Polymeric microspheres as protein transduction reagents

Discovering the function of an unknown protein, particularly one with neither structural nor functional correlates, is a daunting task. Interaction analyses determine binding partners, whereas DNA transfection, either transient or stable, leads to intracellular expression, though not necessarily at physiologically relevant levels. In theory, direct intracellular protein delivery (protein transduction) provides a conceptually simpler alternative, but in practice the approach is problematic. Domains such as HIV TAT protein are valuable, but their effectiveness is protein specific. Similarly, the delivery of intact proteins via endocytic pathways (e.g. using liposomes) is problematic for functional analysis because of the potential for protein degradation in the endosomes/lysosomes. Consequently, recent reports that microspheres can deliver bio-cargoes into cells via a non-endocytic, energy-independent pathway offer an exciting and promising alternative for in vitro delivery of functional protein. In order for such promise to be fully exploited, microspheres are required that (i) are stably linked to proteins, (ii) can deliver those proteins with good efficiency, (iii) release functional protein once inside the cells, and (iv) permit concomitant tracking. Herein, we report the application of microspheres to successfully address all of these criteria simultaneously, for the first time. After cellular uptake, protein release was autocatalyzed by the reducing cytoplasmic environment. Outside of cells, the covalent microsphere-protein linkage was stable for ≥90 h at 37°C. Using conservative methods of estimation, 74.3% ± 5.6% of cells were shown to take up these microspheres after 24 h of incubation, with the whole process of delivery and intracellular protein release occurring within 36 h. Intended for in vitro functional protein research, this approach will enable study of the consequences of protein delivery at physiologically relevant levels, without recourse to nucleic acids, and offers a useful alternative to commercial protein transfection reagents such as Chariot™. We also provide clear immunostaining evidence to resolve residual controversy surrounding FACS-based assessment of microsphere uptake

Anti-seizure gene therapy for focal cortical dysplasia

Focal cortical dysplasias are a common subtype of malformation of cortical development, which frequently presents with a spectrum of cognitive and behavioural abnormalities as well as pharmacoresistant epilepsy. Focal cortical dysplasia type II is typically caused by somatic mutations resulting in mammalian target of rapamycin (mTOR) hyperactivity, and is the commonest pathology found in children undergoing epilepsy surgery. However, surgical resection does not always result in seizure freedom, and is often precluded by proximity to eloquent brain regions. Gene therapy is a promising potential alternative treatment and may be appropriate in cases that represent an unacceptable surgical risk. Here, we evaluated a gene therapy based on overexpression of the Kv1.1 potassium channel in a mouse model of frontal lobe focal cortical dysplasia. An engineered potassium channel (EKC) transgene was placed under control of a human promoter that biases expression towards principal neurons (CAMK2A) and packaged in an adeno-associated viral vector (AAV9). We used an established focal cortical dysplasia model generated by in utero electroporation of frontal lobe neural progenitors with a constitutively active human Ras homolog enriched in brain (RHEB) plasmid, an activator of mTOR complex 1. We characterized the model by quantifying electrocorticographic and behavioural abnormalities, both in mice developing spontaneous generalized seizures and in mice only exhibiting interictal discharges. Injection of AAV9-CAMK2A-EKC in the dysplastic region resulted in a robust decrease (∼64%) in the frequency of seizures. Despite the robust anti-epileptic effect of the treatment, there was neither an improvement nor a worsening of performance in behavioural tests sensitive to frontal lobe function. AAV9-CAMK2A-EKC had no effect on interictal discharges or behaviour in mice without generalized seizures. AAV9-CAMK2A-EKC gene therapy is a promising therapy with translational potential to treat the epileptic phenotype of mTOR-related malformations of cortical development. Cognitive and behavioural co-morbidities may, however, resist an intervention aimed at reducing circuit excitability

Using the hierarchy of intervention effectiveness to improve the quality of recommendations developed during critical patient safety incident reviews

Our Canadian multi-site academic health sciences centre uses a standardized process to review critical patient safety incidents and develop recommendations to prevent incident reoccurrence. We recognized an opportunity to enhance recommendation development by integrating the Hierarchy of Intervention Effectiveness (HIE), a human factors framework, into the incident review process. This project aimed to increase the proportion of system-focused recommendations from critical incident reviews from 16 to 30% over 16 months. A multi-intervention strategy included (1) standardizing the incident analysis review template; (2) earmarking time for recommendation development during reviews; (3) providing participants with just-in-time education and tools; and (4) initiating HIE-based recommendation classification during incident reviews. Statistical process control p-Chart analysis showed an increase in system-focused recommendations from 16 to 30% over 16 months. The HIE promotes system-level change to prevent critical incidents, which other organizations may benefit from incorporating in their patient safety reviews

