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

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

Demographic Aspects of Human Hydatidosis in Egypt

Background: Cystic echinococcosis or hydatidosis is caused by the larval stage of the tapeworm Echinococcus granulosus. It is one of the most important parasitic zoonoses. Objective: The aim of the work was to determine the prevalence of the disease in humans and its clinical presentation, also targeted to identify risk factors predisposing infections.Subjects and Methods: Thirty-one cystic echinococcosis cases were collected to determine the presence of hydatid cysts in human. Confirmation of hydatid cyst infection was done by radiological assessment and direct parasitological detection of hydatid cyst through PAIR technique or pathological confirmation of surgical removal cyst. A questionnaire survey to identify risk factors of transmission was also carried out. Results: The highest percent of infection with hydatid cyst was in age group 21-30 years (29.04%), followed by age group 31-40 years and 41-50 years that attained (25.81%) for each. Females attained the higher percent of infection (58.06%) in comparison to males (41.94 %).The highest percent of cystic echinococcosis was recorded for farmers (32.26 %) followed by housewives (29.03%). 67.75 % of cystic echinococcosis was for rural resident and (32.25%) was for urban resident.Conclusion: It could be concluded that hydatid disease is still an endemic problem and is a course of challenge to all the medical practitioners. The study has established that hydatidosis is prevalent in humans in Egypt and that the risk factors for its transmission exist

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

Wave Interaction Of Partially Immersed Semicircular Breakwater Suspended On Piles Using Flow-3D

Paper presented at 2018 Canadian Society of Mechanical Engineers International Congress, 27-30 May 2018.This paper experimentally investigates the wave transmission through partially immersed semicircular breakwater suspended on two rows of circular piles. Different wave and structure parameters have been investigated such as; incident wave height, wave length, wave period, breakwater draft and the clear gap between the supporting piles. Multiple Polynomial Regression (MPR) model is used to develop an empirical equation to calculate the transmission coefficient (kt) as a representative of the transmitted wave energy. In addition, a Flow-3D numerical study is carried out to simulate the transmitted wave hydrodynamics and verified against the experimental results. The numerical analysis shows good agreement with experimental results with an acceptable value of NRMSE. The results show that the transmission coefficient (kt) decreases by about 31% with increasing the relative wave length from 0.12 to 0.22 and wave steepness from 0.006 to 0.021. Also, the transmitted coefficient (kt) decreases by about 25% with increasing the relative immersion depths from 0.25m to 0.5m and increases by about 16% for increasing of pile porosity value from 0.5 to 0.83 respectively

Molecular Brightness Approach for FRET Analysis of Donor-Linker-Acceptor Constructs at the Single Molecule Level: A Concept

In this report, we have developed a simple approach using single-detector fluorescence autocorrelation spectroscopy (FCS) to investigate the Förster resonance energy transfer (FRET) of genetically encoded, freely diffusing crTC2.1 (mTurquoise2.1-linker-mCitrine) at the single molecule level. We hypothesize that the molecular brightness of the freely diffusing donor (mTurquoise2.1) in the presence of the acceptor (mCitrine) is lower than that of the donor alone due to FRET. To test this hypothesis, the fluorescence fluctuation signal and number of molecules of freely diffusing construct were measured using FCS to calculate the molecular brightness of the donor, excited at 405 nm and detected at 475/50 nm, in the presence and absence of the acceptor. Our results indicate that the molecular brightness of cleaved crTC2.1 in a buffer is larger than that of the intact counterpart under 405-nm excitation. The energy transfer efficiency at the single molecule level is larger and more spread in values as compared with the ensemble-averaging time-resolved fluorescence measurements. In contrast, the molecular brightness of the intact crTC2.1, under 488 nm excitation of the acceptor (531/40 nm detection), is the same or slightly larger than that of the cleaved counterpart. These FCS-FRET measurements on freely diffusing donor-acceptor pairs are independent of the precise time constants associated with autocorrelation curves due to the presence of potential photophysical processes. Ultimately, when used in living cells, the proposed approach would only require a low expression level of these genetically encoded constructs, helping to limit potential interference with the cell machinery