Norgen Biotek Corporation (Canada)

MicroRNAs (miRNAs), ~22 nt single-stranded non-coding RNAs (ncRNAs) abundant in the human brain and retina, have emerged as significant post-transcriptional regulators of messenger RNA (mRNA) abundance and complexity in the human central nervous system (CNS) in aging, health, and disease. Of the 2050 different miRNAs in the human body so far identified, only about 25-30 are abundant in either the brain or the retina, underscoring the high selection pressure carried by RNA sequences located within these select ncRNAs (1-7). It is noteworthy to point out that: (i) that brain neocortex and retina share a common neuroectodermal origin; (ii) that brain and retina share a subfamily of specific miRNA species; and (iii) that the multilayered assemblies of both neural and retinal cells are targeted by pathogenic processes that drive progressive pro-inflammatory neurodegeneration (5-9). Indeed, pathologically upregulated miRNAs common to both the prototypic age-related inflammatory degeneration of the brain in Alzheimer's disease (AD) and of the retina in age-related macular degeneration (AMD) appear to be associated with deficits in the expression of messenger RNA (mRNA) and gene families involved in the innate-immune response, inflammation, neurotrophism, synaptogenesis, and amyloidogenesis (Figure In this "Opinion" paper for the Frontiers in Neurology Special Research Topic, we will highlight some of the most recent work in this research area, with emphasis on a family of five up-regulated pro-inflammatory miRNAs -miRNA-9, miRNA-34a, miRNA-125b, miRNA-146a, and miRNA-155 -that are emerging as key mechanistic contributors to the AD and AMD process.

BackgroundIsolation of pure RNA from woody perennials, especially fruit crops such as grapevine rich in complex secondary metabolites, has remained very challenging. Lack of effective RNA isolation technology has resulted in difficulties in viral diagnosis and discovery as well as studies on many biological processes of these highly important woody plants. It is imperative to develop and refine methodologies with which large amounts of pure nucleic acids can be readily isolated from woody perennials.MethodsWe compared five commonly used RNA isolation kits in isolating total RNA from twelve species of woody perennials. We made modifications to select RNA isolation systems to simplify and improve their efficiency in RNA isolation. The yield and quality of isolated RNAs were assessed via gel electrophoresis and spectrophotometric measurement. We also performed RT-PCR and RT-qPCR to detect several major viruses from grapevines.ResultsTwo of the kits were shown to be the best in both the yield and quality of the isolated RNA from all twelve woody species. Using disposable extraction bags for tissue homogenization not only improved the yield without affecting quality, but also made the RNA isolation technology simpler, less costly, and suitable for adoption by many potential users with facility limitations. This system was successfully applied to a wide range of woody plants, including fruit crops, ornamentals and timber trees. Inclusion of polyvinylpyrrolidone in the extraction buffer drastically improved the performance of the system in isolating total RNA from old grapevine leaves collected later in the season. This modification made our system highly effective in isolating quality RNA from grapevine leaves throughout the entire growing season. We further demonstrated that the resulting nucleic acid preparations are suitable for detection of several major grapevine viruses with RNA or DNA genomes using PCR, RT-PCR and qPCR as well as for assays on plant microRNAs.ConclusionsThis improved RNA isolation system would have wide applications in viral diagnostics and discovery, studies on gene expression and regulation, transcriptomics, and small RNA biology in grapevines. We believe this system will also be useful in diverse applications pertaining to research on many other woody perennials and recalcitrant plant species.Electronic supplementary materialThe online version of this article (doi:10.1186/s12985-015-0376-3) contains supplementary material, which is available to authorized users.

Peptide phage display, a powerful method for ligand identification, was used to identify new peptide ligands for epidermal growth factor receptor. A-431 cells expressing epidermal growth factor receptor were used as the matrix in a cell-based subtractive biopanning approach using a 7-mer peptide displaying phage library. Two novel peptide ligands were identified and tested for their affinities and functional effects on epidermal growth factor receptor. The identified peptides were able to inhibit the epidermal growth factor-induced phosphorylation of epidermal growth factor receptor in a concentration-dependent manner. The results of affinity binding experiments showed that the natural ligand, that is epidermal growth factor, was able to inhibit competitively the binding of peptide-bearing phage to epidermal growth factor receptor expressing A-431 cells. Molecular modeling studies were used to calculate the free energies for the binding of peptides to the receptor-binding site as well as proposing the interaction modes for this binding. The calculated values for the binding energies were found to be similar to our experimental data and those of previously reported studies.