Reverse sequence polymerization-induced self-assembly in aqueous media: a counter-intuitive approach to sterically-stabilized diblock copolymer nano-objects

Polymerization-induced self-assembly (PISA) is a powerful platform technology for the efficient synthesis of block copolymer nanoparticles in many types of solvents, including water. In PISA, a soluble precursor block is used to grow a second insoluble block, which leads to in situ self-assembly of the block copolymer chains. Thus, in the case of aqueous PISA, the water-soluble block is always prepared first because this confers steric stabilization. Herein, we challenge this paradigm by demonstrating that amphiphilic diblock copolymer chains can be prepared in water by preparing the hydrophobic block first via reversible addition–fragmentation chain transfer (RAFT) polymerization. This counter-intuitive reverse sequence PISA formulation utilizes an ionic RAFT agent to conduct the RAFT aqueous dispersion polymerization of 2-hydroxypropyl methacrylate (HPMA), which results in the formation of charge-stabilized PHPMA latex particles of ∼500 nm diameter. Initial attempts to chain-extend these hydrophobic PHPMA chains with water-miscible monomers such as glycerol monomethacrylate (GMA) were unsuccessful, with only uncontrolled free radical polymerization being observed in the aqueous phase. However, using a water-immiscible monomer such as isopropylideneglycerol methacrylate (IPGMA) enabled the synthesis of charge-stabilized PHPMA-PIPGMA latex particles. Subsequent acid hydrolysis of the PIPGMA block led to the in situ formation of sterically-stabilized PHPMA-PGMA diblock copolymer spheres. Alternatively, dissolution of the precursor PHPMA latex in a methanol/water binary mixture enables RAFT solution polymerization of water-miscible monomers such as GMA or N,N′-dimethylacrylamide (DMAC) to be achieved with good control. The resulting amphiphilic diblock copolymer chains then undergo self-assembly in aqueous solution after removal of the methanol co-solvent. Finally, this reverse sequence PISA protocol can also be applied to other vinyl monomers such as 2-methoxyethyl methacrylate (MOEMA) or diacetone acrylamide (DAAM), which significantly broadens its scope

Biogeochemical signatures in the lichen Hypogymnia physodes in the mid Urals

Abstract Multi-element content and uranium (U) isotopes were investigated in the lichen Hypogymnia physodes (native and transplants) sampled across a 60-km transect, centred on Karabash smelter town, from Turgoyak Lake (SW) to Kyshtym (NE) to investigate the origin of U. Kyshtym was the site of a major nuclear accident in 1957. 234 U/ 238 U activity ratios in native thalli sampled during July 2001 were within the natural isotopic ratio in minerals. Uranium/thorium (U/Th) ratios were higher in native thalli towards the NE (average 0.73) than those in the SW (average 0.57). Element signatures in native thalli and transplants suggest U was derived from fossil fuel combustion from Karabash and sources lying further to the east. Systematic and significant U enrichment indicative of a nuclear fuel cycle source was not detected in any sample. Element signatures in epiphytic lichen transplants and native thalli provide a powerful method to evaluate U deposition

One-pot synthesis of {Mo6 I8 }4+ -doped polystyrene microspheres via a free radical dispersion copolymerisation reaction:{Mo6 I8 }4+ -doped polystyrene microspheres via free radical copolymerisation

Molybdenum octahedral clusters, when incorporated into an appropriate polymer matrix, are considered as promising agents for a range of biological applications. This work describes the one-pot synthesis, morphology and cellular toxicity of nano-sized polystyrene beads doped with luminescent cluster complexes [(Mo6X8)(NO3)6]2- (X=Cl, Br or I). Specifically, the particles were obtained by free radical dispersion copolymerisation of styrene and methacrylic acid or 4-vinylpyridine in the presence of the cluster complexes. The effects of the cluster loading in the reaction mixture on both the content of the final material and number-average molar mass of the copolymers were evaluated

Some factors affecting the productivity of Damascus goats in Cyprus

Thesis (M.S.)--Agriculture, A.U.B.Includes bibliographical references

Notes on some species of the genus Amathia (Bryozoa, Ctenostomata)

Volume: 52Start Page: 307End Page: 35