The Experts below are selected from a list of 254202 Experts worldwide ranked by ideXlab platform
Wei Zheng - One of the best experts on this subject based on the ideXlab platform.
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high throughput cell based Assay for identification of glycolate oxidase inhibitors as a potential treatment for primary hyperoxaluria type 1
Scientific Reports, 2016Co-Authors: Noel Southall, Mengqiao Wang, Miao Xu, Yan Long, Sonia Fargue, Xin Hu, John C Mckew, Christopher J Danpure, Wei ZhengAbstract:Glycolate oxidase (GO) and alanine:glyoxylate aminotransferase (AGT) are both involved in the peroxisomal glyoxylate pathway. Deficiency in AGT function causes the accumulation of intracellular oxalate and the primary hyperoxaluria type 1 (PH1). AGT enhancers or GO inhibitors may restore the abnormal peroxisomal glyoxylate pathway in PH1 patients. With stably transformed cells which mimic the glyoxylate metabolic pathway, we developed an indirect glycolate cytotoxicity Assay in a 1,536-well plate format for high throughput screening. This Assay can be used to identify compounds that reduce indirect glycolate-induced cytotoxicity by either enhancing AGT activity or inhibiting GO. A pilot screen of 4,096 known compounds identified two membrane permeable GO inhibitors: dichromate salt and colistimethate. We also developed a GO Enzyme Assay using the hydrogen peroxide-Amplex red reporter system. The IC50 values of potassium dichromate, sodium dichromate, and colistimethate sodium were 0.096, 0.108, and 2.3 μM in the GO Enzyme Assay, respectively. Further Enzyme kinetic study revealed that both types of compounds inhibit GO activity by the mixed linear inhibition. Our results demonstrate that the cell-based Assay and GO Enzyme Assay developed in this study are useful for further screening of large compound libraries for drug development to treat PH1.
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synthesis and characterization of a new fluorogenic substrate for alpha galactosidase
Analytical and Bioanalytical Chemistry, 2009Co-Authors: Zhenda Shi, Omid Motaba, Ehud Goldi, Gary L Griffiths, Noel Southall, Christophe P Austi, Ke Liu, Ellen Sidransky, Wei ZhengAbstract:Alpha-galactosidase A hydrolyzes the terminal alpha-galactosyl moieties from glycolipids and glycoproteins in lysosomes. Mutations in α-galactosidase cause lysosomal accumulation of the glycosphingolipid, globotriaosylceramide, which leads to Fabry disease. Small-molecule chaperones that bind to mutant Enzyme proteins and correct their misfolding and mistrafficking have emerged as a potential therapy for Fabry disease. We have synthesized a red fluorogenic substrate, resorufinyl α-d-galactopyranoside, for a new α-galactosidase Enzyme Assay. This Assay can be measured continuously at lower pH values, without the addition of a stop solution, due to the relatively low pK a of resorufin (~6). In addition, the Assay emits red fluorescence, which can significantly reduce interferences due to compound fluorescence and dust/lint as compared to blue fluorescence. Therefore, this new red fluorogenic substrate and the resulting Enzyme Assay can be used in high-throughput screening to identify small-molecule chaperones for Fabry disease.
Theo Nikiforov - One of the best experts on this subject based on the ideXlab platform.
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a microchip based Enzyme Assay for protein kinase a
Analytical Biochemistry, 1999Co-Authors: Claudia B Cohen, Emily Chindixon, Sang Jeong, Theo NikiforovAbstract:A microchip-based Enzyme Assay for protein kinase A is described. The microchips were prepared by standard photolithographic techniques. The Assay reagents were placed in wells on the microchips, and electroosmosis was used to transport aliquots of these reagents into the network of etched channels, where the enzymatic reaction takes place. Protein kinase A catalyzes the transfer of a phosphate group from ATP to the serine residue of the heptapeptide LeuArgArgAlaSerLeuGly (Kemptide). The outcome of the enzymatic reaction was assessed by performing an on-chip electrophoretic separation of the fluorescently labeled peptide substrate and product. All liquid-handling steps were performed by controlling the electroosmotically driven flow from reagent and buffer wells using electrical current. On-chip dilutions of the peptide substrate, ATP and H-89, a known protein kinase A inhibitor, were performed and the kinetic constants (K(m), K(i)) of these compounds were determined. This prototype Assay demonstrates the usefulness of the microchips for performing enzymatic Assays for which fluorogenic substrates cannot easily be designed.
Deeksha Bali - One of the best experts on this subject based on the ideXlab platform.
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development of a fluorometric microtiter plate based Enzyme Assay for arylsulfatase b mps vi using dried blood spots
Molecular genetics and metabolism reports, 2014Co-Authors: Anirudh J Ullal, David S Millington, Deeksha BaliAbstract:Abstract Mucopolysaccharidosis type VI or Maroteaux–Lamy syndrome is an autosomal recessive lysosomal storage disorder caused by deficiency of arylsulfatase B (ARS-B) Enzyme activity. It results in mild to severe multi-organ system failure from accumulation of undigested glycosaminoglycans (GAGs); dermatan sulfate and chondroitin-4-sulfate. We have developed a single-step Enzyme Assay using a fluorescent substrate and dried blood spots to measure ARS-B activity to identify disease patients. This Assay is robust, reproducible, specific and convenient to perform.
