Modification of Lipid Microenvironments on Solid Support Structures for Use in Transmembrane Protein Assays

Gamma-Secretase (γ-secretase) is a transmembrane protease of increasing interest, which has been shown to have significant connections to both cancer and Alzheimer’s disease. γ-secretase cleaves both Notch-1, a transmembrane signaling protein, and Amyloid precursor protein (APP), a transmembrane protein whose cleavage may result in the formation of β-amyloid plaques in the brain. Notch-1 and APP are widely studied proteins that have substantial impacts on the development and proliferation of cancer and Alzheimer’s disease, respectively. Notch-1 partakes in the signaling of apoptosis in damaged and mutated cells, thus its cleavage by γ-secretase within the plasma membrane has ramifications on cell growth and proliferation. However, the APP molecule is the key protein in the metabolic pathway that produces small amyloid fragments. These fragments, in undesirable conditions, have the propensity to aggregate and form, as stated above, amyloid plaques, depending on the fragment length. These plaques have been long believed to inhibit neuronal function if they are not degraded or removed from the intracellular space, specifically in the brain. Due to these widespread mental and physical health impacts, isolation and modulation of the cleavage of such proteins in intact, controlled bilayers in a highly reproducible, and potentially high-throughput, process is a key goal in understanding these and a vast array of intramembrane proteases for the development of pharmaceutical therapies. The work presented looks to the development of one such platform, yielding crucial spatial and temporal information within these complex lipid microenvironments. Synthetic, biomimetic membranes were studied and manipulated to develop biologically relevant systems in which to resuspend isolated proteins. A formulation of sphingomyelin, 1,2-Dioleoyl-sn-phosphatidylcholine (DOPC), and cholesterol was chosen due to its attributes in resembling fundamental lipodomics within a human brain cell. It is shown that this canonical formulation and subsequent formulations with added complex mixtures, yield a lipid system that retains visible phase separation to a quantifiable degree. These lipid formulations, when fused with solid silica support structures such as planar surfaces or silica microbeads, allows for the reconstitution of the three of proteins of interest. These assay and high throughput platforms are essential to understanding key functions and potential modulations of these protein pathways, however this approach does not fully replicate the biological environment these proteins experience within an active cell. Two approaches are shown in this work to increase the biological relevancy of these platforms. Tethering of the solid support structures with a series of polyethylene glycol (PEG) polymers culminating in a functionalized capping moiety that can yield overall increases to protein mobility, and added functionality of the platform. Additionally, added dopants of more complex lipid components into the basic lipid membrane analogue shows the ability to increase complexity of the formulation and closes the gap between the synthetic membrane and the protein’s true biological lipid environment. These platforms are highly robust and rugged in nature and lend themselves to be useful in future high-throughput screening and functional assay processes in pharmaceutical research. The coupling of both planar surface support structures and micro bead structures in tandem can be analyzed through confocal, super-resolution, and atomic force microscopy, leading to a fuller understanding of these complex spatial reaction-diffusion systems prevalent within human cells. The systems developed in this research, apart from being tested with the aforementioned proteins, are not protein-specific and thus could yield a viable platform on which to test any number of isolated transmembrane proteins in a highly reproducible manner

Herpesvirus Infections of the Central Nervous System

There are over 200 herpesvirus species, of which 10 affect humans. Each of these 10 herpesviruses has a unique clinical syndrome, but common to all is their ability to cause infection and pathology in the central nervous system. In this article, we discuss the epidemiology, clinical presentation, diagnostic modalities, treatment, sequelae, and availability of vaccination of each of the following herpesviruses: herpes simplex virus 1 and 2, varicella zoster virus, human cytomegalovirus, human herpesvirus 6A, 6B, and 7, Epstein-Barr virus, human herpesvirus 8, and simian herpesvirus B

Legitimacy driven change at the World Anti-Doping Agency

The effectiveness of the World Anti-Doping Agency as an international non-governmental organisation with a mission to regulate anti-doping policy has been challenged before by doping scandals in sport. Historically, anti-doping policy development has been primarily reactive, determined by the need for dominant organisations to maintain power rather than to protect athletes. The purpose of this paper is to explore reactive anti-doping policy change from a multi-level legitimacy perspective. Using multi-level legitimacy theory and the concept of legitimacy challenges, it is argued that reactive policy change is motivated by a need to manage perceived organisational legitimacy. The recent exposure of systematic doping in Russia is used as an example to support this analysis. These findings are discussed in the context of current criticisms of anti-doping policy

Dubowitz Syndrome: A Review and Implications for Cognitive, Behavioral, and Psychological Features

Dubowitz syndrome is a rare autosomal recessive disorder characterized by micorcephaly, short stature, abnormal faces, and mild to severe mental retardation. Growth retardation occurs both intrauterine and postnatal. Behavioral characteristics include hyperactivity, short attention span, and aggressiveness. Behavior problems include difficulty feeding, sleep disturbance, and bedwetting. Individuals with the disorder have displayed shyness, fear of crowds, and dislike of loud noises. A high-pitched or hoarse voice is common. Deficits have been found in speech and language skills, reasoning and memory skills, self-help skills, and psychomotor functioning. Ocular, dental, cutaneous, skeletal, cardiovascular, gastrointestinal, neurological, immunological, and hematological medical difficulties have been noted. Approximately 148 cases have been described in the literature. The cause of the disorder remains unknown, however, research suggests genetic origin. Past research emphasizes physical characteristics and medical complications. There is a lack of cognitive, behavioral, and psychological information available regarding the disorder. This article presents a review of the literature and provides assessment and treatment implications for the cognitive, behavioral, and psychological aspects of Dubowitz syndrome