Functional Characterization of the Obesity-Linked Variant of the ß3-Adrenergic Receptor.

Adrenergic receptor ß3 (ADRß3) is a member of the rhodopsin-like G protein-coupled receptor family. The binding of the ligand to ADRß3 activates adenylate cyclase and increases cAMP in the cells. ADRß3 is highly expressed in white and brown adipocytes and controls key regulatory pathways of lipid metabolism. Trp64Arg (W64R) polymorphism in the ADRß3 is associated with the early development of type 2 diabetes mellitus, lower resting metabolic rate, abdominal obesity, and insulin resistance. It is unclear how the substitution of W64R affects the functioning of ADRß3. This study was initiated to functionally characterize this obesity-linked variant of ADRß3. We evaluated in detail the expression, subcellular distribution, and post-activation behavior of the WT and W64R ADRß3 using single cell quantitative fluorescence microscopy. When expressed in HEK 293 cells, ADRß3 shows a typical distribution displayed by other GPCRs with a predominant localization at the cell surface. Unlike adrenergic receptor ß2 (ADRß2), agonist-induced desensitization of ADRß3 does not involve loss of cell surface expression. WT and W64R variant of ADRß3 displayed comparable biochemical properties, and there was no significant impact of the substitution of tryptophan with arginine on the expression, cellular distribution, signaling, and post-activation behavior of ADRß3. The obesity-linked W64R variant of ADRß3 is indistinguishable from the WT ADRß3 in terms of expression, cellular distribution, signaling, and post-activation behavior.

Development of Rapid Aptamer-Based Screening Assay for the Detection of Covid-19 Variants.

The development of a colorimetric severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection assay with the WHO published ASSURED criteria is reported, in which the biosensor should have the following characteristics of (i) being affordable for low-income communities, (ii) sensitive, (iii) specific, (iv) user-friendly to be used by non-skilled personnel, (v) rapid and robust, (vi) equipment-free, and (vii) delivered to the end-users as a simple and easy to use point-of-care tool. Early viral infection detection prevents virus spread and controls the epidemic. We herein report the development of a colorimetric assay in which SARS-COV-2 variants can be detected by colorimetric observation of color on the sensing cotton swab surface. Using the developed biosensor assay, it is possible to discriminate between the various SARS-CoV-2 variants with a LOD of 100 ng/mL within 4 min including sample preconcentration and incubation step. The results illustrated the development of a SARS-CoV-2 colorimetric biosensor that can be mass produced, with low-reagent cost, and can be read-out visually in the field by nonskilled personnel.

Development of a Rapid Immuno-Based Screening Assay for the Detection of Adenovirus in Eye Infections.

Despite progress in fighting infectious diseases, human pathogenesis and death caused by infectious diseases remain relatively high worldwide exceeding that of cancer and cardiovascular diseases. Human adenovirus (HAdV) infects cells of the upper respiratory tract causing flu-like symptoms that are accompanied by pain and inflammation. Diagnosis of HAdV is commonly achieved by conventional methods such as viral cultures, immunoassays, and polymerase chain reaction (PCR) techniques. However, there are a variety of problems with conventional methods including slow isolation and propagation, inhibition by neutralizing antibodies, low sensitivity of immunoassays, and the diversity of HAdV strains for the PCR technique. Herein, we report the development and evaluation of a novel, simple, and reliable nanobased immunosensing technique for the rapid detection of human adenoviruses (HAdVs) that cause eye infections. This rapid and low-cost assay can be used for screening and quantitative tests with a detection limit of 102 pfu/mL in less than 2 min. The sensing platform is based on a sandwich assay that can detect HAdVs visually by a color change. Sensor specificity was demonstrated using other common viral antigens, including Flu A, Flu B, coronavirus (COV), and Middle East respiratory syndrome coronavirus (MERS COV). This cotton-based testing device potentially exhibits many of the desired characteristics of a suitable point-of-care and portable test, which can be carried out by nurses or clinicians especially for low-resource settings.