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novel application of digital microfluidics for the detection of biotinidase deficiency in newborns
Clinical Biochemistry, 2013Co-Authors: Carrie Graham, Ramakrishna Sista, Allen E Eckhardt, David S Millington, Deeksha Bali, Jairus Kleinert, Ning Wu, Vamsee K PamulaAbstract:Objective Newborn screening for biotinidase deficiency can be performed using a fluorometric Enzyme Assay on dried blood spot specimens. As a pre-requisite to the consolidation of different enzymatic Assays onto a single platform, we describe here a novel analytical method for detecting biotinidase deficiency using the same digital microfluidic cartridge that has already been demonstrated to screen for five lysosomal storage diseases (Pompe, Fabry, Gaucher, Hurler and Hunter) in a multiplex format.
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a novel fluorometric Enzyme analysis method for hunter syndrome using dried blood spots
Molecular Genetics and Metabolism, 2012Co-Authors: Adviye A Tolun, Carrie Graham, Qun Shi, Ramakrishna Sista, Tong Wang, Allen E Eckhardt, Vamsee K Pamula, David S Millington, Deeksha BaliAbstract:Mucopolysaccharidosis type II (MPS II) or Hunter syndrome is a lysosomal storage disease caused by deficiency of iduronate-2-sulfatase (IDS). A convenient single-step fluorometric microplate Enzyme Assay has been developed and validated for clinical diagnosis of MPS II using dried blood spots (DBS). The Assay compared well with a recently reported digital microfluidic method, from which it was adapted. Results show that this DBS Assay is robust and reproducible using both technologies.
Adams Alexandra - One of the best experts on this subject based on the ideXlab platform.
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Development of diagnostic Assays for differentiation of atypical Aeromonas salmonicida vapA type V and type VI in ballan wrasse (Labrus bergylta, Ascanius)
'Wiley', 2021Co-Authors: Papadopoulou Athina, Davie Andrew, Monaghan, Sean J, Migaud Herve, Adams AlexandraAbstract:Aeromonas salmonicida (As) is a highly heterogeneous bacterial species, and strains’ host specificity has been reported. Ballan wrasse (Labrus bergylta Ascanius, 1767) is susceptible to atypical As (aAs) vapA type V and type VI in Scotland and Norway. Identification of the bacterium is achieved by culture and molecular techniques; however, the available methods used to distinguish the As types are costly and time‐consuming. This paper describes the development of a PCR and a restriction Enzyme Assay for the detection of aAs vapA type V and type VI in ballan wrasse, respectively. Type V‐specific primers were designed on conserved regions of the vapA gene, and the restriction Enzyme Assay was performed on the PCR products of the hypervariable region of vapA gene for the detection of type VI isolates. Amplification product was produced for type V (254 bp) and restriction bands (368 and 254 bp) for type VI isolates only. In addition, the Assays detected type V and type VI isolates in spiked water samples and type V in diagnostic tissue samples. The Assays are fast, specific and cost‐effective and can be used as specific diagnostic tools for cleaner fish, to detect infectious divergence strains, and to manage and mitigate aAs disease outbreaks through vaccine development.Output Status: Forthcoming/Available Onlin
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Development of diagnostic Assays for differentiation of atypical Aeromonas salmonicida vapA type V and type VI in ballan wrasse (Labrus bergylta, Ascanius)
'Wiley', 2021Co-Authors: Papadopoulou Athina, Davie Andrew, Monaghan, Sean J, Migaud Herve, Adams AlexandraAbstract:Aeromonas salmonicida (As) is a highly heterogeneous bacterial species, and strains’ host specificity has been reported. Ballan wrasse (Labrus bergylta Ascanius, 1767) is susceptible to atypical As (aAs) vapA type V and type VI in Scotland and Norway. Identification of the bacterium is achieved by culture and molecular techniques; however, the available methods used to distinguish the As types are costly and time‐consuming. This paper describes the development of a PCR and a restriction Enzyme Assay for the detection of aAs vapA type V and type VI in ballan wrasse, respectively. Type V‐specific primers were designed on conserved regions of the vapA gene, and the restriction Enzyme Assay was performed on the PCR products of the hypervariable region of vapA gene for the detection of type VI isolates. Amplification product was produced for type V (254 bp) and restriction bands (368 and 254 bp) for type VI isolates only. In addition, the Assays detected type V and type VI isolates in spiked water samples and type V in diagnostic tissue samples. The Assays are fast, specific and cost‐effective and can be used as specific diagnostic tools for cleaner fish, to detect infectious divergence strains, and to manage and mitigate aAs disease outbreaks through vaccine development
Christopher T Seto - One of the best experts on this subject based on the ideXlab platform.
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kinetic delay of cyclization elimination coupled Enzyme Assays analysis and solution
Bioorganic & Medicinal Chemistry Letters, 2011Co-Authors: Christopher T SetoAbstract:Abstract A kinetic analysis of an Enzyme Assay employing a synthetic substrate that produces a detectable signal through a spontaneous organic cyclization/elimination reaction following the enzymatic reaction was conducted. The results from the calculation were used to predict the lag period and provide accurate measurements of the activity of alkaline phosphatase using the fluorogenic substrate ( 1 ).