Assay development for essential enzyme activity in the tegument of live SchistosomesMadhu Sundaraneedi1 , Luke Becker2 , Giovanni Abbenante3 , Alex Loukas2 , Grant Collins1 , Mark Pearson2, 1School of Physical, Environmental and Mathematical Sciences, University of New South Wales, Australia , 2Australian Institute for Tropical Health and Medicine, James Cook University, Australia , 3BMG LABTECH Australia, 12/2017
Schistosomiasis is a parasitic disease that affects over 200 million people in tropical, developing nations, causing severe morbidity and over 300,000 deaths annually. Schistosomiasis is treated with a single drug and no vaccine is available.
We selected three Schistosoma surface-associated enzymes that are indispensable to parasitic survival: alkaline phosphatase; phosphodiesterase SmNPP-5 and an acetylcholinesterase. The activity of these molecules on the surface of live and intact larval and adult Schistosoma can be assayed in real-time of cultured parasites, providing a tool to assess the efficacy of drugs or vaccines targeting these enzymes. The colorimetric assay was read on a FLUOstar® Omega microplate reader.
The versatile instrument supports development of assays and drugs. Apart from its absorbance spectrometer, the FLUOstar Omega is equipped with fluorescence and luminescence detection capabilities allowing fast and reliable endpoint and kinetic measurements in all detection modes.
Calcium retention capacity assay evaluates inhibition of mitochondrial permeability transition poreM. Awais , D. Latawiec , R. Sutton, Liverpool Pancreatitis Research Group, Institute of Translational Medicine, University of Liverpool, UK, 11/2017
Mitochondrial dysfunction is central to the pathogenesis of acute pancreatitis, ischemia-reperfusion injury of the heart, brain and kidney, muscular dystrophies and neurodegeneration. Mitochondrial dysfunction is the result of a sudden increase in permeability of the inner mitochondrial membrane (IMM), via persistent opening of a multi-protein channel known as the mitochondrial permeability transition pore (MPTP). This is followed by uncontrolled proton flow across the IMM and unregulated flux of water, ions and small solutes into and out of the mitochondrial matrix. This results in rupture of the outer mitochondrial membrane (OMM) and eventually cell death by necrosis. Therefore, MPTP is an attractive target for cell death prevention in a host of disease states.
The calcium retention capacity assay challenges isolated mitochondria with spikes of calcium ions. Upon opening of the MPTP, Ca2+ leaks into the assay buffer and increases fluorescence of the membrane-impermeable CalciumGreen™ dye. The Omega multi-mode plate reader has proven excellent robustness for performing the multiple injections as well as reliable fluorescent detection of the assay.
Protein-induced fluorescence enhancement detects protein-nucleic acid interactions in microplatesFulneček J. , Valuchová S. , Petrov A.P. , Tripsianes K. , Říha K., Research group Molecular Biology of Plants, Central European Institute of Technology, Masaryk University, Brno, Czech Republic, 10/2017
Various biological processes depend on the interaction of proteins with nucleic acids. These comprise transcriptional regulation, DNA replication and DNA repair. Conventional methods to study the interaction are either laborious (EMSA, ChIP) or limited to specific targets (EMSA, FP, ChIP).
A novel assay employs the Cy3 fluorophore which increases its fluorescence when being in proximity to proteins: Cy3-labeled oligonucleotides are immobilized on a microplate bottom and the basal fluorescence is measured on a microplate reader. If a protein is added to the microplate well that binds the oligonucleotide, the fluorescence of the fluorophore will increase. This increase can be measured and reports on protein-nucleic acid binding.
This simple and cost-effective assay detects sequence and structure specificities as well as binding constants of nucleic acid:protein liaisions. The change in fluorescence intensity is captured on a huge surface to provide stable measurements. This has been enabled by the well-scan function and high sensitivity of the FLUOstar® Omega.
Quantifying double-stranded DNA with fluorescent dyes: Qubit on BMG LABTECH instrumentsAndrea Krumm , Mark Gröne , Franka Maurer, BMG LABTECH GmbH, Ortenberg, Germany, 07/2017
The quantification of DNA is a standard laboratory activity and pivotal for subsequent applications such as next generation sequencing (NGS). The widely used Qubit™ nucleic acid staining dyes are analyzed on a Qubit™ fluorometer. It measures fluorescence intensity directly in sample preparation tubes and automatically calculates the DNA-concentration based on two standards measured in parallel. This provides an easy, quick and intuitive way to quantitate dsDNA. However, throughput is limited using the Qubit™ fluorometer as only one sample can be analyzed at a time.
This application not presents how the Qubit® dsDNA HS fluorophore can be used to quantitate dsDNA on a BMG LABTECH microplate reader. The Qubit kit read on the FLUOstar® Omega, CLARIOstar® and PHERAstar® resulted in a high linearity (R2 > 0.9999) and quantification of DNA dilutions of 50 pg/μl-100 ng/μl. Detecting Qubit chemistry on BMG LABTECH microplate readers increases throughput while keeping the simplicity of the quantification method.
Detection of plant-synthesized nanoparticles and their antibacterial capacitySalem W. and Schild S., University of Graz , Institute of Molecular Biosciences , BioTechMed-Graz , Austria, 03/2017
Metallic nanoparticles became subject of intensive research because of their potential antibiotic properties. Nanoparticles such as silver, gold or zinc oxide particles are easily and cost-effectively synthesized by blending metal salts with plant extracts that reduce the metal. Different extracts, varying in the plant or the part of the plant used for the extract, are currently investigated in regard to their capacity to form nanoparticles and their antimicrobial efficacy. The formation of nanoparticles can be verified by UV-Vis spectroscopy due to surface plasmon resonance of the particles that lead to a characteristic spectrum defined by the underlying metal and particle size. Subsequent analysis of nanoparticles on microbial growth is typically tested by methods based on absorbance changes.
Here, we present how the spectrometer-based BMG LABTECH instruments are used to quickly confirm Ag and ZnO nanoparticle formation and their inhibitory effect on the diarrhea-causing bacteria Vibrio cholerae and enterotoxic Escherichia coli (ETEC).