Development and Applications of Coarse-Grained Models for RNA

In contrast to proteins, much less attention has been focused on the development of computational models for describing RNA molecules, which are being recognized as playing key roles in many cellular functions. Current atomically detailed force fields are not accurate enough to capture the properties of even simple nucleic acid constructs. In this article, we review our efforts to develop coarse-grained (CG) models that capture the underlying physics for the particular length scale of interest. Two models are discussed. One of them is the three interaction site (TIS) model, in which each nucleotide is represented by three beads corresponding to sugar, phosphate, and base. The other is the self-organized polymer (SOP) model, in which each nucleotide is represented as a single interaction center. Applications of the TIS model to study the complexity of hairpin formation and the effects of crowding in a shifting equilibrium between two conformations in human telomerase pseudoknot are described. The work on crowding illustrates a direct link to the activity of telomerase. We use the SOP model to describe the response of the Tetrahymena ribozyme to force. The simulated unfolding pathways agree well with single molecule pulling experiments. We also review predictions for the unfolding pathways for the Azoarcus ribozyme. The success of the CG applications to describe dynamics in RNA gives hope that more complex processes involving RNA-protein interactions can be tackled using variants of the proposed models.     

Cooperative enhancement of deoxyribozyme activity by chemical modification and added cationic copolymer

Deoxyribozymes (DNAzymes) having RNA-cleaving activity have widely been explored as tools for therapeutic and diagnostic purposes. Both the chemical cleaving step and the turnover step should be improved for enhancing overall activity of DNAzymes. We have shown that cationic copolymer enhanced DNAzyme activity by increasing turnover efficacy. In this paper, effectｓ of the copolymer on DNAzymes modified with locked nucleic acids (LNA) or 2′-O-methylated (2′-OMe) nucleic acids were studied. The copolymer increased activity of these chemically modified DNAzymes. More than 30-fold enhancement in multiple-turnover catalytic activity was observed with 2′-OMe-modified DNAzyme in the presence of the copolymer. DNAzyme catalytic activity was successfully enhanced by cooperation of the added copolymer and chemical modification of DNAzyme.     

Quorum-sensing Cascades Governing Bacterial Multicellular Communities

Quorum sensing (QS) is an efficient mode of intercellular communication between bacteria. This mode is regulated by self-produced small chemical signals, activating dedicated receptors once accumulated. Numerous architecturally complex QS cascades are cardinal for governing bacterial behaviors, such as pathogenicity, luminescence, and bacterial competence. Importantly, QS cascades are essential for the formation of bacterial multicellular communities. Once informed via QS cascades, motile cells often organize themselves into conspicuous multicellular structures that carry out specialized tasks. This review focuses on the major QS systems, playing an active role in the rise of complex bacterial communities in different bacterial models.     

A novel developed method based on single primer isothermal amplification for rapid detection of Alicyclobacillus acidoterrestris in apple juice

Alicyclobacillus acidoterrestris (A. acidoterrestris), as one of the most major spoilage-causing species within the Alicyclobacillus genus, can survive and multiply in the pasteurization process. Nowadays, A. acidoterrestris has become worldwide issue in the fruit juice industry due to its spore-forming and thermo-acidophilic features. A novel single primer isothermal amplification assay (SPIA) was developed for the rapid detection of A. acidoterrestris in apple juice. This assay was designed to target the 16S rRNA gene with a DNA/RNA chimeric primer. Detection of gene amplification was accomplished by amplification curve, turbidity and addition of single strand DNA binding dye SYBR Green II allowing visualization under ultra violet light. The specificity of the assay was confirmed using 7 strains of A. acidoterrestris and 30 strains of non-A. acidoterrestris. The SPIA was highly sensitive and the detection limit was as low as 4.8 CFU/mL. Furthermore, the apple juice inoculated with 61 CFU/mL of A. acidoterrestris could be detected as positive. The novel SPIA with visualization results was successfully applied for the detection of A. acidoterrestris and exhibited high specificity and sensitivity, making it a powerful tool for the detection of A. acidoterrestris in fruit juice industry and being conveniently applied in developing countries with limited resource.     

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.     

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.