gujarat (India), it is being grown for land reclamation and for socio-economic benefits. The long coastline in this state also promotes the growth of a large number of halophytes. Exploiting the genetic resource of Jatropha and halophytes for drought and salt-induced gene is an important area of research. For the isolation of genes and to study the molecular mechanism a good qualitative and quantitative RNA is a prerequisite. Jatropha leaves have latex, and therefore isolating RNA using guanidine thiocyanate or cetyltrimethylammonium bromide did not yield desirable quality of RNA. This paper reports a very simple and economical protocol for the isolation of good quality RNA from Jatropha and a few halophytes. The sodium dodecyl sulphate was used as a detergent for lysis of plant cells in the extraction buffer along with bentonite, which inhibits the ribonuclease’s activity. The addition of water saturated phenol in mortar-pestle, during grinding, facilitated better homogenisation of the tissues. Absolute RNA precipitation was obtained with the help of 2-butoxyethanol. Further this RNA was used successfully in preparation of complementary DNA and subsequently used for gene isolation.     

A Long Noncoding RNA ZEB1-AS1 Promotes Tumorigenesis and Predicts Poor Prognosis in Glioma

Emerging studies show that long noncoding RNAs (lncRNAs) have important roles in carcinogenesis. lncRNA ZEB1 antisense 1 (ZEB1-AS1) is a novel lncRNA, whose clinical significance, biological function, and underlying mechanism remains unclear in glioma. Here, we found that ZEB1-AS1 was highly expressed in glioma tissues, being closely related to clinical stage of glioma. Moreover, patients with high ZEB1-AS1 levels had poor prognoses, with the evidence provided by multivariate Cox regression analysis indicating that ZEB1-AS1 expression could serve as an independent prognostic factor in glioma patients. Functionally, silencing of ZEB1-AS1 could significantly inhibit cell proliferation, migration, and invasion, as well as promote apoptosis. Knockdown of ZEB1-AS1 significantly induced the G0/G1 phase arrest and correspondingly decreased the percentage of S phase cells. Further analysis indicated that ZEB1-AS1 could regulate the cell cycle by inhibiting the expression of G1/S transition key regulators, such as Cyclin D1 and CDK2. Furthermore, ZEB1-AS1 functioned as an important regulator of migration and invasion via activating epithelial to mesenchymal transition (EMT) through up-regulating the expression of ZEB1, MMP2, MMP9, N-cadherin, and Integrin-β1 as well as decreasing E-cadherin levels in the metastatic progression of glioma. Additionally, forced down-regulation of ZEB1-AS1 could dramatically promote apoptosis by increasing the expression level of Bax and reducing Bcl-2 expression in glioma. Taken together, our data suggest that ZEB1-AS1 may serve as a new prognostic biomarker and therapeutic target of glioma.     

A Comparison of Fresh Frozen vs. Formalin-Fixed,Paraffin-Embedded Specimens of Canine Mammary Tumors via Branched-DNA Assay

Mammary neoplasms are the tumors most affecting female dogs and women. Formalin-fixed, paraffin-embedded (FFPE) tissues are an invaluable source of archived biological material. Fresh frozen (FF) tissue is considered ideal for gene expression analysis. However, strategies based on FFPE material offer several advantages. Branched-DNA assays permit a reliable and fast workflow when analyzing gene expression. The aim of this study was to assess the comparability of the branched-DNA assay when analyzing certain gene expression patterns between FF and FFPE samples in canine mammary tumors. RNA was isolated from 109 FFPE samples and from 93 FF samples of different canine mammary tissues. Sixteen (16) target genes (Tp53; Myc; HMGA1; Pik3ca; Mcl1; MAPK3; FOXO3; PTEN; GATA4; PFDN5; HMGB1; MAPK1; BRCA2; BRCA1; HMGA2; and Her2) were analyzed via branched-DNA assay (b-DNA). ACTB, GAPDH, and HPRT1 were used as data normalizers. Overall, the relative gene expression of the two different origins of samples showed an agreement of 63%. Still, care should be taken, as FFPE specimens showed lower expression of the analyzed targets when compared to FF samples. The fact that the gene expression in FFPE proved to be lower than in FF specimens is likely to have been caused by the effect of storage time. ACTB had the best performance as a data normalizer.     

