Fluorescence microspectroscopy for testing the dimerization hypothesis of BACE1 protein in cultured HEK293 cells

Alzheimer’s Disease (AD) is a neurodegenerative disorder that results from the formation of beta-amyloid plaques in the brain that trigger the known symptoms of memory loss in AD patients. The beta-amyloid plaques are formed by the proteolytic cleavage of the amyloid precursor protein (APP) by the proteases BACE1 and gamma-secretase. These enzyme-facilitated cleavages lead to the production of beta-amyloid fragments that aggregate to form plaques, which ultimately lead to neuronal cell death. Recent detergent protein extraction studies suggest that BACE1 protein forms a dimer that has significantly higher catalytic activity than its monomeric counterpart. In this contribution, we examine the dimerization hypothesis of BACE1 in cultured HEK293 cells using complementary fluorescence spectroscopy and microscopy methods. Cells were transfected with a BACE1-EGFP fusion protein construct and imaged using confocal, and differential interference contrast to monitor the localization and distribution of intracellular BACE1. Complementary fluorescence lifetime and anisotropy measurements enabled us to examine the conformational and environmental changes of BACE1 as a function of substrate binding. Using fluorescence correlation spectroscopy, we also quantified the diffusion coefficient of BACE1-EGFP on the plasma membrane as a means to test the dimerization hypothesis as a fucntion of substrate-analog inhibitition. Our results represent an important first towards examining the substrate-mediated dimerization hypothesis of BACE1 in live cells

The Impact of Disease Registries on Advancing Knowledge and Understanding of Dementia Globally

To help address the increasing challenges related to the provision of dementia care, dementia registries have emerged around the world as important tools to gain insights and a better understanding of the disease process. Dementia registries provide a valuable source of standardized data collected from a large number of patients. This review explores the published research relating to different dementia registries around the world and discusses how these registries have improved our knowledge and understanding of the incidence, prevalence, risk factors, mortality, diagnosis, and management of dementia. A number of the best-known dementia registries with high research output including SveDem, NACC, ReDeGi, CREDOS and PRODEM were selected to study the publication output based on their data, investigate the key findings of these registry-based studies. Registries data contributed to understanding many aspects of the disease including disease prevalence in specific areas, patient characteristics and how they differ in populations, mortality risks, as well as the disease risk factors. Registries data impacted the quality of patients’ lives through determining the best treatment strategy for a patient based on previous patient outcomes. In conclusion, registries have significantly advanced scientific knowledge and understanding of dementia and impacted policy, clinical practice care delivery

MULTISCALE SPECTROSCOPY OF DIFFUSING MOLECULES IN CROWDED ENVIRONMENTS

Living cells are known to be crowded with organelles, biomembranes, and macromolecules such as proteins, DNA, RNA, and actin filaments. It is believed that such macromolecular crowding affect biomolecular diffusion, protein-protein and protein-substrate interaction, and protein folding. In this contribution, I will discuss our recent results on rotational and translational diffusion of small and large molecules in crowded environments using time-resolved anisotropy and fluorescence correlation spectroscopy methods. In these studies, rhodamine green and enhanced green fluorescent protein are used as fluorescent probes diffusing in buffers enriched with biomimetic crowding agents such as Ficoll-70, bovine serum albumin (BSA), and ovalbumin. Controlled experiments on pure and glycerol-rich buffers were carried out as environments with variable, homogeneous viscosity. Our results indicate that the microviscosity differs from the corresponding bulk viscosity, depending on the nature of crowding agents (i.e., proteins versus polymers), the concentration of crowding agents and spatio-temporal scaling of our experimental approach. Our findings provide a foundation for fluorescence-based studies of diffusion and binding of biomolecules in the crowded milieu of living cells

Erythropoietin and a nonerythropoietic peptide analog promote aortic endothelial cell repair under hypoxic conditions: role of nitric oxide

The cytoprotective effects of erythropoietin (EPO) and an EPO-related nonerythropoietic analog, pyroglutamate helix B surface peptide (pHBSP), were investigated in an in vitro model of bovine aortic endothelial cell injury under normoxic (21% O2) and hypoxic (1% O2) conditions. The potential molecular mechanisms of these effects were also explored. Using a model of endothelial injury (the scratch assay), we found that, under hypoxic conditions, EPO and pHBSP enhanced scratch closure by promoting cell migration and proliferation, but did not show any effect under normoxic conditions. Furthermore, EPO protected bovine aortic endothelial cells from staurosporine-induced apoptosis under hypoxic conditions. The priming effect of hypoxia was associated with stabilization of hypoxia inducible factor-1α, EPO receptor upregulation, and decreased Ser-1177 phosphorylation of endothelial nitric oxide synthase (NOS); the effect of hypoxia on the latter was rescued by EPO. Hypoxia was associated with a reduction in nitric oxide (NO) production as assessed by its oxidation products, nitrite and nitrate, consistent with the oxygen requirement for endogenous production of NO by endothelial NOS. However, while EPO did not affect NO formation in normoxia, it markedly increased NO production, in a manner sensitive to NOS inhibition, under hypoxic conditions. These data are consistent with the notion that the tissue-protective actions of EPO-related cytokines in pathophysiological settings associated with poor oxygenation are mediated by NO. These findings may be particularly relevant to atherogenesis and postangioplasty restenosis

Characterization of the Inlet Port Flow under Steady-State Conditions Using PIV and POD

The current study demonstrates an experimental investigation of the tumble flow structures using Particle Image Velocimetry (PIV) under steady-state conditions considering the central vertical tumble plane. The experiments were carried out on a four-valve, pent-roof Gasoline Direct Injection (GDI) engine head at different valve lifts and with a pressure difference of 150 mmH2O across the intake valves. Furthermore, the Proper Orthogonal Decomposition (POD) analytical technique was applied to PIV-measured velocity vector maps to characterize the flow structures at various valve lifts, and hence the different rig tumble values. The results show that at low valve lifts (1 to 5 mm), 48.9 to 46.6% of the flow energy is concentrated in the large (mode 1) eddies with only 8.4 to 11.46% in mode 2 and 7.2 to 7.5 in mode 3. At high valve lifts, it can be clearly seen that some of the energy in the large eddies of mode 1 is transferred to the smaller flow structures of modes 2 and 3. This can be clearly seen at valve lift 10 mm where the values of the flow energy were 40.6%, 17.3%, and 8.0% for modes 1, 2, and 3, respectively