Preparation of Group I Introns for Biochemical Studies and Crystallization Assays by Native Affinity Purification

The study of functional RNAs of various sizes and structures requires efficient methods for their synthesis and purification. Here, 23 group I intron variants ranging in length from 246 to 341 nucleotides—some containing exons—were subjected to a native purification technique previously applied only to shorter RNAs (<160 nucleotides). For the RNAs containing both exons, we adjusted the original purification protocol to allow for purification of radiolabeled molecules. The resulting RNAs were used in folding assays on native gel electrophoresis and in self-splicing assays. The intron-only RNAs were subjected to the regular native purification scheme, assayed for folding and employed in crystallization screens. All RNAs that contained a 3′ overhang of one nucleotide were efficiently cleaved off from the support and were at least 90% pure after the non-denaturing purification. A representative subset of these RNAs was shown to be folded and self-splicing after purification. Additionally, crystals were grown for a 286 nucleotide long variant of the Clostridium botulinum intron. These results demonstrate the suitability of the native affinity purification method for the preparation of group I introns. We hope these findings will stimulate a broader application of this strategy to the preparation of other large RNA molecules

Genome-wide screens identify Toxoplasma gondii determinants of parasite fitness in IFNγ-activated murine macrophages

Macrophages play an essential role in the early immune response against Toxoplasma and are the cell type preferentially infected by the parasite in vivo. Interferon gamma (IFNγ) elicits a variety of anti-Toxoplasma activities in macrophages. Using a genome-wide CRISPR screen we identify 353 Toxoplasma genes that determine parasite fitness in naїve or IFNγ-activated murine macrophages, seven of which are further confirmed. We show that one of these genes encodes dense granule protein GRA45, which has a chaperone-like domain, is critical for correct localization of GRAs into the PVM and secretion of GRA effectors into the host cytoplasm. Parasites lacking GRA45 are more susceptible to IFNγ-mediated growth inhibition and have reduced virulence in mice. Together, we identify and characterize an important chaperone-like GRA in Toxoplasma and provide a resource for the community to further explore the function of Toxoplasma genes that determine fitness in IFNγ-activated macrophages

Technology as an Enabler of the Global Branding of Retail Financial Services

The author argues that, though there is little doubt that technology has transformed the delivery of retail financial services in the past decade, this transformation also has implications for the global branding of financial services. The article presents a conceptual framework that illustrates how new distribution and processing technologies on the supply-side and changes in consumer attitudes to banking on the demand-side have driven the global branding of retail financial services. On the supply side, the change process has occurred through the removal of geographic and cost barriers to global distribution, which have enabled insurance companies, supermarkets, utilities, Internet-only banks, and other new players to enter traditional banking markets. Technology has created globally accessible internal information management systems, reduced the need for physical branch networks, stimulated greater brand awareness through interactive Web sites, fuelled the expansion of multinational banks and consolidation through mergers, and ultimately has eroded product differentiation and distribution channels as sources of competitive advantage. The combination of these supply-side elements increases the need for, and the effectiveness of, global brands. On the demand side, the change process has been driven by global convergence in key benefits sought by banking consumers in developed countries. The move to electronic delivery of retail financial services has increased competition and consumer sophistication, and consumers have become less loyal, more informed, and more willing to switch providers and use multiple providers in order to maximize the value of the unique mix of savings, loan, and transactions services they seek. Yet, at the same time, the ongoing need for security and reliability when performing financial transactions means consumers place value on brands that have established images as trusted parties. The result is an increased consumer preference for globally recognized brands and a willingness to purchase financial services from firms other than traditional financial institutions, provided the brand name is a trusted one. The author concludes that for managers of firms providing retail financial services, the major implication of these technology-induced changes in supply and demand is the need for customer-focused marketing strategies