Identification of Autophagy in the Pine Wood Nematode Bursaphelenchus xylophilus and the Molecular Characterization and Functional Analysis of Two Novel Autophagy-Related Genes,BxATG1 and BxATG8

The pine wood nematode, Bursaphelenchus xylophilus, causes huge economic losses in pine forests, has a complex life cycle, and shows the remarkable ability to survive under unfavorable and changing environmental conditions. This ability may be related to autophagy, which is still poorly understood in B. xylophilus and no autophagy-related genes have been previously characterized. In this study, transmission electron microscopy was used to confirm that autophagy exists in B. xylophilus. The full-length cDNAs of BxATG1 and BxATG8 were first cloned from B. xylophilus, and BxATG1 and BxATG8 were characterized using bioinformatics methods. The expression pattern of the autophagy marker BxATG8 was investigated using in situ hybridization (ISH). BxATG8 was expressed in esophageal gland and hypodermal seam cells. We tested the effects of RNA interference (RNAi) on BxATG1 and BxATG8. The results revealed that BxATG1 and BxATG8 were likely associated with propagation of nematodes on fungal mats. This study confirmed the molecular characterization and functions of BxATG1 and BxATG8 in B. xylophilus and provided fundamental information between autophagy and B. xylophilus.     

Coarse-Grained and Atomistic MD Simulations of RNA and DNA Folding

Although the main features of the protein folding problem are coming into clearer focus, the microscopic viewpoint of nucleic acid folding mechanisms is only just beginning to be addressed. Experiments, theory, and simulations are pointing to complex thermodynamic and kinetic mechanisms. As is the case for proteins, molecular dynamics (MD) simulations continue to be indispensable tools for providing a molecular basis for nucleic acid folding mechanisms. In this review, we provide an overview of biomolecular folding mechanisms focusing on nucleic acids. We outline the important interactions that are likely to be the main determinants of nucleic acid folding energy landscapes. We discuss recent MD simulation studies of empirical force field and Go-type MD simulations of RNA and DNA folding mechanisms to outline recent successes and the theoretical and computational challenges that lie ahead.     

Dendrimer as nanocarrier for drug delivery

Dendrimers are novel three dimensional, hyperbranched globular nanopolymeric architectures. Attractive features like nanoscopic size, narrow polydispersity index, excellent control over molecular structure, availability of multiple functional groups at the periphery and cavities in the interior distinguish them amongst the available polymers. Applications of dendrimers in a large variety of fields have been explored. Drug delivery scientists are especially enthusiastic about possible utility of dendrimers as drug delivery tool. Terminal functionalities provide a platform for conjugation of the drug and targeting moieties. In addition, these peripheral functional groups can be employed to tailor-make the properties of dendrimers, enhancing their versatility. The present review highlights the contribution of dendrimers in the field of nanotechnology with intent to aid the researchers in exploring dendrimers in the field of drug delivery.     

Quantum‐dots‐based detection of hepatitis C virus (HCV) NS3 using RNA aptamer on chip

BACKGROUND: Over 170 million people, more than 3% of the world's population, suffer from the hepatitis C virus (HCV) infection and the rate of death from liver‐related mortality to HCV has increased. In respect of this, the development of assays for biological imaging should be urgently considered as an essential factor in diagnosis. RESULTS: A novel HCV‐detecting technique using a nanoparticle‐supported aptamer probe was demonstrated. With the aid of nanoparticle quantum dots (QDs) with carboxyl group as an imaging probe, and 5′‐end‐amine‐modified RNA oligonucleotide as a capturing probe, target HCV NS3 was visually detected on chip. The QDs‐based RNA aptamer for HCV NS3 showed high selectivity and specificity against other protein such as BSA. The detection limit of HCV NS3 protein was 5 ng mL^?1 level. CONCLUSION: With a novel strategy for protein–aptamer interaction, the feasibility of applying QDs‐based fluorescent detection technique to HCV viral protein assay for the development of a protein biochip was demonstrated. This scheme of QDs‐mediated imaging with a target‐oriented specific RNA aptamer for the detection of infectious HCV diseases provides an efficient strategy and a promising new platform for monitoring applications. Copyright © 2010 Society of Chemical Industry