Development of rapid colorimetric assay for the detection of Influenza A and B viruses.

The flu viruses are respiratory pathogens which, according to the World Health Organization (WHO), infect 5-10% of the world population resulting in 3-5 million cases of severe illness and 290,000 to 650,000 annual deaths. Early diagnosis and therapeutic intervention can ameliorate symptoms of infection and reduce mortality. The conventional diagnosis of viral infections, including flu viruses, has evolved over the years with diverse approaches, however, there are inherent short comings associated with these testing. There is an urgent need for rapid and low-cost diagnostic assays, due to the enormous annual burden of influenza diseases and its associated mortality. In this study, novel, low cost and easy to use colorimetric flu virus biosensor assay was developed. The sandwich assay format was utilized using antibodies immobilized onto cotton swabs, for the rapid detection of flu A and B viruses. These swabs serve as sample collection, analytes pre-concentration as well as sensing tool. The proof of concept was established for this assay in buffer and mucus samples. The limit of detection (LOD) of the colorimetric assay was 0.04 ng mL-1 for Flu A and Flu B respectively and with linear dynamic range between 0.04 ng ml-1 to 40 ng ml for both viruses in mucous samples. The assay can be performed at the patient's bed side by minimally skilled hospital personnel without the need for instrumentation. Cross-reactivity assays testing was done using Flu viruses specific activated swabs reacted with other common respiratory viral pathogens' antigen, in order to assess the specificity of the swabs.

Single-cell Analysis of ß2-Adrenergic Receptor Dynamics by Quantitative Fluorescence Microscopy.

BACKGROUND: G protein-coupled receptors (GPCRs) represent the largest family of surface proteins and are involved in the regulation of key physiological processes. GPCRs are characterized by seven transmembrane domains, an extracellular N-terminus and an intracellular C-terminus. Cellular response of these receptors to their ligands is largely determined by their surface expression and postactivation behavior including expression, desensitization and resensitization. OBJECTIVE: To develop a quantitative fluorescence Microscopy assay to study ß2- Adrenergic receptor expression and desensitization. METHOD: ß2-Adrenergic receptor cDNA was engineered to put an HA tag at the extracellular N-terminus and GFP Tag at the intracellular C-terminus. GFP fluorescence serves as a measure of total cellular expression; whereas staining with CY3 conjugated anti-HA antibodies without permeabilizing the cells represents the surface expression of ß2-AR. The images are quantified and amount of CY3 (surface) and GFP (total) fluorescence for each cell determined using image processing software. RESULTS: The method is sensitive and allows for the simultaneous measurement of surface and total expression of ß2-AR. CONCLUSION: A highly accurate method is described for measuring ß2-AR surface and total expression based on single-cell quantitative immunofluorescence. The method can be used to determine agonist-induced desensitization and resensitization process as well as receptor kinetics like endocytosis and exocytosis of ß2-Adrenergic receptor and can be applied to essentially any other GPCR.

Development of a Simple, Fast, and Cost-Effective Nanobased Immunoassay Method for Detecting Norovirus in Food Samples.

This study presents a quick, low-cost, and easy technique for the detection of norovirus in several food samples, including cucumber, lettuce, and chicken. The developed sandwich immunoassay method depends on employing nanotechnology for the detection step. Lactoferrin immobilized on activated Q-tips cotton swabs was used as a general capturing reagent to bind viruses from the sample surface. The cotton swabs were then submerged in a gold nanoparticle solution, which had previously decorated with a specific antibody for noroviruses. Positive samples retained the red color of the gold nanoparticles on the surface of Q-tips, even after washing, while the negative control samples easily lost their color through washing. The results confirmed that the developed assay has superior sensitivity and selectivity with a LOD between 10 and 53 pfu/mL for all tested samples. In light of the difficulty, complexity, and high cost of the methods recently used for detecting viruses in food samples, this method presents a promising reliable technique that can be employed for the rapid detection of norovirus in food samples with an acceptable accuracy.