Co- and Post-Translational Protein Folding in the ER.

The biophysical rules that govern folding of small, single-domain proteins in dilute solutions are now quite well understood. The mechanisms underlying co-translational folding of multidomain and membrane-spanning proteins in complex cellular environments are often less clear. The endoplasmic reticulum (ER) produces a plethora of membrane and secretory proteins, which must fold and assemble correctly before ER exit - if these processes fail, misfolded species accumulate in the ER or are degraded. The ER differs from other cellular organelles in terms of the physicochemical environment and the variety of ER-specific protein modifications. Here, we review chaperone-assisted co- and post-translational folding and assembly in the ER and underline the influence of protein modifications on these processes. We emphasize how method development has helped advance the field by allowing researchers to monitor the progression of folding as it occurs inside living cells, while at the same time probing the intricate relationship between protein modifications during folding.

Simple, fast and efficient iTOP-mediated delivery of CRISPR/Cas9 RNP in difficult-to-transduce human cells including primary T cells.

The advent of the CRISPR/Cas9 system has transformed the field of human genome engineering and has created new perspectives in the development of innovative cell therapies. However, the absence of a simple, fast and efficient delivery method of CRISPR/Cas9 into primary human cells has been limiting the progress of CRISPR/Cas9-based therapies. Here, we describe an optimized protocol for iTOP-mediated delivery of CRISPR/Cas9 in various human cells, including primary T cells, induced pluripotent stem cells (hiPSCs), Jurkat, ARPE-19 and HEK293 cells. We compare iTOP to other CRISPR/Cas9 delivery methods, such as electroporation and lipofection, and evaluate the corresponding gene-editing efficiencies and post-treatment cell viabilities. We demonstrate that the gene editing achieved by iTOP-mediated delivery of CRISPR/Cas9 is 40-95 % depending on the cell type, while post-iTOP cell viability remains high in the range of 70-95 %. Collectively, we present an optimized workflow for a simple, high-throughput and effective iTOP-mediated delivery of CRISPR/Cas9 to engineer difficult-to-transduce human cells. We believe that the iTOP technology® could contribute to the development of novel CRISPR/Cas9-based cell therapies.

Potential drug-drug interactions in prescriptions dispensed in community pharmacies in Greece.

OBJECTIVES: To evaluate the nature, type and prevalence of potential drug-drug interactions (DDIs) in prescriptions dispensed in community pharmacies in Thessaloniki, Greece. Secondary objectives included the classification of DDIs as per pharmacotherapeutic class of the medications and the investigation of the relationship between medical specialties and the frequency of potential DDIs, as well as the relationship between DDIs and prescription size. Setting DDIs are a common cause of adverse drug reactions (ADRs) among patients using multiple drug therapy. In Greece a reliable computerized surveillance system for monitoring potential DDIs is not yet fully established. As a result, the prevalence of such DDIs in prescriptions dispensed by community pharmacies in Greece is unknown. METHODS: We conducted a prospective, descriptive study. Over a 3-month period (November 2007-January 2008), a total of 1,553 handwritten prescriptions were collected from three community pharmacies in Thessaloniki, Greece. The prescriptions were processed using the Drug Interactions Checker within the www.drugs.com database. The identified potential DDIs were categorized into two classes, major and moderate, according to their level of clinical significance. MAIN OUTCOME MEASURES: Overall 213 prescriptions had one or more potential DDIs and a total of 287 major and moderate DDIs were identified. Potential DDIs were identified in 18.5% of all prescriptions. Major DDIs were identified in 1.9% of all prescriptions and represented 10.5% of all DDIs detected, whereas moderate DDIs were identified in 16.6% of all prescriptions and represented 89.5% of all DDIs detected. The rate of DDIs increased with prescription size. The most common drug involved in major DDIs was amiodarone which interacts with potassium-wasting diuretics, digoxin, simvastatin and acenocoumarol. CONCLUSIONS: Our results indicate that patients in Greece are at risk of ADRs caused by medications due to potential DDIs. An appropriate surveillance system for monitoring such interactions should be implemented and physicians should be more aware of potentially harmful DDIs. Pharmacists can contribute to the detection and prevention of drug-related injuries, especially of clinically meaningful DDIs that pose a potential risk to patient safety.

Characterization of CNPY5 and its family members.

The Canopy (CNPY) family consists of four members predicted to be soluble proteins localized to the endoplasmic reticulum (ER). They are involved in a wide array of processes, including angiogenesis, cell adhesion, and host defense. CNPYs are thought to do so via regulation of secretory transport of a diverse group of proteins, such as immunoglobulin M, growth factor receptors, toll-like receptors, and the low-density lipoprotein receptor. Thus far, a comparative analysis of the mammalian CNPY family is missing. Bioinformatic analysis shows that mammalian CNPYs, except the CNPY1 homolog, have N-terminal signal sequences and C-terminal ER-retention signals and that mammals have an additional member CNPY5, also known as plasma cell-induced ER protein 1/marginal zone B cell-specific protein 1. Canopy proteins are particularly homologous in four hydrophobic alpha-helical regions and contain three conserved disulfide bonds. This sequence signature is characteristic for the saposin-like superfamily and strongly argues that CNPYs share this common saposin fold. We showed that CNPY2, 3, 4, and 5 (termed CNPYs) localize to the ER. In radioactive pulse-chase experiments, we found that CNPYs rapidly form disulfide bonds and fold within minutes into their native forms. Disulfide bonds in native CNPYs remain sensitive to low concentrations of dithiothreitol (DTT) suggesting that the cysteine residues forming them are relatively accessible to solutes. Possible roles of CNPYs in the folding of secretory proteins in the ER are discussed.